PL191529B1 - Nowa odmiana proteazy, cząsteczka DNA kodująca nową odmianę proteazy, wektor zawierający tę cząsteczkę oraz karma dla zwierząt i kompozycje zawierające nową odmianę proteazy - Google Patents
Nowa odmiana proteazy, cząsteczka DNA kodująca nową odmianę proteazy, wektor zawierający tę cząsteczkę oraz karma dla zwierząt i kompozycje zawierające nową odmianę proteazyInfo
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- PL191529B1 PL191529B1 PL340600A PL34060098A PL191529B1 PL 191529 B1 PL191529 B1 PL 191529B1 PL 340600 A PL340600 A PL 340600A PL 34060098 A PL34060098 A PL 34060098A PL 191529 B1 PL191529 B1 PL 191529B1
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- positions
- substitution
- amino acid
- subtilisin
- residue
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Abstract
1. Odmiana proteazy, znamienna tym, ze zawiera podstawienie aminowasowe w pozycji reszty odpowiadajacej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadajacej nastepujacym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jesli odmiana obejmuje podstawienie jednoczesnie w pozycjach 103 i 76, to wystepuje równiez pod- stawienie w przynajmniej jednej z pozycji innych niz odpowiadajace nastepujacym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274. 9. Wektor ekspresyjny, znamienny tym, ze obejmuje sekwencje regulatorowe funkcjonalnie polaczone z sekwencja DNA kodujaca odmiane proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadajacej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadajacej nastepujacym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jesli odmiana obejmuje podstawienie jednoczesnie w pozycjach 103 i 76, to wystepuje równiez pod- stawienie w przynajmniej jednej z pozycji innych niz odpowiadajace nastepujacym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie sekwencja regulatorowa obejmuje promotor, sekwencje operatora, sekwencje kodujaca miejsce wiazania rybosomu z mRNA oraz sekwencje regulujace terminacje transkrypcji i translacji. PL PL PL PL PL PL PL
Description
Przedmiotem wynalazku jest nowa odmiana proteazy, cząsteczka DNA kodująca nową odmianę proteazy, wektor ekspresyjny obejmujący DNA kodujący nową odmianę proteazy i komórka gospodarza zawierająca wektor ekspresyjny obejmujący DNA kodujący nową odmianę proteazy oraz karma dla zwierząt i kompozycja do czyszczenia i kompozycja do obróbki tekstyliów zawierające nową odmianę proteazy.
Proteazy serynowe stanowią podgrupę hydrolaz karbonylowych. Obejmują one klasę różnorodnych enzymów o szerokim zakresie specyficzności i funkcji biologicznych, Stroud. R. Sci. Amer., 131: 74-88. Pomimo ich funkcjonalnej różnorodności, katalityczną aktywność proteaz serynowych uzyskano dla przynajmniej dwóch genetycznie odmiennych rodzin enzymów:
1) subtylizyn oraz
2) pokrewnych pochodzącej od ssaków chymotrypsynie i homologicznych proteaz bakteryjnych (np. trypsyna i trypsyna z S. gresius).
Te dwie rodziny proteaz serynowych wykazują niezwykle podobne mechanizmy katalizy, Kraut. J. (1977), Annu. Rev. Biochem., 46: 331-358. Ponadto, chociaż ich zasadnicza budowa nie jest zbliżona, struktura trzeciorzędowa obejmuje zachowaną katalityczną triadę aminokwasową złożoną z seryny, histydyny i asparaginianu.
Subtylizyny są proteazami serynowymi (przybliżona masa cząsteczkowa 27500), wydzielanymi w dużych ilościach przez bakterie z rodzaju Bacillus i inne bakterie. Białkową sekwencję subtylizyny określono dla przynajmniej dziewięciu różnych gatunków Bacillus, Markland F. S. i in. (1983), Hoppe-Seyler's Z. Physiol. Chem., 364: 1537-1540.
Przedstawiono trójwymiarową strukturę krystalograficzną subtylizyn z Bacillus amyloliquefaciens, Bacillus licheniformis oraz kilku naturalnych odmian B. lentus. Te badania wykazały, że chociaż subtylizyna nie jest genetycznie spokrewniona z ssaczymi proteazami serynowymi, to ma podobną strukturę miejsc aktywnych.
Rentgenowskie badania struktury krystalicznej subtylizyny zawierającej kowalencyjnie związane inhibitory peptydowe (Robertus J. D., i in. (1972), Biochemistry. 11: 2439-2449) lub kompleksy (Robertus J. D., i in.(1976), J. Biol. Chem., 251: 1097-1103) również dostarczyły informacji o aktywnych pozycjach i przypuszczalnej lokalizacji wiązania substratów subtylizyny.
Ponadto, wykonano wiele badań kinetycznych i chemicznych odmian subtylizyny, jak opisano w pracach Svendsen, B. (1976), Carlsberg. Res. Commun., 41: 237-291; Markland F. S., tamże).
Istnieje przynajmniej jedno doniesienie o eksperymencie, w którym boczny łańcuch metioniny w pozycji reszty 222 subtylizyny poddano konwersji działaniem nadtlenku wodoru, uzyskując sulfotlenek metioniny (Stauffer, D. C., i in. (1965) J. Biol. Chem. 244: 5333-5338).
Przeprowadzono także oraz obszerne badania techniką mutagenezy punktowej (Wells i Estell (1988) TIBS 13: 291-297).
Przedmiotem wynalazku jest nowa odmiana proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12,13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274.
Korzystnie odmiana proteazy zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 i 236 oraz w przynajmniej jednej z pozycji: 1, 9, 12, 61, 62, 68, 76, 97, 98, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270 i 275 albo podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 i 245 oraz w przynajmniej jednej z pozycji: 1, 9, 12, 61, 62, 68, 76, 97, 98, 97, 98, 101, 102, 104,
PL 191 529 B1
109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270 i 275.
W innym korzystnym rozwiązaniu odmiana proteazy według zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103, 236 i 245 oraz w przynajmniej jednej z pozycji: 1, 9, 12, 61, 62, 68, 76, 97, 98, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270 i 275.
Korzystnie odmiana proteazy zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 159.
W korzystnym rozwiązaniu odmiana proteazy pochodzi od subtylizyny z Bacillus, korzystniej od subtylizyny z Bacillus lentus.
Inną odmianą wynalazku jest cząsteczka DNA, która zawiera kodony kodujące odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom wtej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274.
Kolejną odmianą wynalazku jest wektor ekspresyjny, który obejmuje sekwencje regulatorowe funcjonalnie połączone z sekwencją DNA kodującą odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom wtej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie sekwencja regulatorowa obejmuje promotor, sekwencje operatora, sekwencję kodującą miejsce wiązania rybosomu z mRNA oraz sekwencje regulujące terminację transkrypcji i translacji.
Kolejną odmianą wynalazku jest komórka gospodarza znamienna tym, że jest transforomowana wektorem ekspresyjnym obejmującym sekwencje regulatorowe funcjonalnie połączone z sekwencją DNA kodującą odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104,
PL 191 529 B1
107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie szczep Bacillus BG2036, Bacillus subtilis 1168 oraz B. licheniformis oraz B. lentus.
Przedmiotem wynalazku jest ponadto kompozycja do czyszczenia, która jako środek aktywny zawiera odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie w ilości od 0,01% do 5% wagowych w proszkowych lub ciekłych środkach czyszczących.
Innym przedmiotem wynalazku jest karma dla zwierząt, która zawiera odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom wtej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie w ilości od 0,01% do 5% wagowych.
Inną odmianą wynalazku jest kompozycja do obróbki tekstyliów, która zawiera odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie w ilości od 0,01% do 5% wagowych.
Bardziej korzystne odmiany proteaz obejmują zestaw podstawień wybrany z grupy zawierającej pozycje reszt odpowiadające pozycjom w subtylizynie z Bacillus amyloliquefaciens, podanym w tabeli 1.
PL 191 529 B1
Tabel a 1
| 76 | 103 | 104 | 222 | |||||||||
| 76 | 98 | 103 | 104 | |||||||||
| 76 | 78 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 107 | |||||||||
| 4 | 76 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 246 | |||||||||
| 76 | 77 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 183 | 218 | ||||||||
| 16 | 76 | 103 | 104 | 248 | ||||||||
| 1 | 76 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 261 | |||||||||
| 76 | 103 | 104 | 160 | |||||||||
| 76 | 103 | 104 | 216 | |||||||||
| 17 | 76 | 103 | 104 | |||||||||
| 37 | 76 | 103 | 104 | |||||||||
| 76 | 77 | 103 | 104 | 174 | ||||||||
| 38 | 76 | 103 | 104 | |||||||||
| 38 | 76 | 103 | 104 | 237 | ||||||||
| 8 | 76 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 183 | |||||||||
| 19 | 76 | 103 | 104 | |||||||||
| 13 | 76 | 103 | 104 | |||||||||
| 19 | 76 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 184 | |||||||||
| 76 | 103 | 104 | 252 | |||||||||
| 76 | 103 | 104 | 259 | |||||||||
| 76 | 103 | 104 | 251 | |||||||||
| 76 | 86 | 103 | 104 | |||||||||
| 72 | 76 | 103 | 104 | 185 | ||||||||
| 76 | 103 | 104 | 237 | 274 | ||||||||
| 76 | 103 | 104 | 160 | |||||||||
| 76 | 103 | 104 | 228 | |||||||||
| 55 | 76 | 103 | 104 | 240 | ||||||||
| 76 | 103 | 104 | 254 | |||||||||
| 76 | 103 | 104 | 204 | |||||||||
| 76 | 103 | 104 | 204 | |||||||||
| 43 | 76 | 103 | 104 |
PL 191 529 B1 ciąg dalszy tabeli 1
| 76 | 103 | 104 | 159 | |||||||||
| 10 | 76 | 103 | 104 | 177 | ||||||||
| 58 | 76 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 270 | |||||||||
| 76 | 103 | 104 | 185 | |||||||||
| 27 | 76 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 262 | |||||||||
| 76 | 78 | 103 | 104 | |||||||||
| 24 | 76 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 166 | 236 | 251 | |||||||
| 17 | 76 | 103 | 104 | 237 | ||||||||
| 76 | 103 | 104 | 130 | |||||||||
| 76 | 103 | 104 | 109 | |||||||||
| 76 | 99 | 103 | 104 | 204 | ||||||||
| 76 | 103 | 104 | 181 | |||||||||
| 12 | 76 | 103 | 104 | |||||||||
| 76 | 103 | 104 | 212 | 271 | ||||||||
| 76 | 103 | 104 | 252 | 261 | ||||||||
| 76 | 103 | 104 | 242 | |||||||||
| 76 | 103 | 104 | 271 | |||||||||
| 12 | 76 | 103 | 104 | 242 | ||||||||
| 43 | 76 | 103 | 104 | 116 | 183 | |||||||
| 76 | 103 | 104 | 258 | |||||||||
| 76 | 103 | 104 | 271 | |||||||||
| 61 | 76 | 103 | 104 | |||||||||
| 38 | 76 | 103 | 104 | 182 | 263 | |||||||
| 76 | 103 | 104 | 182 | 272 | ||||||||
| 76 | 103 | 104 | 109 | 246 | ||||||||
| 76 | 87 | 103 | 104 | 206 | 249 | 265 | ||||||
| 76 | 103 | 104 | 137 | 238 | 271 | |||||||
| 103 | 104 | 228 | ||||||||||
| 76 | 103 | 104 | 182 | 198 | ||||||||
| 21 | 76 | 103 | 104 | 182 | ||||||||
| 76 | 103 | 104 | 119 | 137 | ||||||||
| 76 | 103 | 104 | 137 | 248 | ||||||||
| 13 | 76 | 103 | 104 | 206 | ||||||||
| 76 | 103 | 104 | 206 |
PL 191 529 B1 ciąg dalszy tabeli 1
| 76 | 103 | 104 | 212 | 258 | ||||||||
| 58 | 76 | 103 | 104 | 271 | ||||||||
| 76 | 103 | 104 | 206 | 261 | ||||||||
| 4 | 76 | 103 | 104 | 206 | ||||||||
| 76 | 77 | 103 | 104 | 206 | ||||||||
| 76 | 103 | 104 | 158 | |||||||||
| 76 | 103 | 104 | 206 | |||||||||
| 4 | 76 | 103 | 104 | 159 | 217 | 251 | ||||||
| 4 | 76 | 103 | 104 | 159 | 217 | 252 | ||||||
| 76 | 77 | 103 | 104 | 133 | 185 | 251 | ||||||
| 76 | 103 | 104 | 159 | 206 | 244 | |||||||
| 4 | 76 | 103 | 104 | 188 | ||||||||
| 4 | 76 | 103 | 104 | 158 | ||||||||
| 76 | 77 | 103 | 104 | 185 | ||||||||
| 76 | 103 | 104 | 206 | 251 | ||||||||
| 48 | 76 | 103 | 104 | 111 | 159 | |||||||
| 68 | 76 | 103 | 104 | 159 | 236 | |||||||
| 42 | 76 | 103 | 104 | 159 | ||||||||
| 12 | 62 | 76 | 103 | 104 | 159 | |||||||
| 42 | 76 | 103 | 104 | 159 | ||||||||
| 76 | 103 | 104 | 146 | 159 | ||||||||
| 76 | 103 | 104 | 159 | 238 | ||||||||
| 76 | 103 | 104 | 159 | 224 | ||||||||
| 76 | 103 | 104 | 212 | 268 | 271 | |||||||
| 76 | 89 | 103 | 104 | |||||||||
| 76 | 87 | 103 | 104 | 212 | 271 | |||||||
| 76 | 103 | 104 | 212 | 245 | 271 | |||||||
| 76 | 103 | 104 | 134 | 141 | 212 | 271 | ||||||
| 76 | 103 | 104 | 212 | 236 | 243 | 271 | ||||||
| 76 | 103 | 104 | 109 | 245 | ||||||||
| 76 | 103 | 104 | 109 | 210 | ||||||||
| 20 | 62 | 76 | 103 | 104 | ||||||||
| 68 | 76 | 103 | 104 | 236 | ||||||||
| 68 | 76 | 103 | 104 | 159 | 236 | 271 | ||||||
| 68 | 76 | 103 | 104 | 159 | 236 | 245 | ||||||
| 68 | 76 | 103 | 104 | 159 | 217 | 236 | 271 | |||||
| 17 | 68 | 76 | 103 | 104 |
PL 191 529 B1 ciąg dalszy tabeli 1
| 68 | 76 | 103 | 104 | |||||||||
| 68 | 76 | 103 | 104 | 159 | 236 | |||||||
| 68 | 75 | 76 | 103 | 104 | 159 | 236 | ||||||
| 68 | 76 | 76 | 103 | 114 | 121 | 159 | 236 | 245 | ||||
| 12 | 68 | 76 | 103 | 104 | 159 | 236 | ||||||
| 68 | 76 | 103 | 104 | 159 | 209 | 236 | 253 | |||||
| 68 | 76 | 103 | 104 | 117 | 159 | 184 | 236 | |||||
| 68 | 76 | 103 | 104 | 159 | 236 | 243 | ||||||
| 68 | 76 | 103 | 104 | 159 | 236 | 245 | ||||||
| 68 | 76 | 103 | 104 | 142 | 159 | |||||||
| 68 | 76 | 103 | 104 | 123 | 159 | 236 | 249 | |||||
| 68 | 76 | 103 | 104 | 159 | 236 | 249 | ||||||
| 76 | 103 | 104 | 222 | 245 | ||||||||
| 12 | 76 | 103 | 104 | 222 | 249 | |||||||
| 76 | 103 | 104 | 173 | 222 | ||||||||
| 76 | 103 | 104 | 222 | 263 | ||||||||
| 21 | 76 | 103 | 104 | 222 | 237 | 263 | ||||||
| 76 | 103 | 104 | 109 | 222 | ||||||||
| 76 | 103 | 104 | 109 | 222 | 271 | |||||||
| 61 | 76 | 103 | 104 | 222 | ||||||||
| 76 | 103 | 104 | 137 | 222 | ||||||||
| 76 | 103 | 104 | 109 | 222 | 248 | |||||||
| 76 | 103 | 104 | 222 | 249 | ||||||||
| 68 | 76 | 103 | 104 | 159 | 236 | 245 | 261 | |||||
| 68 | 76 | 103 | 104 | 141 | 159 | 236 | 245 | 255 | ||||
| 68 | 76 | 103 | 104 | 159 | 236 | 245 | 247 | |||||
| 68 | 76 | 103 | 104 | 159 | 174 | 204 | 236 | 245 | ||||
| 68 | 76 | 103 | 104 | 159 | 204 | 236 | 245 | |||||
| 68 | 76 | 103 | 104 | 133 | 159 | 218 | 236 | 245 | ||||
| 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
| 68 | 76 | 103 | 104 | 159 | 194 | 203 | 236 | 245 | ||||
| 12 | 76 | 103 | 104 | 222 | 245 | |||||||
| 76 | 103 | 104 | 232 | 245 | ||||||||
| 24 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | |||||
| 68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
| 12 | 76 | 103 | 104 | 222 | 244 | 245 |
PL 191 529 B1 ciąg dalszy tabeli 1
| 12 | 76 | 103 | 222 | 210 | 245 | |||||||
| 12 | 76 | 103 | 104 | 130 | 222 | 245 | ||||||
| 22 | 68 | 76 | 103 | 104 | ||||||||
| 68 | 76 | 103 | 104 | 184 | ||||||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | ||||||
| 68 | 103 | 104 | 140 | 159 | 232 | 236 | 245 | 252 | ||||
| 43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
| 43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
| 43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
| 68 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | 275 | |||
| 12 | 76 | 103 | 104 | 130 | 222 | 245 | 248 | 262 | ||||
| 12 | 76 | 103 | 104 | 130 | 215 | 222 | 245 | |||||
| 12 | 76 | 103 | 104 | 130 | 222 | 227 | 245 | 262 | ||||
| 12 | 76 | 103 | 104 | 130 | 222 | 245 | 261 | |||||
| 76 | 103 | 104 | 130 | 222 | 245 | |||||||
| 12 | 76 | 103 | 104 | 130 | 218 | 222 | 245 | 262 | 269 | |||
| 12 | 57 | 76 | 103 | 104 | 130 | 222 | 245 | 251 | ||||
| 12 | 76 | 103 | 104 | 130 | 170 | 185 | 222 | 243 | 245 | |||
| 12 | 76 | 103 | 104 | 130 | 222 | 245 | 268 | |||||
| 12 | 76 | 103 | 104 | 130 | 222 | 210 | 245 | |||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
| 68 | 103 | 104 | 116 | 159 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | |||||
| 10 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 203 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 232 | 236 | 237 | 245 | |||||
| 68 | 76 | 79 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
| 68 | 103 | 104 | 159 | 183 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 174 | 206 | 232 | 236 | 245 | ||||
| 68 | 103 | 104 | 159 | 188 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | |||||
| 68 | 98 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | |||||
| 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
| 68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 |
PL 191 529 B1 ciąg dalszy tabeli 1
| 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | ||||
| 76 | 103 | 104 | 232 | 236 | 245 | 257 | ||||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 275 | ||||
| 76 | 103 | 104 | 257 | 275 | ||||||||
| 68 | 103 | 104 | 159 | 224 | 232 | 236 | 245 | 257 | ||||
| 76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
| 68 | 76 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | ||||
| 68 | 76 | 103 | 104 | 159 | 211 | 232 | 236 | 245 | ||||
| 12 | 68 | 76 | 103 | 104 | 159 | 214 | 232 | 236 | 245 | |||
| 68 | 76 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | ||||
| 12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
| 20 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 259 | |||
| 68 | 87 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | |||
| 68 | 76 | 103 | 104 | 169 | 232 | 236 | 245 | 261 | ||||
| 76 | 103 | 104 | 232 | 236 | 242 | 245 | ||||||
| 68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
| 12 | 48 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
| 76 | 103 | 104 | 232 | 236 | 245 | |||||||
| 76 | 103 | 104 | 159 | 192 | 232 | 236 | 245 | |||||
| 76 | 103 | 104 | 147 | 159 | 232 | 236 | 245 | 248 | 251 | |||
| 12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 272 | |||
| 68 | 76 | 103 | 104 | 159 | 183 | 206 | 232 | 236 | 245 | |||
| .68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 256 | ||||
| 68 | 76 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | ||||
| 27 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
| 68 | 76 | 103 | 104 | 116 | 159 | 170 | 185 | 232 | 236 | 245 | ||
| 61 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 99 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||
| 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||
| 68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 309 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
| 68 | 103 | 104 | 159 | 185 | 232 | 236 | 245 | 248 | 252 |
PL 191 529 B1 ciąg dalszy tabeli 1
| 68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 185 | 210 | 232 | 236 | 245 | 248 | 252 | ||
| 68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 213 | 232 | 236 | 245 | 248 | 252 | ||||
| 68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 216 | 232 | 236 | 245 | 248 | 252 | |||
| 20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 173 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 251 | 252 | |||
| 68 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 55 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 255 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 256 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 257 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 258 | |||
| 8 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 269 | ||
| 68 | 103 | 104 | 116 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | ||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | |||
| 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 232 | 236 | 245 | 248 | 252 | |||||
| 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 18 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 68 | 76 | 101 | 103 | 104 | 159 | 213 | 218 | 232 | 236 | 245 | 260 | |
| 68 | 103 | 104 | 159 | 228 | 232 | 236 | 245 | 248 | 252 | |||
| 33 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
| 68 | 76 | 89 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | |
| 61 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||
| 103 | 104 | 159 | 205 | 210 | 232 | 236 | 245 | |||||
| 61 | 68 | 103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | ||
| 61 | 68 | 103 | 104 | 133 | 137 | 159 | 232 | 236 | 245 | 248 | 252 | |
| 61 | 103 | 104 | 133 | 159 | 232 | 236 | 245 | 248 | 252 |
PL 191 529 B1 ciąg dalszy tabeli 1
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 68 | 103 | 104 | 159 | 218 | 232 | 236 | 245 | 248 | 252 | |||
| 61 | 68 | 103 | 104 | 159 | 160 | 232 | 236 | 245 | 248 | 252 | ||
| 3 | 61 | 68 | 76 | 103 | 104 | 232 | 236 | 245 | 248 | 252 | ||
| 61 | 68 | 103 | 104 | 159 | 167 | 232 | 236 | 245 | 248 | 252 | ||
| 97 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 98 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 99 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 103 | 104 | 106 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 261 | ||||
| 62 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 103 | 104 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||||
| 103 | 104 | 159 | 166 | 232 | 236 | 245 | 248 | 252 | ||||
| 103 | 104 | 159 | 217 | 232 | 236 | 245 | 248 | 252 | ||||
| 20 | 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
| 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
| 103 | 104 | 159 | 206 | 217 | 232 | 236 | 245 | 248 | 252 | |||
| 62 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
| 103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 27 | 103 | 104 | 159 | 2-32 | 236 | 245 | 248 | 252 | ||||
| 38 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 38 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
| 68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | 271 | ||
| 68 | 76 | 103 | 104 | 159 | 209 | 213 | 232 | 236 | 245 | 260 | ||
| 68 | 76 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | ||
| 68 | 76 | 103 | 104 | 159 | 205 | 213 | 232 | 236 | 245 | 260 | ||
| 68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 260 | |||
| 68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||||
| 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | ||||
| 68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 126 | 232 | 236 | 245 |
PL 191 529 B1 ciąg dalszy tabeli 1
| 68 | 103 | 104 | 159 | 205 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | |||||
| 103 | 104 | 159 | 230 | 236 | 245 | |||||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 260 | |||||
| 103 | 104 | 159 | 232 | 236 | 245 | |||||||
| 68 | 103 | 104 | 159 | 174 | 232 | 236 | 245 | 257 | ||||
| 68 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 257 | ||||
| 68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 257 | ||||
| 103 | 104 | 159 | 232 | 236 | 245 | 257 | ||||||
| 68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | 261 | ||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 261 | ||||
| 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||||
| 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | ||||
| 103 | 104 | 159 | 209 | 232 | 236 | 245 | 257 | |||||
| 68 | 76 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | ||
| 12 | 103 | 104 | 159 | 209 | 213 | 232 | 236 | 245 | 260 | |||
| 103 | 104 | 209 | 232 | 236 | 245 | 257 | ||||||
| 103 | 104 | 159 | 205 | 210 | 213 | 232 | 236 | 245 | 260 | |||
| 103 | 104 | 159 | 205 | 209 | 232 | 236 | 245 | 260 | ||||
| 68 | 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | |||
| 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | 257 | |||
| 103 | 104 | 159 | 205 | 209 | 232 | 236 | 245 | 257 | ||||
| 68 | 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | 260 | ||
| 103 | 104 | 159 | 205 | 209 | 210 | 232 | 236 | 245 | ||||
| 103 | 104 | 159 | 209 | 210 | 232 | 236 | 245 | |||||
| 103 | 104 | 159 | 205 | 210 | 232 | 236 | 245 | |||||
| 68 | 103 | 104 | 128 | 159 | 232 | 236 | 245 | |||||
| -48 | 103 | 104 | 159 | 230 | 236 | 245 | ||||||
| 48 | 68 | 103 | 104 | 169 | 209 | 232 | 236 | 245 | ||||
| 48 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 48 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | 261 | |||
| 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
| 12 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
| 101 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
| 98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
| 102 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
| 103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 |
PL 191 529 B1 ciąg dalszy tabeli 1
| 103 | 104 | 159 | 184 | 232 | 236 | 245 | 248 | 252 | ||||
| 103 | 104 | 159 | 232 | 236 | 244 | 245 | 248 | 252 | ||||
| 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | 256 | ||
| 12 | 62 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
| 101 | 103 | 104 | 159 | 185 | 232 | 236 | 245 | 248 | 252 | |||
| 101 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | 248 | 252 | |||
| 101 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
| 98 | 102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 101 | 102 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | ||
| 98 | 102 | 103 | 104 | 159 | 212 | 232 | 236 | 248 | 252 | |||
| 62 | 103 | 104 | 109 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
| 62 | 103 | 104 | 159 | 212 | 213 | 232 | 236 | 245 | 248 | 252 | ||
| 62 | 101 | 103 | 104 | 159 | 212 | 213 | 232 | 236 | 245 | 248 | 252 | |
| 103 | 104 | 159 | 232 | 245 | 248 | 252 | ||||||
| 103 | 104 | 159 | 230 | 245 | ||||||||
| 62 | 103 | 104 | 130 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
| 101 | 103 | 104 | 130 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 101 | 103 | 104 | 128 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 62 | 101 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
| 62 | 103 | 104 | 128 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
| 62 | 103 | 104 | 128 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | ||
| 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||
| 101 | 103 | 104 | 131 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 98 | 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 99 | 101 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 101 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
| 101 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
| 101 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||
| 101 | 103 | 104 | 159 | 205 | 232 | 236 | 245 | 248 | 252 | |||
| 101 | 103 | 104 | 159 | 230 | 236 | 245 | ||||||
| 101 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 248 | 252 | |||
| 76 | 101 | 103 | 104 | 159 | 194 | 232 | 236 | 245 | 248 | 252 | ||
| 101 | 103 | 104 | 159 | 230 | 232 | 236 | 245 | 248 | 252 | |||
| 62 | 103 | 104 | 159 | 185 | 206 | 213 | 232 | 236 | 245 | 248 | 252 | 271 |
PL 191 529 B1
Bardziej korzystne odmiany proteazy zawierają zestaw podstawień wybrany z grupy obejmującej pozycje reszt odpowiadających pozycjom subtylizyny z Bacillus amyloliquefaciens przedstawionym w tabeli 2.
T a b e l a 2
| N76D | S103A | V104l | M222S | |||||||||
| N76D | A98E | S103A | V104l | |||||||||
| N76D | S78T | S103A | Y104I | |||||||||
| N76D | S103A | V104l | I107Y | |||||||||
| V4E | N76D | S103A | V104l | |||||||||
| N76D | S103A | V104l | I246Y | |||||||||
| N76D | N77D | S103A | V104J | |||||||||
| N76D | S103A | V104l | N183D | N218I | ||||||||
| A16T | N76D | S103A | V104l | N248D | ||||||||
| A1E | N76D | S103A | V104l | |||||||||
| N76D | S103A | V104l | N261D | |||||||||
| N76D | S103A | V104l | S160T | |||||||||
| N76D | S103A | Y104I | S216C | |||||||||
| H17Q | N76D | S103A | V104l | |||||||||
| S37T | N76D | S103A | V104l | |||||||||
| N76D | N77D | S103A | V104l | A174V | ||||||||
| T38S | N76D | S103A | V104l | |||||||||
| T38S | N76D | S103A | V104l | K237Q | ||||||||
| I8V | N76D | S103A | V104l | |||||||||
| N76D | S103A | V 1041 | N183D | |||||||||
| R19L | N76D | S103A | V104l | |||||||||
| A13V | N76D | S103A | V104l | |||||||||
| R19C | N76D | S103A | V104l | |||||||||
| N76D | S103A | V104l | N184D | |||||||||
| N76D | S103A | V104l | N252D | |||||||||
| N76D | S103A | V104l | S259C | |||||||||
| N76D | S103A | V104l | K251T | |||||||||
| N76D | P86S | S103A | V104l | |||||||||
| I72V | N76D | S103A | V104l | N185D | ||||||||
| N76D | S103A | V104l | K237E | T274A | ||||||||
| N76D | S103A | V104l | S160L | |||||||||
| N76D | S103A | V104l | A228Y | |||||||||
| P55S | N76D | S103A | V104l | S240T |
PL 191 529 B1 ciąg dalszy tabeli 2
| N76D | S103A | V104l | A254T | |||||||||
| N76D | S103A | I104N | N204T | |||||||||
| N76D | S103A | V104l | N204D | |||||||||
| N43S | N76D | S103A | V104l | |||||||||
| N76D | S103A | V104i | G159D | |||||||||
| R10H | N76D | S103A | V104l | Y177A | ||||||||
| T58S | N76D | S103A | V104l | |||||||||
| N76D | S103A | V104l | A270Y | |||||||||
| N76D | S103A | V104l | N185D | |||||||||
| Κ27Ν | N76D | S103A | V104l | |||||||||
| N76D | S103A | V104l | L262M | |||||||||
| N76D | S78P | S103A | V104l | |||||||||
| S24P | N76D | S103A | V104l | |||||||||
| N76D | S103A | V104l | S166G | Q236R | K251R | |||||||
| H17L | N76D | S103A | V104l | K237E | ||||||||
| N76D | S103A | V104l | S130L | |||||||||
| N76D | S103A | V104l | Q109R | |||||||||
| N76D | S99R | S103A | V104l | N204T | ||||||||
| N76D | S103A | V104l | D181N | |||||||||
| Q12R | N76D | S103A | V104l | |||||||||
| N76D | S103A | V104l | S212P | E271V | ||||||||
| N76D | S103A | V104l | N252K | N261Y | ||||||||
| N76D | S103A | V104l | S242T | |||||||||
| N76D | S103A | V104l | E271Q | |||||||||
| Q12R | N76D | S103A | V104l | S242T | ||||||||
| N43S | N76D | S103A | V104l | N116K | N1831 | |||||||
| N76D | S103A | V104l | G258R | |||||||||
| N76D | S103A | V104l | E271G | |||||||||
| G61R | N76D | S103A | V104l | |||||||||
| T38S | N76D | S103A | V104l | Q182R | Y263H | |||||||
| N76D | S103A | V104l | Q182R | A272S | ||||||||
| N76D | S103A | V104l | Q109R | I246Y | ||||||||
| N76D | S87G | S103A | V104l | Q206R | H249Q | S265G | ||||||
| N76D | S103A | V104l | Q137R | N238Y | E271V | |||||||
| S103A | V104l | A228T | ||||||||||
| N76D | S103A | V104l | Q182R | 1198V | ||||||||
| L21M | N76D | S103A | V104l | Q182R |
PL 191 529 B1 ciąg dalszy tabeli 2
| N76D | S103A | V104l | M119I | Q137R | ||||||||
| N76D | S103A | V104l | Q137R | N248S | ||||||||
| Α13Τ | N76D | S103A | V104) | Q206R | ||||||||
| N76D | S103A | V104l | Q206R | |||||||||
| N76D | S103A | Y104I | 8212P | G258R | ||||||||
| T58S | N76D | S103A | V104l | E271G | ||||||||
| N76D | S103A | V104l | Q206E | N261D | ||||||||
| V4E | N76D | S103A | V104l | Q206E | ||||||||
| N76D | N77D | S103A | V104l | Q206E | ||||||||
| N76D | S103A | V104l | A158E | |||||||||
| N76D | S103A | V104l | Q206E | |||||||||
| V4E | N76D | S103A | V104l | G159D | L217E | K251Q | ||||||
| V4E | N76D | S103A | V104l | G159D | L217E | N252D | ||||||
| N76D | N77D | S103A | V104l | A1SST | N185D | K251T | ||||||
| N76D | S103A | V104l | G159D | Q206E | Y244A | |||||||
| V4E | N76D | S103A | V104l | 8188E | ||||||||
| V4E | N76D | S103A | V104l | A158E | ||||||||
| N76D | N77D | S103A | V104l | N185D | ||||||||
| N76D | S103A | V104l | Q206E | K251T | ||||||||
| A48T | N76D | S103A | V104l | L111M | G159D | |||||||
| V68A | N76D | S103A | V104l | G159D | Q2S6H | |||||||
| L42V | N76D | S103A | V104l | G159D | ||||||||
| Q12H | N62H | N76D | S103A | V104l | G159D | |||||||
| L421 | N76D | S103A | V104l | G159D | ||||||||
| N76D | S103A | Y104I | G146S | G159D | ||||||||
| N76D | S103A | V104l | G159D | N238S | ||||||||
| N76D | S103A | Y104I | G159D | T224A | ||||||||
| N76D | S103A | V104l | S212P | Y268F | E271Y | |||||||
| N76D | E89A | S103A | V104l | |||||||||
| N76D | S87R | S103A | V104l | S212P | E271Y | |||||||
| N76D | S103A | V104l | S212P | Q245L | E271V | |||||||
| N76D | S103A | V104l | T1A4S | S141N | S212P | E271V | ||||||
| N76D | S103A | V104l | S212P | Q236L | N243S | E271V | ||||||
| N76D | S103A | V104l | Q109R | Q245R | ||||||||
| N76D | S103A | V104l | Q109R | P210L | ||||||||
| G20V | N62S | N76D | S103A | V104l | ||||||||
| V68A | N76D | S103A | V104l | Q236H |
PL 191 529 B1 ciąg dalszy tabeli 2
| V68A | N76D | S103A | V104l | G159D | Q236H | E271V | ||||||
| V68A | N76D | S103A | V104l | G159D | Q236H | Q245R | ||||||
| V68A | N76D | S103A | V104l | G159D | L217I | Q236H | E271V | |||||
| H17Q | V68A | N76D | S103A | V104l | ||||||||
| V68A | N76D | S103A | V104l | |||||||||
| V68A | N76D | S103A | V104l | G159D | Q236R | |||||||
| V68A | L75R | N76D | S103A | V104l | G159D | Q236H | ||||||
| V68A | N76D | N76D | S103A | A114Y | Y121I | G159D | Q236H | Q245R | ||||
| Q12R | V68A | N76D | S103A | V104l | G159D | Q236H | ||||||
| V68A | N76D | S103A | V104l | G159D | Y209S | Q236H | T253K | |||||
| V68A | N76D | S103A | V104l | N117K | G159D | N184S | Q236H | |||||
| V68A | N76D | S103A | V104l | G159D | Q236H | N243I | ||||||
| V68A | N76D | S103A | V104l | G159D | Q236H | Q245L | ||||||
| V68A | N76D | S103A | V104l | A142Y | G159D | |||||||
| V68A | N76D | S103A | V104l | N123S | G159D | Q236H | H249Y | |||||
| V68A | N76D | S103A | V104l | G159D | Q236H | H249Q | ||||||
| N76D | S103A | V104l | M222S | Q245R | ||||||||
| N76D | S103A | V104l | Q12R | M222S | H249R | |||||||
| N76D | S103A | V104l | N173R | M222S | ||||||||
| N76D | S103A | V104l | M222S | Y263F | ||||||||
| L21M | N76D | S103A | V104l | M222S | K237R | Y263F | ||||||
| N76D | S103A | V104l | Q109R | M222S | ||||||||
| N76D | S103A | Y104I | Q109R | M222S | E271D | |||||||
| G61R | N76D | S103A | V104l | M222S | ||||||||
| N76D | S103A | V104l | Q137R | M222S | ||||||||
| N76D | S103A | V104l | Q109R | M222S | N248S | |||||||
| N76D | S103A | Y104I | M222S | H249R | ||||||||
| V68A | N76D | S103A | V104i | G159D | Q236H | Q245R | N261D | |||||
| V68A | N76D | S103A | V104l | S141N | G159D | Q236H | Q245R | T255S | ||||
| V68A | N76D | S103A | V104l | G159D | Q236H | Q245R | R247H | |||||
| V68A | N76D | S103A | V104l | G159D | A174V | N204D | Q236H | Q245R | ||||
| V68A | N76D | S103A | V104l | G159D | N204D | Q236H | Q245R | |||||
| V68A | N76D | S103A | V104l | A133V | G159D | N218D | Q236H | Q245R | ||||
| V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | |||||
| V68A | N76D | S103A | V104l | G159D | A194I | Y203A | Q236H | Q245R | ||||
| Q12R | N76D | S103A | V104l | M222S | Q245R | |||||||
| N76D | S103A | Y104I | A232Y | Q245R |
PL 191 529 B1 ciąg dalszy tabeli 2
| S24T | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | ||||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N252K | |||||
| V68A | N76D | S103A | V104l | G159D | T213R | A232V | Q236H | G245R | T260A | |||
| Q12R | N76D | S103A | I104T | M222S | Y244I | Q245R | ||||||
| Q12R | N76D | S103A | M222S | P210T | Q245R | |||||||
| Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | ||||||
| T22K | V68A | N76D | S103A | V104l | ||||||||
| V68A | N76D | S103A | N184D | |||||||||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| V68A | S103A | Y104I | G159D | A232Y | Q236H | Q245R | ||||||
| V68A | S103A | V104l | N140D | G159D | A232V | Q236H | Q245R | N252K | ||||
| N43S | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N252K | ||||
| N43K | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | |||||
| N43D | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N252K | ||||
| V68A | S87G | S103A | V104l | G159D | A232V | Q236H | Q245R | N252K | R275S | |||
| Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | N248S | L262M | ||||
| Q12R | N76D | S103A | I104T | S130T | A215Y | M222S | Q245R | |||||
| Q12R | N76D | S103A | I104T | S130T | M222S | V227A | Q245R | L262S | ||||
| Q12R | N76D | S103A | I104T | S130T | A215T | M222S | Q245R | |||||
| Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | N261D | |||||
| N76D | S103A | I104T | S130T | M222S | Q245R | |||||||
| Q12R | N76D | S103A | I104T | S130T | N218D | M222S | Q245R | L262S | N269D | |||
| Q12R | S57P | N76D | S103A | I104T | S130T | M222S | Q245R | K251Q | ||||
| Q12R | N76D | S103A | I104T | S130T | R170S | N185D | M222S | N243D | O245R | |||
| Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | Y268A | |||||
| Q12R | N76D | S103A | I104T | S130T | M222S | P210S | Q245R | |||||
| V68A | S103A | V104l | G159D | A232Y | Q236H | Q245R | L257V | |||||
| V68A | S103A | V104l | N116D | G159D | A232Y | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | A232Y | Q236H | Q245R | N248D | |||||
| R10C | V68A | S103A | V104l | G159D | A232Y | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | Y203E | A232Y | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | A232Y | Q236H | K237E | Q245R | |||||
| V68A | N76D | 179N | S103A | V104l | G159D | A232Y | Q236H | Q245R | ||||
| V68A | S103A | V104l | G159D | N183D | A232Y | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | A174Y | Q206L | A232Y | Q236H | Q245R | ||||
| V68A | S103A | V104l | G159D | S188C | A232Y | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | A230T | A232Y | Q236H | Q245R |
PL 191 529 B1 ciąg dalszy tabeli 2
| V68A | A98T | S103A | V104l | G159D | A232Y | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | A215T | A232Y | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | A232Y | Q236H | Q245R | N248S | |||||
| V68A | N76D | S103A | V104l | G159D | A232Y | Q236H | Q245R | |||||
| V68A | N76D | S103A | V104l | G159D | P210R | A232Y | Q236H | Q245R | ||||
| V68A | N76D | S103A | V104l | G159D | A232Y | Q236H | Q245R | L257V | ||||
| N76D | S103A | V104l | A232Y | Q236H | Q245R | L257V | ||||||
| V68A | S103A | V104l | G159D | A232Y | Q236H | Q245R | L257Y | R275H | ||||
| N76D | S103A | V104l | L257Y | R275H | ||||||||
| V68A | S103A | V104l | G159D | T224A | A232Y | Q236H | Q245R | L257Y | ||||
| N76D | S103A | V104l | G159D | A232Y | Q236H | Q245R | L257Y | |||||
| V68A | N76D | S103A | V104l | G159D | Y209W | A232V | Q236H | Q245R | ||||
| V68A | N76D | S103A | V104l | G159D | G211R | A232V | Q236H | Q245R | ||||
| V68A | N76D | S103A | V104l | G159D | G211Y | A232V | Q236H | Q245R | ||||
| Q12R | V68A | N76D | S103A | V104l | G159D | Y214L | A232V | Q236H | Q245R | |||
| V68A | N76D | S103A | V104l | G159D | A215R | A232V | Q236H | Q245R | ||||
| Q12R | V68A | N76D | S103A | V1Q4I | G159D | A232V | Q236H | Q245R | ||||
| G20R | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | S259G | |||
| V68A | S87R | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | T260Y | |||
| V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | N261G | ||||
| V68A | N76D | S103A | V104l | G159D | A232Y | Q236H | Q245R | N261W | ||||
| N76D | S103A | V104l | A232Y | Q236H | S242P | Q245R | ||||||
| V68A | N76D | S103A | V104l | G159D | P210L | A232V | Q236H | Q245R | ||||
| Q12R | A48Y | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | |||
| N76D | S103A | V104l | A232Y | Q236H | Q245R | |||||||
| N76D | S103A | V104l | G159D | Y192F | A232V | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | A232Y | Q236H | Q245R | L257Y | R275H | ||||
| N76D | S103A | V104l | L257Y | R275H | ||||||||
| V68A | S103A | V104l | G159D | T224A | A232Y | Q236H | Q245R | L257V | ||||
| N76D | S103A | V104l | G159D | A232Y | Q236H | Q245R | L257V | |||||
| V68A | N76D | S103A | V104l | G159D | Y209W | A232V | Q236H | Q245R | ||||
| V68A | N76D | S103A | V104l | G159D | G211R | A232V | Q236H | Q245R | ||||
| V68A | N76D | S103A | V104l | G159D | G211Y | A232V | Q236H | Q245R | ||||
| Q12R | V68A | N76D | S103A | V104l | G159D | Y214L | A232V | Q236H | Q245R | |||
| V68A | N76D | S103A | VfÓ4l | G159D | A215R | A232V | Q236H | Q245R | ||||
| Q12R | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | ||||
| G20R | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | S259G |
PL 191 529 B1 ciąg dalszy tabeli 2
| V68A | S87R | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | T260Y | |||
| V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | N261G | ||||
| V68A | N76D | S103A | V104l | G159D | A232Y | Q236H | Q245R | N261W | ||||
| N76D | S103A | V104l | A232Y | Q236H | S242P | Q245R | ||||||
| V68A | N76D | S103A | V104l | G159D | P210L | A232V | Q236H | Q245R | ||||
| Q12R | A48V | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | |||
| N76D | S103A | V104l | A232Y | Q236H | Q245R | |||||||
| N76D | S103A | V104l | G159D | Y192F | A232V | Q236H | Q245R | |||||
| N76D | S103A | V104l | Y147I | G159D | A232V | Q236H | Q245R | N248S | K251R | |||
| Q12R | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | A272S | |||
| V68A | N76D | S103A | V104l | G159D | N183K | Q206L | A232V | Q236H | Q245R | |||
| V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | S256R | ||||
| V68A | N76D | S103A | V104l | G159D | Q206R | A232V | Q236H | Q245R | ||||
| K27R | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | ||||
| V68A | N76D | S103A | V104l | N116T | G159D | R170S | N185S | A232V | Q236H | Q245R | ||
| G61E | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| N43D | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | S212P | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | S99N | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | ||
| S103A | V104l | G159D | A232Y | Q236H | Q245R | N248D | N252K | |||||
| V68A | S103A | V104l | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | 0109R | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| G20R | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | Y209F | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | N261 | |||
| V68A | S103A | V104l | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | P210R | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | P210T | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | P210S | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | N185D | P210L | A232V | Q236H | Q245R | N248D | N252K | ||
| V68A | S103A | V104l | G159D | P210L | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | S212A | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | S212E | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | T213S | A232V | G236H | Q245R | N248D | N252K | ||||
| V68A | A103V | V104l | G159D | T213E | A232V | Q236H | Q245R | N248D | N252K |
PL 191 529 B1 ciąg dalszy tabeli 2
| V68A | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | T213G | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | A215Y | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | A215R | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | S216T | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | S216Y | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | S216C | A232V | Q236H | Q245R | N248D | N252K | |||
| G20A | V68A | S103A | V104ł | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | N173D | A232V | Q236K | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | K251Y | N252K | |||
| V68A | S103A | V104l | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252F | ||||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252L | ||||
| P55S | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252F | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | T255Y | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | S256N | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | S256E | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | S256R | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | T260R | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | L257R | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | G258D | |||
| 18V | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | N269D | ||
| V68A | S103A | V104l | N116S | G159D | A232V | Q236H | Q245R | N248D | N252K | T260E | ||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | N261R | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | N261D | |||
| V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | A232V | Q236H | Q245R | N248D | N252K | |||||
| S103A | V104l | G159D | A232S | Q236H | Q245R | N248D | N252K | |||||
| V68A | S103A | V104l | G159D | A232Y | Q236R | Q245R | N248D | N252K | ||||
| N18S | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | A232Y | Q236H | Q245V | N248D | N252K | ||||
| V68A | N76D | S101T | S103A | V104l | G159D | T213R | N218S | A232V | Q236H | Q245R | T260A | |
| V68A | S103A | V104l | G159D | A228Y | A232V | Q236H | Q245R | N248D | N252K | |||
| T33S | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
| V68A | N76D | E89D | S103A | V104l | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | |
| G61E | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
| S103A | V104l | G159D | V205l | P210I | A232V | Q236H | Q245R |
PL 191 529 B1 ciąg dalszy tabeli 2
| G61E | V68A | S103A | V104l | S130A | G159D | A232V | Q236H | Q245R | N248D | N252K | ||
| G61E | V68A | S103A | V104l | A133S | Q137R | G159D | A232V | Q236H | Q245R | N248D | N252K | |
| G61E | S103A | V104l | A133Y | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248G | N262K | ||||
| V68A | S103A | V104l | G159D | N218S | A232V | Q236H | Q245R | N248D | N252K | |||
| G61E | V68A | S103A | V104l | G159D | S 160 | A232V | Q236H | Q245R | N248D | N252K | ||
| S3L | G61E | V68A | N76D | S103A | V104l | A232V | Q236H | Q245R | N248D | N252K | ||
| G61E | V68A | S103A | V104l | G159D | S167F | A232V | Q236H | Q245R | N248D | N252K | ||
| G97E | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| A98D | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| S99E | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| S101E | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| S101G | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| G102A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| S103A | V104l | S106E | G159D | A232V | Q236K | Q245R | N248D | N252K | ||||
| S103A | V104l | Q109E | G159D | A232V | Q236K | Q245R | N248D | N252K | ||||
| S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | N261 | ||||
| S103A | V104l | Q109R | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| N62D | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| S103A | V104l | G159D | N184D | A232V | Q236H | Q245R | N248D | N252K | ||||
| S103A | V104l | G159D | S166D | A232V | Q236H | Q245R | N248D | N252K | ||||
| S103A | V104l | G159D | L217E | A232V | Q236H | Q245R | N248D | N252K | ||||
| G20R | N62D | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
| N62D | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
| S103A | V104l | G159D | Q206R | L217E | A232V | Q236H | Q245R | N248D | N252K | |||
| N62D | S103A | V104l | G159D | Q206R | A232V | Q236H | Q245R | N248D | N252K | |||
| S103A | V104l | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| S103A | V104l | P131Y | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| K27N | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| T38G | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| T38A | N76D | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | T260A | |||
| V68A | N76D | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | T260A | E271G | ||
| V68A | N76D | S103A | V104l | G159D | Y209W | T213R | A232V | Q236H | Q245R | T260A | ||
| V68A | N76D | S103A | V104l | G159D | P210I | T213R | A232V | Q236H | Q245R | T260A | ||
| V68A | N76D | S103A | V104l | G159D | Y205I | T213R | A232V | Q236H | Q245R | T260A | ||
| V68A | N76D | S103A | V104l | G159D | P210I | A232V | Q236H | Q245R | T260A | |||
| V68A | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | T260A |
PL 191 529 B1 ciąg dalszy tabeli 2
| N76D | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | T260A | ||||
| V68A | S103A | V104l | G159D | Y209W | A232V | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | P210I | A232V | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | A230Y | A232V | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | L126F | A232V | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | Y205! | A232V | Q236H | Q245R | |||||
| V68A | S103A | V104l | G159D | P210L | A232V | Q236H | Q245R | |||||
| S103A | V104l | G159D | A230Y | Q236H | Q245R | |||||||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | T260A | |||||
| S103A | V104l | G159D | A232V | Q236H | Q245R | |||||||
| V68A | S103A | V104l | G159D | A174V | A232V | Q236H | Q245R | L257V | ||||
| V68A | S103A | V1041 | G159D | A194S | A232V | Q236H | Q245R | L257V | ||||
| V68A | S103A | V1041 | G159D | Y209W | A232V | Q236H | Q245R | L257V | ||||
| S103A | V1041 | G159D | A232Y | Q236H | Q245R | L257V | ||||||
| V68A | N76D | S103A | V1041 | G159D | T213R | A232V | Q236H | Q245R | T260A | N261W | ||
| V68A | S103A | V1041 | G159D | A232V | Q236H | Q245R | L257V | N261W | ||||
| S103A | V1041 | G159D | T213R | A232V | Q236H | Q245R | T260A | |||||
| S103A | V1041 | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K | ||||
| S103A | Y104I | G159D | Y209W | A232V | Q236H | Q245R | L257V | |||||
| V68A | N76D | S103A | V104l | G159D | P210L | T213R | A232V | Q236H | Q245R | T260A | ||
| Q12R | S103A | V1041 | G159D | Y209W | T213R | A232V | Q236H | Q245R | T260A | |||
| S103A | V1041 | Y209W | A232V | Q236H | Q245R | L257V | * | |||||
| S103A | V1041 | G159D | V205l | P210I | T213R | A232V | Q236H | Q245R | T260A | |||
| S103A | Y104I | G159D | V205l | Y209W | A232V | Q236H | Q245R | T260A | ||||
| V68A | S103A | V1041 | G159D | V205l | Y209W | P210I | A232V | Q236H | Q245R | |||
| S103A | V1041 | G159D | V205l | Y209W | P210I | A232V | Q236H | Q245R | L257V | |||
| S103A | V1041 | G159D | V205l | Y209W | A232V | Q236H | Q245R | L257V | ||||
| V68A | S103A | V1041 | G159D | V205l | Y209W | P210I | A232V | Q236H | Q245R | T260A | ||
| S103A | V1041 | G159D | V205l | Y209W | P210I | A232V | Q236H | Q245R | ||||
| S103A | V1041 | G159D | Y209W | P210I | A232V | Q236H | Q245R | |||||
| S103A | V104l | G159D | V205l | P210I | A232V | Q236H | Q245R | |||||
| V68A | S103A | V104l | S128L | G159D | A232V | Q236H | Q245R | |||||
| A48V | S103A | V104l | G159D | A230Y | Q236H | Q245R | ||||||
| A48V | V68A | S103A | V104l | G159D | Y209W | A232V | Q236H | Q245R | ||||
| A48V | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| A48V | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | L257Y | N261W | |||
| G102A | S103A | V104l | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K |
PL 191 529 B1 ciąg dalszy tabeli 2
| Q12R | G102A | S103A | V104l | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
| S101G | G102A | S103A | V104l | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
| A98L | G102A | S103A | V104l | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
| G102A | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | |||
| S103A | Y104I | P131Y | G159D | A232V | Q236H | Q245R | N248D | N252K | ||||
| S103A | Y104I | G159D | N184S | A232V | Q236H | Q245R | N248D | N252K | ||||
| S103A | Y104I | G159D | N184G | A232V | Q236H | Q245R | N248D | N252K | ||||
| S103A | V104l | G159D | A232Y | Q236H | V244T | Q245R | N248D | N252K | ||||
| S103A | V104l | G159D | A232V | Q236H | Y244A | Q245R | N248D | N252K | ||||
| N62D | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | S256R | ||
| Q12R | N62D | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
| S101G | S103A | V104l | G159D | N185D | A232V | Q236H | Q245R | N248D | N252K | |||
| S101G | S103A | V104l | G159D | Q206E | A232V | Q236H | Q245R | N248D | N252K | |||
| S101G | S103A | V104l | G159D | T213Q | A232V | Q236H | Q245R | N248D | N252K | |||
| A98L | G102A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| S101G | G102A | S103A | V104t | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| A98L | G102A | S103A | V104l | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | ||
| A98L | G102A | S103A | V104l | G159D | S212G | A232V | Q236H | N248D | N252K | |||
| N62D | S103A | V104l | Q109R | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
| N62D | S103A | V104l | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
| N62D | S101G | S103A | V104l | G159D | S212G | T213R | A232V | Q236H | Q245R | N248D | N252K | |
| S103A | V104l | G159D | A232Y | Q245R | N248D | N252K | ||||||
| S103A | V104l | G159D | A230V | Q245R | ||||||||
| N62D | S103A | V104l | S130G | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
| S101G | S103A | V104l | S130G | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| S101G | S103A | V104l | S128G | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| S101G | S103A | V104l | S128L | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| N62D | S101G | S103A | V104l | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
| N62D | S103A | V104l | S128G | G159O | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
| N62D | S103A | V104l | S128L | G159D | T213R | A232V | Q236H | Q245R | N248D | N252K | ||
| S101G | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | T260A | |||
| S101G | S103A | V104l | P131Y | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| A98V | S101G | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| S99G | S101G | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | |||
| S101G | S103A | V104l | G159D | S212G | A232V | Q236H | Q245R | N248D | N252K | |||
| S101G | S103A | Y104I | G159D | Y209W | A232V | Q236H | Q245R | N248D | N252K | |||
| S101G | S103A | Y104I | G159D | P210I | A232V | Q236H | Q245R | N248D | N252K |
PL 191 529 B1 ciąg dalszy tabeli 2
| S101G | S103A | V104l | G159D | Y205I | A232V | Q236H | Q245R | N248D | N252K | |||
| S101G | S103A | V104l | G159D | A230Y | 0236H | Q245R | ||||||
| S101G | S103A | V104l | G159 | A194P | A232V | Q236H | Q245R | N248D | N252K | |||
| N76D | S101G | S103A | V104l | G159D | A194P | A232V | Q236H | Q245R | N248D | N252K | ||
| S101G | S103A | V104l | G159D | A230Y | A232V | Q236H | Q245R | N248D | N252K | |||
| N62D | S103A | V104l | G159D | N185D | 0206E | T213R | A232V | Q236H | Q245R | N248D | N252K | E271Q |
Jeszcze bardziej korzystne odmiany proteazy zawierają zestaw podstawień wybrany z grupy obejmującej pozycje reszt odpowiadających pozycjom subtylizyny z Bacillus amyloliquefaciens przedstawionym w tabeli 3.
T ab el a 3
| 76 | 103 | 104 | 222 | 245 | ||||||||
| 76 | 103 | 104 | 222 | 249 | ||||||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | |||||
| 68 | 76 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 260 | |||
| 22 | 68 | 76 | 103 | 104 | ||||||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | ||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | ||||||
| 68 | 103 | 104 | 140 | 159 | 232 | 236 | 245 | 252 | ||||
| 43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 252 | ||||
| 43 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | |||||
| 12 | 76 | 103 | 104 | 130 | 222 | 245 | 261 | |||||
| 76 | 103 | 104 | 130 | 222 | 245 | |||||||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
| 68 | 76 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | ||||
| 68 | 103 | 104 | 159 | 224 | 232 | 236 | 245 | 257 | ||||
| 76 | 103 | 104 | 159 | 232 | 236 | 245 | 257 | |||||
| 68 | 76 | 103 | 104 | 159 | 211 | 232 | 236 | 245 | ||||
| 12 | 68 | 76 | 103 | 104 | 159 | 214 | 232 | 236 | 245 | |||
| 68 | 76 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | ||||
| 12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
| 20 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 259 | |||
| 68 | 76 | 87 | 103 | 104 | 159 | 232 | 236 | 245 | 260 |
PL 191 529 B1 ciąg dalszy tabeli 3
| 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 261 | ||||
| 12 | 48 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | |||
| 76 | 103 | 104 | 159 | 192 | 232 | 236 | 245 | |||||
| 76 | 103 | 104 | 147 | 159 | 232 | 236 | 245 | 248 | 251 | |||
| 12 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 272 | |||
| 68 | 76 | 103 | 104 | 159 | 183 | 206 | 232 | 236 | 245 | |||
| 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | 256 | ||||
| 68 | 76 | 103 | 104 | 159 | 206 | 232 | 236 | 245 | ||||
| 27 | 68 | 76 | 103 | 104 | 159 | 232 | 236 | 245 | ||||
| 68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
| 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||||
| 68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 109 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 209 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 210 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 212 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 213 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 213 | 232 | 236 | 245 | 248 | 252 | ||||
| 68 | 103 | 104 | 159 | 215 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 216 | 232 | 236 | 245 | 248 | 252 | |||
| 20 | 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 255 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 256 | |||
| 68 | 103 | 104 | 159 | 232 | 236 | 245 | 248 | 252 | 260 | |||
| 68 | 103 | 104 | 159 | 228 | 232 | 236 | 245 | 248 | 252 | |||
| 68 | 76 | 89 | 103 | 104 | 159 | 210 | 213 | 232 | 236 | 245 | 260 | |
| 68 | 103 | 104 | 159 | 218 | 232 | 236 | 245 | 248 | 252 |
Wynalazek jest dodatkowo objaśniony na rysunku, na którym: fig. 1 przedstawia sekwencje DNA i aminokwasową subtylizyny z Bacillus amyloliquefaciens oraz częściową mapę restrykcyjną tego genu; fig. 2 przedstawia konserwowane reszty aminokwasowe subtylizyn z Bacillus amyloliquefaciens (BPN1) i Bacillus lentus (naturalna); oraz fig. 3 A i 3 B przedstawiają sekwencje aminokwasowe czterech subtylizyn w następującym porządku: pierwsza linia dotyczy sekwencji aminokwasowej subtylizyny z Bacillus amyloliquefaciens (zwanej także subtylizyną BPN'), druga linia - z Bacillus subtylis, trzecia - z Bacillus licheniformis, a czwarta - Bacillus lentus (zwanej także subtylizyną 309 w opisie PCT W089/06276). Symbol * oznacza brak specyficznych reszt aminokwasowych w porównaniu do subtylizyny BPN'.
PL 191 529 B1
W niniejszym opisie określenie „proteza” oznacza proteazę naturalną lub rekombinowaną, gdzie proteazy są hydrolazami karbonylowymi, których działanie polega ogólnie na cięciu wiązań białek lub peptydów. Do występujących naturalnie proteaz należą α-aminoacylopeptydohydrolaza, peptydyloaminohydrolaza kwasowa, acyloaminohydrolaza, karboksypeptydazaserynowa, metalokarboksypeptydaza, proteinaza tiolowa, karboksyloproteinaza oraz metaloproteinaza. Należą tu także proteazy serynowe, metalo-, tiolo-oraz kwasowe, a także endo -i egzoproteazy.
Niniejszy wynalazek dotyczy enzymów proteazowych, które nie stanowią odmian naturalnie występującej hydrolazy karbonylowej. Mają one odmienną czynność proteolityczną, stabilność, specyficzność dla substratów, profil pH i/lub wydajność w porównaniu do wyjściowej hydrolazy karbonylowej, z której pochodzi sekwencja aminokwasowa odmiany. Takie odmiany proteazy zawierają sekwencje aminokwasowe nieznane w naturze, a powstałe w wyniku podstawienia wielu reszt aminokwasowych proteazy wyjściowej przez różne aminokwasy. Proteazy wyjściowe mogą być naturalne lub rekombinowane.
Użyteczne odmiany proteaz według wynalazku zawierają podstawienia określonego z dziewiętnastu naturalnie występujących L-aminokwasów w określonych położeniach reszt aminokwasowych. Takich podstawień można dokonać w dowolnej wyjściowej subtylizynie (prokariotycznej, eukariotycznej, pochodzącej od ssaków itd.). W niniejszym opisie aminokwasy określa się za pomocą standardowego kodu jedno- lub trzyliterowego.
W niniejszym opisie określenie „subtylizyna”oznacza subtylizynę naturalną lub rekombinowaną, gdzie subtylizyny są bakteryjnymi lub grzybowymi proteazami, których działanie polega na cięciu wiązań peptydowych w białkach lub peptydach. Różne gatunki drobnoustrojów wytwarzają i wydzielają wiele naturalnych subtylizyn. Sekwencje aminokwasowe tych subtylizyn nie są całkowicie homologiczne, jednakże wykazują one ten sam lub podobny typ aktywności proteolitycznej. Ta klasa proteaz serynowych ma wspólną sekwencjęaminokwasową z triadą katalityczną, która odróżnia je od proteaz pokrewnych chymotrypsynie. Wprawdzie w obu tych grupach proteaz występuje triada złożona z asparaginianu, histydyny i seryny, ale położenie tych reszt jest różne. Odczytując od końca N wkierunku końca C, kolejność dla proteaz spokrewnionych z subtylizyną jest następująca: asparaginian - histydyna - seryna, podczas gdy w przypadku spokrewnionych z chymotrypsyną: histydyna asparaginian - seryna. A zatem subtylizyny według wynalazku są proteazami serynowymi z triadą katalityczną charakterystyczną dla proteaz spokrewnionych z subtylizyną. Przykłady przedstawiono na fig. 3. Wopisie niniejszego wynalazku numeracja aminokwasów w proteazach odpowiada numerom podanym dla sekwencji subtylizyny z dojrzałej Bacillus amyloliquefaciens, przedstawionej na fig. 1.
„Rekombinacyjna subtylizyna” lub „rekombinacyjna proteza” oznacza subtylizynę lub proteazę, w której sekwencja DNA kodująca subtylizynę lub proteazę jest zmodyfikowana tak, aby wytworzyć odmienną (lub zmutowaną) sekwencję DNA, która koduje podstawienie, delecję lub insercję przynajmniej jednego aminokwasu w naturalnie występującej sekwencji aminokwasowej.
Odpowiednie sposoby wytwarzania takich modyfikacji są dostępne w literaturze.
„Subtylizyny nie pochodzące od człowieka” i kodujące je DNA można uzyskać z wielu organizmów prokariotycznych i eukariotycznych. Przykładami organizmów prokariotycznych są bakterie Gram ujemne, takie jak E. coli lub Pseudomonas oraz bakterie Gram dodatnie, jak Micrococcus lub Bacillus; eukariotyczne -drożdże,np. Saccharomyces cerevisiae oraz grzyby, takie jak Aspergillus sp.
„Odmiana proteazy” ma sekwencję aminokwasową pochodzącą z „proteazy wyjściowej”. Doproteaz wyjściowych należą proteazy naturalne i rekombinowane. Sekwencja aminokwasowa odmiany proteazy jest pochodną sekwencji proteazy wyjściowej uzyskaną przez podstawienie, delecje lub insercje przynajmniej jednego aminokwasu w sekwencji wyjściowej. Taka modyfikacja dotyczy „wyjściowej sekwencji DNA”, kodującej sekwencję aminokwasową proteazy wyjściowej niż samego enzymu jako takiego. Odpowiednie sposoby takiej manipulacji wyjściową sekwencją DNA podaje niniejszy opis,jak również znane są z literatury.
Numeracja aminokwasów odpowiada numerom podanym dla sekwencji subtylizyny z dojrzałego Bacillus amyloliquefaciens, przedstawionej na fig. 1. Wynalazek nie ogranicza się jednakże do mutacji tej wybranej subtylizyny, ale dotyczy również proteaz wyjściowych zawierających reszty aminokwasowe w położeniach „ekwiwalentnych” ze zidentyfikowanymi dla subtylizyny z Bacillus amyloliquefaciens. Pozycję uważa się za ekwiwalentną do pozycji z subtylizyny Bacillus amyloliquefaciens w przypadku, jeżeli jest ona albo homologiczna (tj. odpowiada jej na poziomie zasadniczej lub trzeciorzędowej struktury) albo analogiczna do specyficznej reszty bądź jej części z subtylizyny Bacillus amyloliquefaciens (tj. ma taką samą zdolność do wiązania się, reakcji lub interakcjichemicznej).
PL 191 529 B1
W celu ustalenia homologiczności struktury zasadniczej, sekwencję proteazy wyjściowej porównuje się bezpośrednio z zasadniczą sekwencją subtylizyny z Bacillus amyloliquefaciens, a zwłaszcza z zestawem reszt znanych jako niezmienne dla subtylizyn o znanej sekwencji. Przykładowo, na fig. 2 przedstawiono zachowane reszty subtylizyn z B. amyloliquefaciens i B. lentus. Po ich przeprowadzeniu w postać liniową, z uwzględnieniem koniecznych insercji i delecji, aby zachować liniowość (tj. uniknąć eliminacji reszt przez arbitralne delecje i insercje), definiuje się reszty równoważne do poszczególnych aminokwasów w pierwszorzędowej sekwencji subtylizyny z Bacillus amyloliquefaciens.
Zgodność zachowanych reszt korzystnie powinna dotyczyć 100% takich reszt, jednakże zgodność obejmująca więcej niż 75% bądź około 50% jest wystarczająca do zdefiniowania równoważnych pozostałości. Powinna być zachowana triada katalityczna, Asp32 / His64 / Ser221. Wskazano zgodność znacznej ilości proteaz serynowych, grupując je jako subtylazy i subtylizynopodobne.
Przykładowo, na fig. 3 przedstawiono w postaci liniowej sekwencje aminokwasowe z Bacillus amyloliquefaciens, Bacillus subtylis, Bacillus licheniformis (carlsbergensis) i Bacillus lentus, celem wykazania maksymalnej homologiczności tych sekwencji. Porównanie wskazuje, że w każdej sekwencji obecna jest pewna ilość konserwowanych reszt. Reszty te zidentyfikowano na fig. 2 (dla BPN'i B. lentus).
Te konserwowane reszty mogą być użyte do identyfikacji odpowiednich równoważnych reszt aminokwasowych subtylizyny z Bacillus amyloliquefaciens w innych subtylizynach, jak subtylizyna z Bacillus lentus (publikacja PCT nr W089/06279 z 13 lipca 1989), która jest korzystną proteazą wyjściową lub subtylizyna znana jako PB92 (opis patentowy europejski EP 0 328 299), która wykazuje znaczna homologiczność z wyżej wymienioną. Sekwencje aminokwasowe niektórych subtylizyn przedstawiono w postaci liniowej na fig. 3A i 3B w porównaniu z sekwencjami subtylizyny z Bacillus amyloliquefaciens dla wykazania zgodności konserwowanych reszt. Jak widać, w sekwencji Bacillus lentus występuje pewna ilość delecji w porównaniu z subtylizyną z Bacillus amyloliquefaciens. Przykładowo, ekwiwalentnym aminokwasem dla Val165 w subtylizynie z Bacillus amyloliquefaciens jest izoleucyna w subtylizynach z B. lentus i B. licheniformis.
„Reszty ekwiwalentne” można także zdefiniować przez określenie homologiczności na poziomie struktury trzeciorzędowej proteazy wyjściowej, którą określa się metodą krystalografii rentgenowskiej. Jako reszty ekwiwalentne uważa się takie reszty, w których współrzędne atomowe przynajmniej dwóch atomów z głównego łańcucha danej reszty aminokwasowej proteazy wyjściowej i subtylizyny z Bacillus amyloliquefaciens ( N na N, CAna CA, C na C i O na O) znajdują się w zakresie 0,13 nm, a korzystnie 0,1 nm po przeprowadzeniu w postać liniową. Zgodność uzyskuje się po zorientowaniu i ustawieniu najlepszego modelu tak, aby uzyskać maksymalne nałożenie współrzędnych atomowych dla atomów (nie będących atomami wodoru) cząsteczki danej proteazy i subtylizyny z Bacillus amyloliquefaciens. Najlepszym modelem jest taki model krystalograficzny, dla którego uzyskuje się najniższą wartość współczynnika R dla danych krystalograficznych przy najwyższej osiągalnej rozdzielczości.
ιη, ·, ·, η Eh |Fo(h)-Fc(h)|
Współczynnik R = h——-L21
Ph | Fo(h) |
Resztami ekwiwalentnymi, które są funkcjonalnie analogiczne do danej reszty subtylizyny z Bacillus amyloliquefaciens, określa się te aminokwasy proteazy wyjściowej, które mogą przyjąć konformację prowadzącą do zmiany, modyfikacji lub udziału w budowie białka, wiązaniu substratów lub katalizie w sposób zdefiniowany i przypisany danej reszcie subtylizyny z Bacillus amyloliquefaciens. Ponadto są to takie reszty z proteazy wyjściowej (o strukturze trzeciorzędowej określonej metodą krystalografii rentgenowskiej), które zajmują analogiczne pozycje w takim stopniu, że chociaż atomy głównego łańcucha nie spełniają kryterium ekwiwalentności na podstawie homologiczności położenia, to współrzędne atomowe przynajmniej dwóch atomów w łańcuchu bocznym leżą w zakresie 0,13 nm od odpowiednich atomów w łańcuchu bocznym subtylizyny z Bacillus amyloliquefaciens. Współrzędne trójwymiarowej struktury subtylizyny z Bacillus amyloliquefaciens podano w publikacji EPO nr 0 251 446 i można ich używać do wyznaczania reszt równoważnych, jak wyżej podano.
Niektóre z reszt, wykorzystywanych do podstawień, są resztami konserwowanymi, podczas gdy inne - nie. Jeżeli chodzi o reszty niekonserwowane, podstawienia przynajmniej jednego aminokwasu są ograniczone do takich, w których powstają odmiany z sekwencjami aminokwasowymi innymi od istniejących w naturze. W przypadku reszt konserwowanych podstawienia nie powinny prowadzić do powstania sekwencji naturalnych. Odmiany proteazy według wynalazku są odmianami dojrzałymi, jak
PL 191 529 B1 również formami pro- i prepro- tych odmian. Formy prepro- są najkorzystniejsze, gdyż ułatwia to ekspresję, wydzielanie i dojrzewanie odmian proteaz.
Określenie „prosekwencja” dotyczy sekwencji aminokwasowej dołączonej do końca N dojrzałej postaci proteazy, która usunięta, daje „dojrzałą” postać proteazy. Wiele enzymów proteolitycznych występuje w naturze jako produkty proenzymów translacyjnych i, przy braku obróbki potranslacyjnej, ulega ekspresji w taki sposób. Korzystną prosekwencją do wytwarzania odmian proteazy jest domniemana prosekwencją subtylizyny z Bacillus amyloliquefaciens, chociaż można używać także innych prosekwencji proteazowych.
Określenie „sekwencja sygnałowa” lub „presekwencja” odnosi się do każdej sekwencji aminokwasowej dołączonej do końca N proteazy lub proproteazy, która może wziąć udział w wydzielaniu dojrzałych proteaz lub ich proform. Jest to definicja funkcjonalna, oznaczająca włączenie wszystkich sekwencji aminokwasowych kodowanych przez gen kodujący koniec N proteazy, biorących udział wwydzielaniu proteazy w naturalnych warunkach. Niniejszy wynalazek wykorzystuje takie sekwencje, aby wywołać wydzielanie odpowiednich odmian proteaz. Jedna z możliwych sekwencji sygnałowych zawiera pierwszych siedem reszt aminokwasowych sekwencji sygnałowej subtylizyny z Bacillus subtylis połączonych z pozostałą częścią sekwencji sygnałowej subtylizyny z (ATCC21536).
Postać „prepro”odmiany proteazy składa się z dojrzałej postaci proteazy mającej Bacillus lentus presekwencję czynnie sprzężoną z końcem N prosekwencji.
„Wektor ekspresyjny” oznacza konstrukt DNA zawierający sekwencję DNA funkcjonalnie połączoną z odpowiednią sekwencją regulatorową zdolną do kontrolowania ekspresji wspomnianego DNA wkomórce wybranego gospodarza. Takie sekwencje regulatorowe zawierają promotor inicjujący transkrypcję, sekwencję operatora regulacji transkrypcji, sekwencję kodującą miejsce wiązania rybosomu iodpowiedniego mRNA oraz sekwencje regulujące terminację transkrypcji i translacji. Wektor może być plazmidem, cząstką faga lub ewentualnie insertem genomowym. Po transformacji odpowiednich komórek gospodarza, wektormoże ulec replikacji i działać niezależnie od genomu gospodarza, bądź w niektórych przypadkach, integrować się z genomem. W niniejszym opisie określenia „wektor” oraz „plazmid” używane są zamiennie, ponieważ obecnie plazmid jest najczęściej używaną formą wektora. Jednakże stosuje się także inne znane postacie wektorów ekspresyjnych, spełniające równoważne funkcje.
„Komórki gospodarza” według wynalazku mogą być prokariotyczne lub eukariotyczne. Poddaje się je manipulacjom tak, aby uczynić je niezdolnymi do wydzielania enzymatycznie aktywnych endoproteaz. Korzystnym gospodarzem do ekspresji proteaz jest szczep Bacillus BG2036, który ma deficyt enzymatycznie aktywnej obojętnej proteazy i proteazy alkalicznej (subtylizyny). Innymi stosowanymi gospodarzami są szczepy: Bacillus subtylis 1168 i inne wybrane z B. licheniformis, B. lentus, itd.
Komórki gospodarza są transformowane lub transfektowane wektorami otrzymanymi znanymi sposobami rekombinacji DNA. Po transformacji są one zdolne do replikacji wektorów kodujących odmiany proteazy lub ekspresji pożądanych odmian proteaz. W przypadku wektorów kodujących formy pre- lub prepro- odmian proteaz, takie odmiany są zazwyczaj wydzielane z komórki gospodarza do otoczenia tej komórki.
„Funkcjonalnie połączony”, odnośnie zależności dwóch obszarów DNA, oznacza po prostu ich funkcjonalną współzależność. Przykładowo, presekwencja jest funkcjonalnie połączona z peptydem, jeżeli działa jako sekwencja sygnałowa, uczestnicząca w wydzielaniu dojrzałej postaci białka najbardziej prawdopodobnie z odcięciem sekwencji sygnałowej. Promotor jest funkcjonalnie połączony zsekwencją kodującą, jeżeli reguluje jej transkrypcję; miejsce wiązania rybosomu jest funkcjonalnie połączone z sekwencją kodującą, jeżeli znajduje się w położeniu umożliwiającym translację.
Geny kodujące naturalnie występującą proteazę wyjściową można otrzymać ogólnie znanymi sposobami. Należą do nich: syntetyzowanie oznaczonych sond zawierających domniemane sekwencje kodujące obszarów pożądanych proteaz, przygotowanie bibliotek genomicznych z organizmów zekspresją proteazy oraz przeszukiwanie bibliotek celem odszukania pożądanego genu przez hybrydyzację z sondą. Pozytywnie zhybrydyzowane klony są następnie mapowane i sekwencjonowane. Sklonowaną proteazę stosuje się z kolei do transformowania komórek gospodarza w celu uzyskania ekspresji proteazy. Gen proteazy jest dołączany do plazmidu wielokopijnego. Taki plazmid replikuje się w organizmie gospodarza w tym sensie, że zawiera dobrze znane elementy niezbędne do replikacji plazmidu: promotor czynnie sprzężony z danym genem (który może być dostarczony jako jego własny, homologiczny promotor, jeżeli jest rozpoznany, tj. transkrybowany, przez gospodarza), obszar terminacji transkrypcji i poliadenylacji (niezbędny dla stabilności mRNA transkrybowanego przez gospodarza z genu proteazy w pewnych eukariotycznych komórkach gospodarzy), który jest egzogeniczny lub
PL 191 529 B1 dostarczony przez obszar endogenicznego terminatora genu proteazy oraz gen selekcyjny, taki jak gen oporności na antybiotyk, który umożliwia uzyskanie selekcji hodowli komórek gospodarza zainfekowanych plazmidem poprzez hodowlę w pożywce zawierającej antybiotyk. Plazmidy wielokopijne zawierają także początek replikacji odpowiedni dla gospodarza, umożliwiając wytwarzanie dużej ilości plazmidów w cytoplazmie bez ograniczeń chromosomowych.
Wynalazek obejmuje integrację wielu kopii genu proteazy z genomem gospodarza. Ułatwiają to organizmy prokariotyczne i eukariotyczne, które są szczególnie czułe na homologiczną rekombinację.
Gen może pochodzić z naturalnego B. lentus, ewentualnie wytwarza się syntetyczny gen kodujący naturalną lub zmutowaną proteazę wyjściową. W takim przypadku określa się sekwencję DNA i/lub aminokwasową proteazy wyjściowej. Następnie syntetyzuje się wielokrotne, nakładające się fragmenty DNA o pojedynczej nici, które poddaje się hybrydyzacji i łączy celem wytworzenia DNA kodującego wyjściową proteazę. Po sklonowaniu naturalnego lub syntetycznego genu proteazy wykonano szereg modyfikacji prowadzących do wzmocnienia zastosowania genu poza syntezą naturalnej proteazy wyjściowej. Te modyfikacje obejmują produkcję proteaz rekombinowanych, a także produkcję odmian proteaz według wynalazku.
W celu ułatwienia utworzenia odmiany proteazy według wynalazku można zastosować poniżej opisany sposób mutagenezy kasetowej, chociaż możliwe jest zastosowanie również innych sposobów. Najpierw uzyskuje się natywny gen kodujący proteazę, po czym poddaje się go sekwencjonowaniu w całości lub części. Następnie przeszukuje się sekwencję szukając miejsca, w którym zamierza się w kodowanym enzymie dokonać mutacji przynajmniej jednego aminokwasu (delecji, insercji lub podstawienia). W sekwencjach flankujących to miejsce sprawdza się obecność miejsc restrykcyjnych wcelu wymiany krótkiego fragmentu genu na inny oligonukleotyd, który po ekspresji może kodować różne mutanty. Korzystnie, aby ułatwić wymianę fragmentu genu, wykorzystuje się unikalne miejsca restrykcyjne w genie proteazy. Jednakże inne odpowiednie miejsce restrykcyjne, może być wykorzystane, które występuje w genie proteazy niezbyt licznie, pod warunkiem, że uzyskane fragmenty genów będą mogły być połączone w odpowiedniej kolejności. Jeżeli miejsca restrykcyjne nie występują w odpowiedniej odległości od wybranego punktu (od 10 do 15 nukleotydów), uzyskuje sieje przez podstawianie nukleotydów w genie w taki sposób, aby w ostatecznym fragmencie nie została zmieniona ani ramka odczytu, ani kodowane aminokwasy. Mutację w genie prowadzącą do zmiany jego sekwencji na pożądaną, wykonuje się znanymi sposobami przy użyciu startera M13. Lokalizację odpowiednich obszarów flankujących i ocenę niezbędnych zmian w sekwencjach dwóch odpowiednich pozycji restrykcyjnych wykonuje się rutynowo z uwzględnieniem zredukowanego kodu genetycznego, mapy enzymów restrykcyjnych i wielu różnych enzymów. Należy zauważyć, że jeżeli dostępne jest odpowiednie flankujące miejsce restrykcyjne, powyższy sposób może być użyty tylko w stosunku do obszaru flankującego, który nie zawiera tego miejsca.
Po sklonowaniu natywnego lub syntetycznego DNA, miejsca restrykcyjne flankujące sekwencje przeznaczone do mutacji tnie się odpowiednimi enzymami restrykcyjnymi i wiele kaset oligonukleotydowych komplementarnych do N-końca liguje się z genem. Taki sposób upraszcza mutagenezę, gdyż wszystkie oligonukleotydy można syntetyzować tak, aby miały te miejsca restrykcyjne i nie potrzeba żadnych syntetycznych linkerów do utworzenia tych miejsc.
W niniejszym opisie, aktywność proteolityczną definiuje się jako szybkość hydrolizy wiązań peptydowych na miligram aktywnego enzymu. Opracowano wiele sposobów jej mierzenia. Oprócz lub zamiast zmodyfikowanej aktywności proteolitycznej, odmiany enzymów według wynalazku mają inne zmodyfikowane właściwości, określonych wartościami Km, kcat, stosunek kcat/Km oraz/lub zmodyfikowaną specyficzność substratową, oraz/lub zmodyfikowany profil aktywności w zakresie pH. Enzymy można dopasowywać do poszczególnych substratów, których obecność jest antycypowana, na przykład w przygotowaniu peptydów lub do procesów hydrolitycznych, takich jak pranie.
Celem wynalazku jest uzyskanie odmiany proteazy o zmienionej w porównaniu z wyjściową proteazą, korzystnie lepszej wydajności prania, w przynajmniej jednej recepturze środka piorącego i przynajmniej jednych warunkach prania.
Warunki prania obejmują dobór środka piorącego, ilość wody, temperatury wody oraz czasu prania. W różnych obszarach używa się środków piorących w różnych stężeniach. Przykładowo zawartość środka piorącego w wodzie z płukania wynosi w Europie: 4500-5000 ppm, w Japonii: około 667 ppm, a w Ameryce Północnej, zwłaszcza Stanach Zjednoczonych: około 975 ppm.
Układ o niskiej zawartości środka piorącego zawiera ten składnik w ilości mniejszej od 800 ppm. Przykładem są tu japońskie środki piorące o zawartości około 667 ppm składnika środka piorącego.
PL 191 529 B1
W układach o średniej zawartości środka piorącego wynosi ona od 800 ppm do 2000 ppm. Jako przykłady można wymienić środki piorące z Ameryki Północnej zawierające około 975 ppm środka piorącego w kąpieli pralniczej, a także brazylijskie - z zawartością około 1500 ppm.
Układ o dużej zawartości środka piorącego obejmują środki piorące o zawartości składników piorących w ilości powyżej 2000 ppm w kąpieli pralniczej. Dotyczy to kąpieli pralniczych typowych dla Europy, zawierających od 4500 ppm do 5000 ppm środka piorącego.
W środkach piorących latynoamerykańskich występuje duża ilość fosforanowych polepszaczy pienistości, a ilość składnika piorącego wynosi od 1500 ppm do 6000 ppm, co pozwala zaliczyć je do układów o średnim, bądź wysokim stężeniu środków piorących.
Przedstawione przykłady wskazują, że w skali świata zawartość środków piorących w kąpielach pralniczych jest znacznie zróżnicowana. Stężenia określano doświadczalnie. Na przykład, w Stanach Zjednoczonych, w typowej maszynie mieści się 64,4 l kąpieli pralniczej, a zatem dla uzyskania stężenia około 975 ppm środka piorącego należy dodać około 62,79 g tego składnika. Jest to typowa ilość wprowadzana przez klienta korzystającego z miarki dołączanej do opakowania. Ponadto stosuje się także różne temperatury prania, przy czym przykładowo w Japonii są one niższe niż w Europie.
Na podstawie wyników przeszukiwania odmian proteaz uważa się, że mutacje obserwowane wsubtylizynie z Bacillus amyloliquefaciens są istotne dla czynności proteolitycznej, wydajności i/lub stabilności tych enzymów oraz ich przydatności do celów pralniczych.
Wiele odmian proteaz według wynalazku jest przydatnych jako składniki środków piorących lub środków higieny osobistej, jak szampony i płyny kosmetyczne. W ich skład wchodzą także odpowiednie środki powierzchniowo czynne, mające charakter niejonowy, anionowy, kationowy lub amfoteryczny. Odpowiedni skład środka piorącego przedstawia przykład 7 w opisie patentowym Stanów Zjednoczonych Ameryki Północnej nr 5204 015. Odmiany proteaz według wynalazku znajdują zastosowanie również do innych celów, jak mydła stałe lub płynne, płyny do mycia naczyń, płyny do mycia soczewek kontaktowych, hydroliza peptydów, oczyszczanie ścieków, włókiennictwo, produkcja białek itd. Zwiększona wydajność w składzie środka piorącego oznacza lepsze usuwanie plam z substancji podatnych nadziałanie enzymów, jak trawa lub krew. Proteazy według wynalazku można wprowadzać w ilości od 0,01% do 5% (korzystnie 0,1% do 0,5%) wagowych do proszkowych lub ciekłych środków piorących, mających pH od 6,5 do 12,0. Takie środki piorące mogą zawierać także inne enzymy, jak znane proteazy, amylazy, celulazy, lipazy lub endoglikozydazy oraz wypełniacze aktywne i stabilizatory.
Dodatek proteaz według wynalazku do zwykłych kompozycji czyszczących nie ogranicza ich zastosowania. Innymi słowy, każda temperatura i wartość pH odpowiednia dla danego środka piorącego, będzie także odpowiednia dla kompozycji z tymi proteazami, jeśli pH znajduje się w określonym zakresie, a temperatura nie przekracza wartości, przy której zachodzi denaturacja proteazy. Proteazy te mogą być także składnikami czyszczących kompozycji bezdetergentowych.
Wynalazek obejmuje w kompozycjach zawierających proteazy według wynalazku, także inne typowe składniki kompozycji do czyszczenia, jak wybielacze, środki powierzchniowo czynne, wypełniacze, enzymy i katalizatory bielenia. Przy tworzeniu składu należy brać pod uwagę wzajemną kompatybilność składników.
Ilość dodatków w kompozycji do czyszczenia wynosi od 0,01% do 99,9%, korzystnie od 1% do 95%, a zwłaszcza korzystnie od 1% do 80%.
Poniższych przykładów nie należy traktować jako ograniczenia wynalazku.
Przykład 1
Uzyskano dużą ilość odmian proteazy i oczyszczono je znanymi sposobami. Wszystkich mutacji dokonano w subtiiizynie GG36 z Bacillus lentus. Odmiany przedstawiono w tabeli 3, powyżej.
Przykład 2
Badano wydajność dużej ilości odmian proteaz uzyskanych w przykładzie 1, w dwóch rodzajach środków do prania i warunków prania, stosując sposób opisany w „An improved method of assaying for a preferred enzyme and/or preferred detergent composition”, U. S. Serial No. 60/068,796.
W tabeli 4 wymieniono badane odmiany proteaz i wyniki badań w dwóch różnych środkach piorących. Wyniki w kolumnie A dotyczą 0,67 g/l filtrowanego środka piorącego Ariel Ultra (Procter & Gamble, Cincinnati, OH, USA) w roztworze o mieszanej twardości wapniowo/magnezowej, w ilości 3 gramów na 4 litry oraz 0,3 ppm enzymu w każdej studzience w temperaturze 20°C; w kolumnie B, dane dotyczą 3,38 g/l filtrowanego środka piorącego Ariel Futur (Procter & Gamble, Cincinnati, OH, USA) w roztworze o mieszanej twardości wapniowe/ magnezowej w wysokości 15 gramów na 4 litry oraz 0.3 ppm enzymu w każdej studzience w temperaturze 40°C.
PL 191 529 B1
Tab el a 4
| A | B | |||||||||||
| N76D | S103A | Y104I | 1 | 1 | ||||||||
| S103A | V104l | A228T | 0.56 | 1.11 | ||||||||
| V68A | S103A | V104l | G159D | A232V | 0236H | Q245R | N252K | 1.41 | 1.85 | |||
| V68A | S103A | V104l | G159D | A232Y | Q236H | Q245R | N248D | N252K | 2.77 | 1.20 | ||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | 2.26 | 1.67 | ||||
| V68A | S103A | V104l | N140D | G159D | A232V | Q236H | Q245R | N252K | 2.96 | 1.42 | ||
| N43S | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N252K | 1.91 | 1.80 | ||
| N43K | V68A | S103A | V1OAI | G159D | A232V | Q236H | Q245R | 2.05 | 1.78 | |||
| N43D | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N252K | 2.00 | 1.34 | ||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | L257Y | 2.38 | 1.67 | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | 2.83 | 0.53 | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | K237E | Q245R | 2.87 | 0.20 | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N252S | 2.56 | 1.41 | |||
| V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | L257Y | R275H | 3.97 | 0.47 | ||
| V68A | S103A | V104l | G159D | T224A | A232V | Q236H | Q245R | L257Y | 3.35 | 1.28 | ||
| G61E | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | 3.77 | 0.09 | |
| N43D | V68A | S103A | V104l | G159D | A232V | Q236H | Q245R | N248D | N252K | 3.50 | 0.47 | |
| V68A | S103A | V104l | G159D | S212P | A232V | Q236H | Q245R | N248D | N252K | 2.81 | 1.46 | |
| N76D | A98E | S103A | V104l | 1.56 | 0.28 | |||||||
| V4E | N76D | S103A | V104l | 1.22 | 0.33 | |||||||
| N76D | N77D | S103A | V104l | 1.13 | 0.36 | |||||||
| A16T | N76D | S103A | V104l | N248D | 1.22 | 0.43 | ||||||
| A1E | N76D | S103A | V104l | 1.12 | 0.32 | |||||||
| N76D | S103A | V104l | N261 D | 1.54 | 0.33 | |||||||
| N76D | S103A | V104l | S216C | 1.04 | 0.13 | |||||||
| N76D | N77D | S103A | V104l | A174V | 1.09 | 0.35 |
PL 191 529 B1 ciąg dalszy tabeli 4
| T38S | N76D | S103A | V104l | K237Q | 1.11 | 0.55 | ||||||
| N76D | S103A | V104l | N1830 | 1.50 | 0.25 | |||||||
| R19L | N76D | S103A | V104l | 1.11 | 0.48 | |||||||
| R19C | N76D | S103A | V104l | 1.05 | 0.19 | |||||||
| N76D | S103A | V104l | N184D | 1.32 | 0.29 | |||||||
| N76D | S103A | V104l | N252D | 1.19 | 0.53 | |||||||
| N76D | S103A | V104l | S259C | 0.92 | 0.12 | |||||||
| N76D | S103A | V104l | K251T | 1.31 | 0.43 | |||||||
| N76D | P86S | S103A | V104l | 1.00 | 0.98 | |||||||
| I72V | N76D | S103A | V104l | N1B5D | 1.70 | 0.37 | ||||||
| N76D | S103A | V104l | K237E | T274A | 1.12 | 0.16 | ||||||
| N76D | S103A | V104l | A228Y | 1.13 | 0.99 | |||||||
| N76D | S103A | V104l | G159D | 1.88 | 0.23 | |||||||
| H17L | N76D | S103A | V104l | K237E | 1.29 | 0.28 | ||||||
| N76D | S103A | V104l | S130L | 0.52 | 0.71 | |||||||
| N76D | S103A | V104l | Q109R | 0.23 | 1.26 | |||||||
| N76D | S99R | S103A | V104l | N204T | 0.21 | 0.87 | ||||||
| N76D | S103A | V104l | D181N | 0.24 | 1.07 | |||||||
| Q12R | N76D | S103A | V104l | 0.61 | 1.31 | |||||||
| N76D | S103A | V104l | S212P | E271Y | 0.69 | 1.35 | ||||||
| N76D | S103A | V104l | N252K | N261Y | 0.37 | 1.02 | ||||||
| N76D | S103A | Y104I | S242T | 0.98 | 0.92 | |||||||
| N76D | S103A | V104l | E271Q | 0.63 | 1.25 | |||||||
| Q12R | N76D | S103A | V104l | S242T | 049 | 1.32 | ||||||
| N43S | N76D | S103A | V104l | N116K | N1831 | 039 | 1.10 | |||||
| N76D | S103A | V104l | G258R | 034 | 1.17 | |||||||
| N76D | S103A | V104l | E271G | 0.57 | 1.25 | |||||||
| N76D | S103A | V104l | Q182R | 1198 V | 022 | 0.95 | ||||||
| L21M | N76D | S103A | V1Q4I | Q182R | 0.24 | 0.98 | ||||||
| N76D | S103A | V104l | M119I | Q137R | 0.13 | 0.91 | ||||||
| N76D | S103A | V104l | Q137R | N248S | 0.16 | 1.02 | ||||||
| A13T | N76D | S103A | V104l | Q206R | 0.31 | 1.01 | ||||||
| N76D | S103A | Y104I | Q206R | 0.33 | 1.02 | |||||||
| N76D | S103A | V104l | S212P | G258R | 0.38 | 1.06 | ||||||
| T58S | N76D | S103A | V104l | E271G | 0.84 | 1.26 | ||||||
| N76D | S103A | V104l | Q206E | N261D | 1.97 | 0.04 | ||||||
| V4E | N76D | S103A | V104l | Q206E | 1.51 | 0.05 |
PL 191 529 B1 ciąg dalszy tabeli 4
| N76D | N77D | S103A | V104l | Q206E | 1.40 | 0.04 | ||||||
| N76D | S103A | V104l | A158E | 1.95 | 0.16 | |||||||
| N76D | S103A | V104l | Q206E | 2.41 | 0.88 | |||||||
| N76D | N770 | S103A | V104l | A133T | N185D | K251T | 1.34 | 0.03 | ||||
| N76D | S103A | V104l | Q206E | N261 D | 1.78 | 0.04 | ||||||
| N76D | S103A | V104l | G159D | Q206E | V244A | 2.16 | 0.04 | |||||
| V4E | N76D | S103A | V104l | S188E | 1.91 | 0.04 | ||||||
| V4E | N76D | S103A | V104l | A158E | 2.06 | 0.04 | ||||||
| N76D | S103A | V104l | Q206E | K251T | 1.73 | 0.06 | ||||||
| A48T | N76D | S103A | V104l | L111M | G159D | 2.04 | 0.16 | |||||
| V68A | N760 | S103A | V104l | G159D | Q236H | 3.20 | 0.09 | |||||
| L42V | N76D | S103A | V104l | G159D | 1.83 | 0.17 | ||||||
| Q12H | N62 H | N76D | S103A | V104l | G159D | 1.42 | 0.14 | |||||
| L42I | N76D | S103A | V104l | G159D | 1.86 | 0.18 | ||||||
| N76D | S103A | V104l | G146S | G159D | 1.87 | 0.19 | ||||||
| N76D | S103A | V104l | G159D | N238S | 1.90 | 0.15 | ||||||
| N76D | S103A | V104l | G159D | T224A | 1.61 | 0.07 | ||||||
| N76D | S103A | V104l | S212P | Y268F | E271V | 0.44 | 1.42 | |||||
| N76D | S87R | S103A | V104l | S212P | E271Y | 0.39 | 2.03 | |||||
| N76D | S103A | V104l | S212P | Q245L | E271V | 0.62 | 1.79 | |||||
| N76D | S103A | V104l | Q109R | Q245R | 0.11 | 1.78 | ||||||
| N76D | S103A | V104l | Q109R | P210L | 0.12 | 1.21 | ||||||
| G20V | N62S | N76D | S103A | V104l | 1.63 | 0.78 | ||||||
| V68A | N76D | S103A | V104l | Q236H | 2.37 | 0.44 | ||||||
| V68A | N76D | S103A | V104l | G159D | Q236H | E271V | 2.97 | 0.45 | ||||
| V88A | N76D | S103A | V104l | G159D | Q236H | Q245R | 3.00 | 0.61 | ||||
| V68A | N76D | S103A | V104l | G159D | L217I | Q236H | E271V | 2.71 | 0.12 | |||
| H17Q | V68A | N76D | S103A | V104l | 2.46 | 0.38 | ||||||
| V68A | N76O | S103A | V104l | 2.46 | 0.61 | |||||||
| V68A | N76D | S103A | V104l | G159D | Q236R | 3.31 | 0.11 | |||||
| V68A | L75R | N76D | S103A | V104l | G159D | Q236H | 3.06 | 0.14 | ||||
| V68A | N76D | S103A | V104l | A114Y | V121l | G159D | Q236H | Q245R | 3.11 | 0.40 | ||
| Q12R | V68A | N76D | S103A | V104l | G159D | Q236H | 3.12 | 0.34 | ||||
| V68A | N76D | S103A | V104l | G159D | Y209S | Q236H | T253K | 3.18 | 0.03 | |||
| V68A | N76D | S103A | V104l | N117K | G159D | N184S | Q236H | 2.78 | 0.06 | |||
| V68A | N76D | S103A | V104l | Q236H | 2.49 | 0.57 | ||||||
| V68A | N76D | S103A | V104l | G159D | Q236H | Q245L | 3.37 | 0.03 |
PL 191 529 B1 ciąg dalszy tabeli 4
| V68A | N76D | S103A | Y104I | N123S | G159D | Q236H | H249Y | 3.11 | 0.03 | |||
| V68A | N76D | S103A | Y104I | G159D | Q236H | H249Q | 3.15 | 0.04 | ||||
| V68A | N76D | S103A | Y104I | G159D | Q236H | Q245R | N261 D | 3.31 | 0.03 | |||
| V68A | N76D | S103A | Y104I | S141N | G159D | Q236H | Q245R | T255S | 3.26 | 0.62 | ||
| V68A | N76D | S103A | Y104I | G159D | Q236H | Q245R | R247H | 2.78 | 0.03 | |||
| V68A | N76D | S103A | Y104I | G159D | A 174 V | N204D | Q236H | Q245R | 3.28 | 0.02 | ||
| V68A | N760 | S103A | Y104I | G159D | N204D | Q236H | Q245R | 3.34 | 0.02 | |||
| V68A | N76D | S103A | V104l | A133V | G159D | N218D | Q236H | Q245R | 3.28 | 0.03 | ||
| V68A | N76D | S103A | Y104I | G159D | A232V | Q236H | Q245R | 2.91 | 0.58 | |||
| V68A | N76D | S103A | Y104I | G159D | A194I | Y203A | Q236H | Q245R | 2.86 | 0.13 | ||
| V68A | N76D | S103A | Y104I | G159D | T213R | A232V | Q236H | Q245R | T260A | 1.30 | 1.73 | |
| T22K | V68A | N76D | S103A | V104l | 1.83 | 1.13 | ||||||
| V68A | N76D | S103A | V104l | G159D | P210R | A232V | Q236H | Q245R | 1.28 | 1.54 | ||
| V68A | N76D | S103A | Y104I | G159D | A232V | Q236H | Q245R | L257V | 3.72 | 0.8 | ||
| N76D | S103A | V104l | A232Y | Q236H | Q245R | L257V | 0.6 | 1.5 | ||||
| N76D | S103A | V104l | L257Y | R275H | 1.91 | 0.15 | ||||||
| N76D | S103A | V104l | G159D | A232Y | Q236H | Q245R | L257V | 1.92 | 1.09 | |||
| V68A | N76D | S103A | V104l | G159D | Y209W | A232V | Q236H | Q245R | 3.57 | 0.99 | ||
| V68A | N76D | S103A | V104l | G159D | G211R | A232V | Q236H | Q245R | 1.74 | 1.76 | ||
| VB8A | N76D | S103A | V104l | G159D | G211Y | A232V | Q236H | Q245R | 3.15 | 1.06 | ||
| Q12R | V68A | N76D | S103A | V104l | G159D | Y214L | A232V | Q236H | Q245R | 2.33 | 1.92 | |
| V68A | N76D | S103A | V104l | G159D | A215R | A232V | Q236H | Q245R | 1.67 | 1.45 | ||
| Q12R | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | 2.16 | 1.72 | ||
| G20R | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | S259G | 2.77 | 1.59 | |
| V68A | N76D | S87R | S103A | V104l | G159D | A232V | Q236H | Q245R | T260Y | 2.62 | 1.49 | |
| V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | N261G | 2.92 | 0.68 | ||
| V68A | N78D | S103A | V104l | G159D | A232V | Q236H | Q245R | N261 W | 2.17 | 1.37 | ||
| N76D | S103A | V104l | A232V | Q236H | S242P | Q245R | 0.48 | 1.2 | ||||
| V68A | N76D | S103A | V104l | G159D | P210L | A232V | Q236H | Q245R | 2.92 | 0.76 | ||
| Q12R | A48V | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | 2.09 | 1.86 | |
| N76D | S103A | V104l | A232V | Q236H | Q245R | 0.51 | 1.44 | |||||
| N76D | S103A | Y104I | G159D | Y192F | A232V | Q236H | Q245R | 1,60 | 1.14 | |||
| N76D | S103A | V104l | Y147I | G159D | A232V | Q236H | Q245R | N248S | K251R | 1.35 | 1.29 | |
| Q12R | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | A272S | 1.92 | 1.81 | |
| V68A | N76D | S103A | V104l | G159D | N183K | Q206L | A232V | Q236H | Q245R | 1.17 | 1.53 | |
| V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | S256R | 2.01 | 1.72 | ||
| V68A | N76D | S103A | V104l | G159D | Q206R | A232V | Q236H | Q245R | 2.09 | 1.62 |
PL 191 529 B1 ciąg dalszy tabeli 4
| K27R | V68A | N76D | S103A | V104l | G159D | A232V | Q236H | Q245R | 3.00 | 1.08 | ||
| V68A | N76D | S103A | V104l | N116T | G159D | R170S | N185S | A232V | Q236H | Q245R | ND | ND |
| N76D | S103A | V104l | M222S | Q245R | 1.01 | 1.23 | ||||||
| Q12R | N76D | S103A | V104l | M222S | H249R | 0.57 | 1.65 | |||||
| N76D | S103A | V104l | N173R | M222S | 0.86 | 0.46 | ||||||
| N76D | S103A | V104l | M222S | Y263F | 1.24 | 0.77 | ||||||
| L21M | N76D | S103A | V104l | M222S | K237R | Y263F | 1.18 | 0.76 | ||||
| N76D | S103A | V104l | Q109R | M222S | 0.52 | 1.16 | ||||||
| N76D | S103A | V104l | Q109R | M222S | E271D | 0.56 | 1.12 | |||||
| G61R | N76D | S103A | V104l | M222S | 0.43 | 0.96 | ||||||
| N76D | S103A | V104l | Q137R | M222S | 0.42 | 1.25 | ||||||
| N76D | S103A | V104l | M222S | H249R | 1.15 | 1.01 | ||||||
| Q12R | N76D | S103A | V104l | M222S | Q245R | 0.53 | 1.46 | |||||
| N76D | S103A | V104l | A232Y | Q245R | 0.69 | 1.56 | ||||||
| Q12R | N76D | S103A | I104T | M222S | V244l | Q245R | 0.66 | 1.74 | ||||
| Q12R | N76D | S103A | V104l | M222S | P210T | Q245R | 0.52 | 1.56 | ||||
| Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | 0.70 | 1.61 | ||||
| Q12R | N76D | S103A | I104T | S130T | A215V | M222S | Q245R | 0.79 | 1.85 | |||
| Q12R | N76D | S103A | I104T | S130T | M222S | Y227A | Q245R | L262S | 0.78 | 1.56 | ||
| Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | N261 D | 1.25 | 1.30 | |||
| N76D | S103A | I104T | S130T | M222S | Q245R | 1.29 | 1.30 | |||||
| Q12R | S57P | N76D | S103A | I104T | S130T | M222S | Q245R | K251Q | 1.44 | 0.16 | ||
| Q12R | N76D | S103A | I104T | S130T | R170S | N185D | M222S | N243D | Q245R | 2.01 | 0.04 | |
| Q12R | N76D | S103A | I104T | S130T | M222S | Q245R | Y268A | 0.77 | 1.60 | |||
| Q12R | N76D | S103A | I104T | S130T | M222S | P210S | Q245R | 0.73 | 1.66 | |||
| V68A | N76D | S103A | Y104I | G159D | A232Y | Q236H | Q245R | 2.09 | 0.86 |
Pr zy kła d 3
W tabeli 5 podano wyniki badania proteaz z przykładu 1 w czterech różnych środkach piorących. Badania wykonano, jak w przykładzie 2. Do badań, których wyniki przedstawiono w poszczególnych kolumnach użyto następujących środków piorących: kolumna A - 0,67 g/l filtrowanego środka piorącego Ariel Ultra (Procter & Gamble, Cincinnati, OH, USA) w roztworze o mieszanej twardości wapniowo/magnezowej w wysokości 3 gramów na 4 litry oraz 0,3 ppm enzymu w każdej studzience w temperaturze 20°C; kolumna B - 3,38 g/l filtrowanego środka piorącego Ariel Futur (Procter & Gamble, Cincinnati, OH, USA) w roztworze o mieszanej twardości wapniowo/magnezowej w wysokości 15 gramów na 4 litry oraz 0,3 ppm enzymu w każdej studzience w temperaturze 40°C; kolumna C - 3,5 g/l środka piorącego HSP1 (Procter & Gamble, Cincinnati, OH, USA) w roztworze o mieszanej twardości wapniowo/magnezowej w wysokości 8 gramów na 4 litry oraz 0,3 ppm enzymu w każdej studzience w temperaturze 20°C; kolumna D - 1,5 ml/l środka piorącego Tide KT (Procter & Gamble, Cincinnati, OH, USA) w roztworze o mieszanej twardości wapniowo/magnezowej w wysokości 3 gramów na 4 litry oraz 0,3 ppm enzymu w każdej studzience w temperaturze 20°C.
PL 191 529 B1
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| Ω | a | a | o | a | o | X | Ω | Ω | n | n | ΓΊ | n | Ω | Q | ||||
| 00 | 00 | 00 | 00 | 00 | 00 | io | 00 | 00 | 00 | 00 | 00 | CN | 00 | 00 | 00 | |||
| Ν’ | Tf | Ν’ | Tf- | LO | ||||||||||||||
| CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||
| Z | Z | z | z | Z | z | o | z | Z | z | z | Z | Z | z | Z | Z | |||
| X. | X | IX | X | x | x | a: | X | X | X | X | X | X | Q | X | X | ct | ||
| CN | m | io | LO | LO | O | LO | CD | LO | LO | LO | LO | LO | 00 | IO | LO | iO | ||
| ιο | Ν’ | 5T | sf | CO | 5T | 5Γ | Ν’ | N- | ||||||||||
| CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||
| Z | o | o | a | o | σ | σ | o | o | o | σ | O | σ | Z | o | a | σ | ||
| Ω | I | X | X | X | X | X | > | X | X | X | X | X | X | X | X | X | ||
| 00 | CD | CO | CD | CD | CD | CN | CD | CD | co | CD | co | iO | CD | co | CD | |||
| 5f | CO | co | CO | CO | CO | CO CN | CO | CO | co | CO | co | Ν’ | CO | co | CO | |||
| CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||||
| Z | o | o | o | o | σ | o | < | σ | σ | o | o | σ | σ | σ | σ | σ | ||
| a: | > | >- | > | > | >- | >- | _J | >- | > | > | > | >- | X | > | > | > | ||
| CN | CN | CN | CN | CN | CN | o | CN | CN | CN | CN | CN | CD | CN | CN | CN | |||
| CO | CO | CO | CO | CO | CO | CO | CO | CO | CO | CO | CO | CO | CO | CO CN | ||||
| CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||||
| a | < | < | < | < | < | < | 0- | < | < | < | < | < | σ | < | < | < | ||
| x | § | o | Ω | U- | a | X | ω | —1 | o | o | 111 | UJ | > | UJ | >- | |||
| 05 | 05 | 05 | 05 | lo | o | io | o | CN | CN | CN | co | CN | co | LO | ||||
| O | O | 00 | 00 | CO | ||||||||||||||
| CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||||||||
| o | > | O | o | > | Z | CL | z | 0. | W | 00 | W | P | < | 1- | < | |||
| > | a | Q£ | Ω | a | Ω | Ω | Ω | Ω | a | o | Ω | co | n | n | n | |||
| CN | 05 | Ν’ | 05 | 05 | 05 | 05 | 05 | 05 | 05 | 05 | 05 | 05 | 05 | 05 | ||||
| CO | lO | O | O | io | LO | lO | iO | LO | LO | LO | IO | LO | LO | lO | LO | |||
| CN | ||||||||||||||||||
| < | o | O | > | o | o | o | o | O | o | O | o | o | 1- | o | o | o | ||
| Q 05 | Ν' | $ | Ν’ | N | Ν’ | Ν’ | Ν' | |||||||||||
| ο | o | O | o | o | O | o | O | o | O | o | O | o | o | o | o | o | ||
| > | o | > | > | co | > | > | > | > | > | > | > | > | > | > | > | > | > | |
| < | < | < | < | < | < | < | < | < | < | <r | <r | < | >- | < | < | |||
| CO | co | co | < | co | CO | co | co | co | co | co | co | CO | co | co | co | co | ||
| ο | O | o | o | co £ | o | O | o | o | o | o | o | o | o | o | o | o | o | |
| 00 | > | ω | 00 | w | w | co | ω | w | w | w | w | w | w | < | w | ώ | ||
| Ω | 3A | < | < | X | < | < | < | < | < | < | < | < | < | < | < | < | < | |
| CD | O | 00 Ϊ | 00 | 00 | oo | DO | 00 | 00 | 00 | oo | 00 | DO | 00 | oo | 00 | co | ||
| CD i | CD | CD | to | to | to | CD | CD | CD | to | to | to | to | co | CD | ||||
| Z | 00 | > | > | o | > | > | > | > | > | > | > | > | > | > | > | > | > |
PL 191 529 B1 ciąg dalszy tabeli 5
| α | co | r~- | OT | CN | LO | ΓΝ | LO | CN | CN | LO | co | LO | oo | Ν' | CD | N. | ||||
| CN | ot | oo | «5 | Ν' | OT | Ν' | co | CN | r- | CD | OT | OT | O | t£5 | co | r- | CD | IO | Ν’ | |
| τ- | T- | *- | CN | CN | O | CN | CD | ί- | «- | csi | O | i- | CN | O | O | CN | csi | |||
| co | K. | OT | Ν' | CN | o | CD | 00 | OT | CD | CO | lO | O | r~ | CN | CD | |||||
| Ν; | ot | N· | O | CN | CN | Ν' | <D | N | OT | LO | CD | N | cD | oo | CD | CD | CD | Ν’ | io | CD |
| *- | T- | «- | »“ | »— | V | O | Ί1-*- | ł- | x— | 5- | π— | τ- | ||||||||
| 00 | co | CD | CD | <D | co | O | cD | CO | CD | r- | CN | OT | Π-- | O | CD | co | LO | |||
| tO | CD | CD | cD | N | N | O | OT | CD | N | r- | LO | Ν’ | Ν’ | tO | Ν' | LO | OT | CD | CN | |
| *“ | O | O | *- | O | τ- | o | 1“ | 1- | T- | O | V- | 1- | O | Ί- | ci | |||||
| <Ν | CN | «O | Ν' | CD | <O | CN | LO | 00 | Ν' | CD | CD | CN | Ν' | O | CD | |||||
| <Ν | co | CN | N | OT | LO | O | (O | N | CD | CN | LO | LO | O | O | CD | CD | oo | |||
| CN | «- | CN | CN | O | CN | CN | CN | CN | csi | ΓΝ | »- | csi | τ- | *- | csi | csi | csi | |||
| Z | ||||||||||||||||||||
| CN | ||||||||||||||||||||
| LO | ||||||||||||||||||||
| CN | ||||||||||||||||||||
| Z | ||||||||||||||||||||
| Ζ | Z | Z | Z | Z | * | LL | > | z | III | rr | rr | tt | Q | X | V | n | ||||
| <Ν | CN | CN | CN | CN | CN | CN | LO | tp | cp | CD | co | CN | 40 | |||||||
| m | OT | CO | LO | io | LO | LO | OT | OT | OT | LO | iO | co | LO | |||||||
| CN | <N | CN | CN | CN | CN | CN | CM | CN | CN | CN | CN | CN | CN | CN | ||||||
| <L | ć. | Λ | ć. | Z | Z | H | OT | OT | W | H | l | 0 | Z | Z | Z | |||||
| α | a | a | O | D | □ | U- | _1 | Q | Z | z | Z | Z | z | Z | z | Z | o | er | ||
| oo | co | 00 | co | CO | co | CN | CN | 00 | CN | CN | CN | CN | CN | CN | CN | CN | CN | oo | LO | |
| Ν' | N | ’Τ | Ν' | Ν’ | N | CO | LO | N | LO | LO | LO | LO | LO | LO | Ό | LO | IO | Ν' | Ν' | |
| CN | CN | CN | CN | CN | CN | <N | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |
| Z | z | Z | Λ | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | α | |
| Di | Di | oi | oi | Di | Di | n | n | (£ | n | n | n | n | n | n | o | Q | Z | n | rr | T |
| OT | CO | LO | LO | OT | OT | co | co | LO | 00 | co | co | co | 00 | oo | co | co | CN | co | LO | CD |
| Ν' | N | Ν' | Ν' | Ν' | Ν' | Ν' | Ν’ | N | Tt | N | N | N | N- | N- | Ν' | N1 | LO | Ν' | Ν' | CD |
| CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN |
| O | <J | o | o | σ | o | Z | Z | o | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | o | o |
| I | X | T | X | X | X | rr | rr | T | rr | rr | cc | rr | rr | rr | rr | rr | Q | > | T | > |
| CD | CD | co | to | co | co | ot | OT | (O | OT | OT | LO | LO | LO | LO | LO | to | co | LO | CD | |
| CD | CD | CD | CD | CD | CD | Ν' | V | CD | Ν' | N | Ν' | N | Ν' | Ν' | Ν' | Ν' | N | Ν' | CD | CD |
| CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN |
| υ | o | O | O | α | O | o | σ | o | o | o | σ | σ | σ | σ | σ | σ | Z | σ | σ | |
| > | > | > | > | > | > | X | X | > | X | X | X | X | X | X | X | X | X | X | > | n |
| CN | CN | CN | CN | CN | CN | co | (O | CN | CŁJ | CD | CD | CD | CD | to | Hi | co | LO | to | OT | |
| CD | cD | rD | CD | CD | CD | CD | CD | CD | CD | CD | CD | CD | CD | CD | Ν' | CD | LO | |||
| CN | CN | CN | CN | CN | ΓΝ | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||||
| < | < | < | < | •i | o | o | < | o | a | o | o | o | σ | o | 0 | a | α | 0 | ||
| oi | ł— | > | o | o | rr | > | > | O | > | > | > | > | > | > | > | > | X | > | > | < |
| OT | CD | co | CD | CD | co | CN | CN | OT | CN | CN | CN | CN | CN | CN | CN | CN | Φ | CN | oo | O |
| c* | o | CD | CD | OT | σ) | CD | CD | CD | CD | CD | CD | CD | CD | CD | CN | CD | ||||
| cn | CN | CN | CN | CN | (sj | CN | CN | CN | CN | CN | CN | CN | CN | CN | cy | ry | ||||
| < | ot | ot | ot | Z | O | < | < | 0 | < | < | < | < | < | < | < | < | U | < | < | OT |
| o | o | o | □ | □ | Q | Q | n | ΓΊ | n | n | n | n | n | n | n | > | n | n | ||
| ot | <n | 05 | O> | OT | OT | OT | OT | OT | OT | OT | OT | OT | OT | OT | OT | CN | OT | OT | ||
| OT | co | LO | m | LO | LO | LO | LO | o | LO | LO | LO | LO | LO | LO | LO | LO | CD | LO | LO | O |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | > | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | > | |
| 041 | 3 | 3 | 3 | N O | Ν' O | Ν' O | Ν' O | νεο | Ν' O | 3 | 3 | 3 | 3 | Ν' O | Ν' σ | Ν' O | Ν' O | N O | Ν’ O | 03A |
| > | > | > | > | > | > | > | > | OT | > | > | > | > | > | > | > | > | > | > | > | W |
| s | 3A | s | s | $ | $ | s | 3A | < | < co | < CD | S | S | 3A | s | $ | s | i | < | ||
| o | O | o | o | O | o | o | O | 00 | o | O | O | O | O | o | o | o | o | o | o | 00 |
| tp | ||||||||||||||||||||
| ot | w | w | w | w | ot | w | OT | > | V) | OT | OT | OT | OT | OT | OT | OT | W | OT | OT | > |
| < | < | < | < | < | < | < | < | cn | < | < | < | < | < | < | <C | <T | LU | |||
| <o | oo | co | co | co | w | 00 | 00 | LO | 00 | 00 | OO | co | CO | oo | 40 | CO | w | |||
| CD | CD | SP | co | tp | (D | SP | CD | u> | ID | (O | CD | CD | CD | co | «0 | CD | CD | CD | ||
| > | > | > | > | > | > | > | > | Q_ | > | > | > | > | > | > | > | > | > | > | > | o |
PL 191 529 B1 ciąg dalszy tabeli 5
| σ> | r- | 8 | id | «3 | ID | 00 | id | ID | O | CM | o | co | Ol | o | co | cO | 3 | |||
| CM | CM | CM | <M | CM | M- | uD | r- | O) | co | CD | i*- | M- | ID | co | ||||||
| CM | *“ | T- | CM | T- | r- | «- | T- | CM | CM | CM | 1- | CM | CM | |||||||
| ΙΟ | <T> | CM | r- | CO | ||||||||||||||||
| CO | co | CO | co | x— | M· | Cł | O | Q | O | a | O | o | n | O | a | O | O | n | O | |
| — | o | *“ | *- | *“ | »- | z | Z | Z | Z | z | Z | z | z | Z | z | Z | z | Z | z | Z |
| h- | o | O | CM | O | co | r- | o | O) | 00 | oo | r- | o | co | D | iD | <31 | co | CD | ||
| σι | D | m | l·- | co | co | o | co | h- | ω | to | CM | co | <o | m | O | to | ||||
| o | »“ | »- | ▼- | *- | o | o | o | O | O | ó | o | O | i- | -f- | o | 1- | CM | CM | ci | 1- |
| o | h- | o | CM | CM | o | o | h- | CM | 1— | o | uf) | a> | CM | iD | ID | CM | co | |||
| T- | <O | co | [— | co | tf> | CM | CO | r- | O) | tfł | CM | CO | co | CM | h- | M- | M- | |||
| CM | <r- | CM | 1- | CM | M- | co | -T | co | CM | c\i | CO | CM | co | M- | M1 | id | CO | l< | iD | |
| < | ||||||||||||||||||||
| O | ||||||||||||||||||||
| CD | ||||||||||||||||||||
| CM | ||||||||||||||||||||
| H | ||||||||||||||||||||
| X | Y | γ | id | |||||||||||||||||
| U) | CM | CM | CM | |||||||||||||||||
| 'ł | in | ID | ŁD | |||||||||||||||||
| CM | CM | CM | CM | |||||||||||||||||
| O | Z | Z | Z | |||||||||||||||||
| id | γ | id | I | Y | n | n | n | |||||||||||||
| CM | CM | CM | <D | CM | GO | GO | 00 | |||||||||||||
| in | LD | iD | co | UD | M* | Μ- | M | |||||||||||||
| CM | CM | CM | CM | CM | CM | CM | CM | |||||||||||||
| Z | Z | Z | O | Z | Z | Z | Z | |||||||||||||
| Q | Si | Q | O | > | O | X | X | X | Y | Y | Y | Z | Y | Y | X | Y | Y | id | ||
| OT | CM | GO | 03 | CM | co | io | Li) | ID | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | |
| •M· | id | fl· | CO | M· | M- | M- | M- | ID | ID | ID | ID | ID | ID | iD | iD | <D | ID | ID | D | |
| CM | CM | CM | CM | CM | <M | CM | CM | CM | CM | CM | CM | CM | CM | <M | CM | CM | CM | CM | CM | CXI |
| Z | Z. | Z | Z | < | Z | σ | o | a | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z |
| IX | o | (X | X | X | X | z | Z | z | a | n | n | Q | a | a | a | o | Y | a | n | n |
| 10 | ao | LO | kO | co | tf> | 8 | 8 | ID | oo | 03 | oo | 00 | ao | oo | co | co | CM | co | co | oo |
| M· | M- | M· | M- | M- | to | M- | Μ | ID | M | |||||||||||
| CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM |
| O | Z | U | σ | H | σ | y | y | a | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z |
| I | X | T | Z | z | > | > | > | X | X | X | X | X | X | X | X | n | X | X | X | |
| co | iD | co | co | o | 8 | CM | CM | ID | UD | ID | ID | ID | ID | ID | D | CO | ID | ID | ID | |
| co | M- | co | <*> | CO CM | co cm | CO | Μ- | TT | *3 | rf | TT | M- | M- | 5t | ||||||
| CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | <M | CM | CM | <M | CM | CM | ||
| O | U | u | y | Ł | O | i | < | < | y | u | u | υ | o | y | σ | y | Z | y | u | y |
| > | Z | > | ·> | O | > | > | LL | Z | z | z | Z | z | Z | Z | X | Z | T | Z | ||
| 232 | co co | 232 | CM CO | (31 ID | CM CO | co | M o | r·- (D | 8 | CO CO | 8 | 8 | 8 | CD CO | <D CO | (D CO | ID Μ- | <D CO | CD CO | to ΓΟ |
| CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | CM | ||||||
| < | o | < | < | 0 | < | co | > | > | u | CO | u | y | o | o | O | σ | o | σ | O | σ |
| a | > | CO | a | a | Q | < | Q | > | > | > | > | > | > | > | > | z | > | > | > | |
| σ> | CM | 00 | O) | 3 | σι | σι | co | 2 | CM | CM | CM | CM | CM | CM | CM | CM | CO | CM | ΓΜ | CM |
| UD | CO | kfi | ID | m | O | ID | CO | (O | CO | CO | CO | CO | CO | CO | co | <O | (O | CO | ||
| CM | CM | CM | CM | CM | CM | CM | <M | <M | CM | 3 | <M | |||||||||
| 0 | < | Z | 0 | > | 0 | 0 | <0 | 0 | < | < | < | < | < | < | < | < | u | 3 | < | |
| Q | o | <r | a | Q | a | Q | Cl | a | o | o | > | Q | O | a | ||||||
| co | cn | en | 3 | 3 | a | <n | O) | 05 | cn | cn | <31 | <31 | <7) | <3ł | rt> | |||||
| co | m | Ul | a | o | (D | O | iD | ID | ID | iD | lO | <D | iD | iD | co | ID | ID | co | ||
| < | □ | δ | > | ώ | > | > | Z | > | o | o | a | o | C5 | u | u | u | 3 | o | o | Z |
| 1041 | 1041 | 1041 | I03A | E89D | I03A | I03A | ś § | I03A | 1041 | 1041 .. 1 | 1041 | 1041 | 1041 | 1041 | I06E | LU σι o | Q <31 ID | X g | 041 | | 1590 |
| > | > | > | CO | ca | <0 | co | > | > | > | > | > | > | ω | σ | o | σ | > | O | ||
| < | < | < | UJ | < | < | < | < | < | < | |||||||||||
| co | ro | co | □ | O | <C | co | cO | co | co | co | co | 3 | 3 | ’Τ | co | |||||
| o | o | o | CD | CD | CO | O | O | O | O | o | O | o | O | o | O | |||||
| T— | Γ*- | r- | co | |||||||||||||||||
| co | co | co | > | Z | z | > | 0 | > | w | CD | 0 | <D | 0 | ω | > | > | > | > | ω | > |
| LLJ | < | < | < | < | 111 | LU | LU | □ | LU | UJ | o | 2A | s | s | s | a | < co | |||
| 00 | BO | co | oo | —l | r*- | s | s | O | o | O | o | o | CM | o | ||||||
| CD | CD | CO | CM | CD | co | CD | co | CD | O) | CD | ||||||||||
| O | > | > | 0 | > | > | 0 | co | 0 | 0 | < | ω | <0 | CD | 0 | w | w | w | ω | Z | W |
PL 191 529 B1 ciąg dalszy tabeli 5
| κ | D3 | o | s | o | ot | o | CD | OT | l·- | CN | Tf | CD | cn | CN | OT | co | 3 | cn | r*. | |
| ο | ο | (O | T- | OT | Tf | OO | ot | Tf | CD | Tf | cn | CO | cn | to | O | co | ||||
| to | tO | CN | CN | T- | CN | CN | T- | T- | CN | T- | T- | T- | OT | T- | T- | T- | CN | CO | có | CN |
| Ο | Ω | Ω | Ω | Ω | Ω | Ω | Q | n | O | a | Π | O | n | n | n | n | n | n | n | Ω |
| Ζ | Ζ | Z | Z | z | Z | Z | Z | z | Z | z | Z | Z | z | z | z | z | z | z | z | Z |
| ot | co | CN | co | 00 | Tf | CO | OT | CO | co | Tf | r~- | to | σ | o | o | h- | ||||
| CN | cn | OD | OT | co | Tf | o | O | t~ | to | o | co | r- | di | Tf | OT | ou | OT | CN | co | |
| τ- | ο | CN | *“ | CN | CN | CN | CN | CN | T- | CN | CN | CN | o | «- | Ó | o | -- | csi | CN | |
| CM | co | r- | CO | to | OT | CN | 3 | Tf | O | CO | t— | Tf | Tf | f- | CN | CD | oo | |||
| τ~ | co | t— | co | O | O | co | CN | oo | co | CO | O | r- | Oł | •4 | ’Τ | CO | co | OT | ||
| ιη | co | co | Tf | co | Tf | CN | CN | CN | CN | CN | CN | CN | f- | OT | CN | Tf | OT | OT | CN | |
| σ | ||||||||||||||||||||
| A- | ||||||||||||||||||||
| CN | ||||||||||||||||||||
| LU | ||||||||||||||||||||
| V | ||||||||||||||||||||
| CN | ||||||||||||||||||||
| OT | ||||||||||||||||||||
| CN | ||||||||||||||||||||
| Z | ||||||||||||||||||||
| ¥ | (X | Ω | ||||||||||||||||||
| CN | CO | IN | CU | CN | ||||||||||||||||
| IO | OT | OT | 4· | OT | ||||||||||||||||
| CN | CN | CN | tN | CN | ||||||||||||||||
| Z | OT | Z | Z | Z | ||||||||||||||||
| Ω | Z | ki | * | o | IX | V | * | Ω | ||||||||||||
| co | CN | CN | CN | CN | ao | OT | CN | CN | CN | CN | CN | CN | co | |||||||
| Tf | ot | OT | OT | OT | 4- | Tf | OT | OT | OT | OT | OT | OT | ||||||||
| CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||||||
| Z | Z | Z | Z | Z | Z | O | Z | Z | Z | Z | Z | Z | Z | |||||||
| 5Χ | 5Χ | cx | Ω | Ω | Ω | X | V | ¥ | V | * | n | IX | X | n | n | o | n | n | n | rr |
| CN | CN | LO | 00 | oO | oO | CN | CN | CN | CN | CN | co | OT | <o | oo | 03 | oo | oo | oo | co | OT |
| m | ot | Tf | Ν’ | Tf | Tf | ot | ot | OT | to | LO | Tf | Tf | co | Tf | Tf | Tf | Tf | Tf | ||
| CN | CN | CN | CN | CN | ΓΝ | CN | CN | CN | CN | CN | CN | CN | <N | CN | CN | CN | CN | CN | CN | CN |
| Ζ | Z | σ | Z | Z | Z | Z | Z | Z | Z | Z | Z | O | O | Z | Z | Z | Z | Z | Z | ϋ |
| Ω | O | X | £X | [X | (X | Ω | Ω | a | a | n | rr | X | > | 0£ | ir | LX | (X | rr | rr | X |
| m | co | co | OT | to | ot | OO | OO | co | 00 | co | OT | £ | OT | OT | OT | OT | OT | OT | to | |
| ν· | f | co | •=f | 3· | Tf | Tf | Tf | Ν' | Tf | Tf | co CN | Tf | Tf | Tf | Ν’ | tO | ||||
| ί\| | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |
| Ζ | Z | o | σ | u | U | Z | Z | Z | Z | Z | u | a | a | O | o | O | o | σ | σ | |
| α: | IX | > | X | X | X | cx | ix | X | ix | IX | X | (Y | X | X | X | X | X | X | > | |
| OT | ot | CN | co | CD | £ | OT | to | IO | U) | U) | co | CN | to | to | to | to | to | to | ||
| S | xr | CO | co | co | Tf | Tf | Tf | Tf | Tf | CO | CO | to | to | σ> | σ) | to | to | |||
| (Ν | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | tN | tN | CN | CN | tN | |||
| α | O | < | u | U | O | U | U | O | a | a | O | < | 1- | O | O | O | O | σ | a | < |
| τ | X | rr | > | > | > | X | H | < | X | I | > | tx | LU | > | > | > | > | > | > | o |
| to | £ | CO | CN | CN | CN | co | Tf | (O | to | CN | co | cn | ΓΝ | CN | CN | |||||
| co | co | CO | Tf | N- | co | to | co | σ | co CN | to CN | (O | CO | co | to CN | ||||||
| CN | CN | A2 | cn | <N | CN | CN | CN | CN | CN | cn | tN | CN | CN | CN | ||||||
| σ | O | H | < | < | O | > | > | U | U | < | H | U | < | < | < | < | < | < | OT | |
| > | > | Ω | rx | LU | (X | > | X | X | > | cx | Ω | n | n | LU | σ | o | n | o | n | |
| CN | CN | OT | (O | co | CN | £ | £ | CN | CN | co | OT | OT | OT | <n | (T) | OT | CN | OT | ||
| ίΟ | cO | ot | o | co | co | CO | OT | 00 | 00 | o | OT | OT | OT | |||||||
| cn | C\| | CN | 3 | CN | CN | CN | CN | CN | cn | CN | ||||||||||
| < | < | O | H | U | < | U | O | < | < | P | □ | Z | Z | o | P | o | o | OT | o | |
| Ω | LU | Ω | X | Ω | o | >- | > | Ω | Ω | Ω | Ω | Ω | Ω | n | O | |||||
| to CO | r- | Tf O | OT IO | to o | σι to | 3 | CN CO | CN co | 03 to | OT «Ο | cn OT | 3 | u> OT | 03 OT | OT OT | OT OT | 3 | 3 | OT OT | 3 |
| CN | CN | CN | CN | |||||||||||||||||
| ω | l | > | O | U | O | Z | < | < | o | O | o | > | o | O | O | o | > | > | O | > |
| Ο σ> ot | 159D | I03A | I04I | 159D | I04I | 159D | Ω σ> OT | 159D | I04I | I04.I | 1041 | I03A | ,04, J | 1041 | 1041 | 1041 | I03A | νεο. | 1041 | I03A |
| Ο | O | OT | > | O | > | O | O | O | > | > | > | OT | > | > | > | > | OT | OT | > | OT |
| < | <r | <r | <r | <T | < | < | £ | < | ||||||||||||
| Tf | Ω | co | I04 | co | Tf | co | co | CO | Ω | co | <o | CN | CN | CN | ||||||
| ο | o | CN | o | O | o | o | a | o | o | o | N62 | o | o | o | o | O | O | O | O | |
| > | > | Z | ω | > | w | > | > | > | M | OT | w | OT | OT | OT | OT | o | O | OT | O | |
| $ | s | IX | Ω | s | Ω | a | 3A | s | Z | O | Ω | X | n | O | o | o | _J | O | 2A | rr |
| ο | o | o | CN co z | o | CN CD Z | o | O | o | r* | co | N62 | CN | CN CO Z | o | o | o | co | o | O | CN |
| ω | ω | o | ot | OT | CO | (Λ | 2 | 1- | σ | OT | OT | OT | < | OT | o | σ |
PL 191 529 B1 ciąg dalszy tabeli 5
| 2.25 | CO o CN | m CN CN | 2.34 | CO CO | I1-49 | ao U3 CN | 5 | 0 03 | to p | 1X3 03 CN | r- r- | 1X3 N | 3.05 | co 0 | O CN | 0 | co | 03 p | 1X3 p | 03 CN | 0 | 1.25 |
| α | O | α | 0 | O | O | Cl | Cl | Cl | Cl | Cl | O | 0 | Cl | Cl | a | O | Q | η | a | O | n | a |
| z | Z | z | Z | Z | Z | Z | z | z | Z | Z | Z | Z | Z | Z | z | Z | Z | z | z | Z | z | z |
| r~ | CD | <O | O | 10 | CN | 03 | 03 | co | h- | co | n- | 03 | co | 03 | CD | 0 | 0 | |||||
| ν- | o | Ν' | r- | T | 03 | K | 03 | CN | r*- | CO | 03 | O | CO | N- | 03 | CN | 03 | 1X3 | 03 | CO | ||
| ο | CN | CN | CN | CN | CN | 0 | «- | «- | 0 | O | O | r- | 1- | ł- | ł- | łr- | t- | »- | O | |||
| CN | 03 | 03 | <0 | CN | cn | O | r- | cX3 | r- | CO | CN | CO | 03 | 1X3 | n- | O | 1X3 | <0 | O) | |||
| Ο | CO | O | O) | r*~ | *— | Ν' | ix) | o> | σ> | T— | 0 | CN | 00 | h- | Ν' | 03 | 1X3 | 3) | ^r | |||
| *t | co | CN | CN | CN | CN | 03 | CN | T- | CN | 03 | CN | 03 | CN | Ί- | CN | T- | 1- | ł- | T- | »- | CN | |
| Ni | id | Z | Z | Z | Z | Z | Νί | |||||||||||||||
| CN | IN | CN | CN | CN | CN | CN | CN | |||||||||||||||
| cn | m | UC3 | in | 1X3 | 1X3 | 1X3 | 1X3 | |||||||||||||||
| CN | CN | CN | CN | CN | CN | CN | CN | |||||||||||||||
| z. | z | Z | Z | Z | Z | Z | Z | |||||||||||||||
| o | O | * | 0 | O | Z | Z | a | 0 | a | 'Z | id | Z | id | id | Ν' | 0 | ||||||
| 03 | oo | CN | ao | CO | CN | CN | CN | co | co | to | CN | CN | CN | CN | CN | CN | CN | CN | CO | |||
| s | Ν | 1x3 | N- | U) | U) | 1X3 | Ν' | 5F | 1X3 | 1X3 | 1X3 | 1X3 | 1X3 | 1X3 | 1X3 | 1X3 | ||||||
| CN | CN | CN | CN | CN | CN | <N | CN | CN | CN | cn | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||
| Z | Z | z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | Z | |||
| Ct | Ct | O | QC | a | n | n | 0 | ft | CC | Cd | 0 | n | n | n | Π | a | n | 0 | tt | |||
| cX3 | iX3 | 00 | U3 | m | 00 | co | co | 1X3 | CO | 1X3 | CO | 00 | 00 | CO | co | CO | CO | co | lO | |||
| 3 | Ν’ | N- | Ν’ | •'J | Ν' | Ν’ | Ν' | Ν' | Ν' | Ν' | ’ί | Ν' | Ν' | Tf | ||||||||
| CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||
| σ | σ | Z | σ | O | Z | Z | Z | σ | U | σ | Z | Z | Z | Z | Z | z | Z | Z | 0 | |||
| X | X | ar | X | X | et | cd | cd | I | X | X | et | cd | [£ | rr | rr | rr | rr | it | X | |||
| <g | 10 | CD | co | U) | IX) | 1X3 | co | co | co | 1X3 | 1X3 | 1X3 | 1X3 | 1X3 | 1X3 | 1x3 | 1X3 | CD | ||||
| o3 | o5 | Ν’ | 03 | 03 | ''T | Ν' | N | 03 | 03 | O) | <r | T | Ν' | Ν' | Ν' | N | ’Τ | 03 | ||||
| CN | CN | CN | CN | CN | (N | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | CN | |||
| α | U | O | O | O | O | σ | σ | σ | O | O | O | σ | σ | σ | σ | σ | σ | 0 | 0 | |||
| >- | >- | X | > | X. | > | X | X | X | >- | > | >- | X | X | X | X | X | X | X | et | X | > | |
| CN | CN | CO | CN | CN | CN | co | CX) | co | CN | CN | CN | <0 | co | CD | U) | «) | «) | CO | 1X3 | co | ||
| 03 | 03 | 03 | 03 | IX) | 03 | 03 | 03 | 03 | 03 | 03 | 03 | 03 | 03 | 03 | 03 | O) | O) | O) | Ν' | 03 | 03 | |
| CN | CM | CN | CN | CN | <N | CN | CN | CN | CN | CN | <N | CN | CN | CN | CN | CN | CN | CN | CN | CN | ||
| < | < | a | < | Z | < | U | 0 | 0 | < | < | < | O | O | σ | σ | σ | a | O | 0 | 0 | ||
| O | 0 | > | ct | Cl | cd | > | > | > | Od 03 | Cd | cd | > | > | > | > | > | > | > | X | >- | a. | |
| CN | CN | CN | 03 | co | 03 | CN | CN | CN | 03 | 03 | CN | CN | CN | CN | CN | CN | CN | CN | ||||
| 03 | N- | 03 | 03 | 03 | CN | 03 | 03 | 03 | 03 | 03 | 03 | 03 | 03 | 03 | 03 | |||||||
| CN | CN | <N | CN | CN | CN | CN | p | cn | H | CN | CN | CN | CM | <N | CN | CN | CN | CN | ||||
| w | en | < | H | z | ł- | < | < | ł— | H | < | < | < | < | < | < | < | O | < | < | |||
| Q | o | ft | 0 | Cd | QC | Cl | Cl | O | O | cc | a | Cl | O | O | n | 0 | § | >- | Q_ | a | ||
| cn | c» | cn | 10 | 10 | cn | 03 | CJł | 0 | cn | <33 | cn | 03 | 03 | 03 | O | O | O) | |||||
| cn | cn | cC3 | 3 | ’Τ | 123 | 1x3 | 1X3 | 1X3 | 1X3 | 1X3 | U) | 1X3 | 1X3 | 1X3 | O | O | 03 | O) | 1X3 | |||
| CN | CN | CN | CN | CN | ||||||||||||||||||
| 0 | 0 | H | 0 | 0 | U | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | en | > | 0. | < | 0 | ||
| 0 | er | >- | >- | 0 | 0 | 0 | _J | 0 | _l | > | ci | Cl | Cl | O | a | Q | ||||||
| 't | 3- | cn | 03 | CN | 0 | n | 0 | CD | CD | 5 | CO | CO | cn | cn | O) | O) | O) | O) | ||||
| o | 0 | U3 | O | 03 | 03 | 03 | 03 | CN | CN | 0 | CN | CN | 03 | 0 | 0 | IX) | U) | «) | uf» | 1X3 | 1X3 | P |
| CN | ||||||||||||||||||||||
| > | > | 0 | σ | < | < | cn | en | en | CO | > | CO | CO | CŁ | > | > | 0 | 0 | 0 | 0 | 0 | 0 | > |
| < | < | Cl | 0 | < | < | <r | < | |||||||||||||||
| 03 O | 03 O | 3 | Ν’ σ | 03 cX3 | iX3 | ’Τ o | »T 0 | Ν' o | 3 | 03 O | Ν' O | 3 | 3 | 03 O | 03 O | 3 | 3 | 3 | 3 | 3 | «ί- ο | 03 O |
| W | W | > | > | 0 | 0 | > | > | > | > | CO | > | > | > | (0 | <n | > | > | > | > | > | > | cn |
| s | < CN | s | s | s | $ | s | $ | 0 | < 03 | < 03 | S | 0 | 0 | $ | s | < 03 | < 03 | < 03 | < 03 | 0 | ||
| O | 0 | 0 | O | O | 0 | 0 | 0 | 0 | 0 | O | O | 0 | O | o | 0 | 0 | O | O | O | O | 0 | |
| 0 | 0 | W | w | > | > | cn | en | cn | en | en | CO | en | <0 | en | en | cn | en | en | cn | en | cn | en |
| _l | 0 | s | 0 | s | s | O | 0 | 0 | 0 | O | 0 | a | 0 | > | 0 | 0 | 0 | 0 | 0 | 0 | 0 | O |
| 00 | O | 0 | CN | 0 | 0 | CN | 0 | 0 | 0 | CN | CN | CN | 0 | co | 03 | 0 | 0 | 0 | 0 | 0 | 0 | CO |
| cn | CD | to | CI3 | CX3 | CO | 03 | O) | h- | ||||||||||||||
| < | en | 0 | Z | w | cn | Z | w | w | co | Z | Z | Z | <0 | < | en | cn | cn | cn | cn | cn | « | Z |
PL 191 529 B1
Lista sekwencji <110> Poulose, Ayrookaran J.
Schellenberger, Volker Kellis, Jr,, James T.
Paech, Christian Nadherny, Joannę Naki, Donald P.
<120> Nowa odmiana proteazy, cząsteczka DNA kodująca nową odmianę proteazy, wektor zawierający tę cząsteczkę oraz karma dla zwierząt i kompozycje zawierające nową odmianę proteazy <130> GC502-2 <140> 09/178,155 <141> 1998-10-23 <150> 08/956,323 <151> 1997-10-23 <150> 08/956,564 <151> 1997-10-23 <150> 08/956,324 <151> 1997-10-23 <160> 6 <170> FastSEO do Windows Version 3.0 <210> 1 <211> 1497 <212> DNA <213> B. amyloliąuefaciens <220>
<221> CDS <222> (96) ... (1245) <400> 1 ggtctactaa aatattattc catactatac aattaataca cagaataatc tgtctattgg 60 ttattctgca aatgaaaaaa aggagaggat aaaga gtg aga ggc aaa aaa gta 113
Met Arg Gly Lys Lys Val 1 5
| tgg | atc | agt | ttg | ctg | ttt | gct | tta | gcg | tta | atc | ttt | acg | atg | gcg | ttc |
| 161 | |||||||||||||||
| Trp | Ile | Ser | Leu 10 | Leu | Phe | Ala | Leu | Ala 15 | Leu | Tle | Phe | Thr | Met 20 | Ala | Phe |
| ggc | agc | aca | tcc | tct | gcc | eag | gcg | gca | ggg | aaa | tca | aac | ggg | gaa | aag |
| 209 | |||||||||||||||
| Gly | Ser | Thr 25 | Ser | Ser | Ala | Gin | Ala 30 | Ala | Gly | Lys | Ser | Asn 35 | Gly | Glu | Lys |
| aaa | tat | att | gtc | ggg | ttt | aaa | cag | aca | atg | agc | acg | atg | agc | gcc | gct |
| 257 | |||||||||||||||
| Lys | Tyr | Ile | Val | Gly | Phe | Lys | Gin | Thr | Met | Ser | Thr | Met | Ser | Ala | Ala |
PL 191 529 B1
| 40 | 45 | 50 | |||||||||||||
| aag | aag | aaa | gat | gtc | att | tct | gaa | aaa | ggc | ggg | aaa | gtg | caa | aag | caa |
| 305 | |||||||||||||||
| Lys | Lys | Lys | Asp | Val | Ile | Ser | Glu | Lys | Gly | Gly | Lys | Val | Gin | Lys | Gin |
| 55 | 60 | 65 | 70 | ||||||||||||
| ttc | aaa | tat | gta | gac | gca | gct | tca | gtc | aca | tta | aac | gaa | aaa | gct | gta |
| 353 | |||||||||||||||
| Phe | Lys | Tyr | Val | Asp | Ala | Ala | Ser | val | Thr | Leu | Asn | Glu | Lys | Ala | Val |
| 75 | 80 | 85 | |||||||||||||
| aaa | gaa | ttg | aaa | aaa | gac | ccg | agc | gtc | gct | tac | gtt | gaa | gaa | gat | cac |
| 401 | |||||||||||||||
| Lys | Glu | Leu | Lys | Lys | Asp | Pro | Ser | Val | Ala | Tyr | Val | Glu | Glu | Asp | His |
| 90 | 95 | 100 | |||||||||||||
| gta | gca | cat | gcg | tac | gcg | cag | tcc | gtg | cct | tac | ggc | gta | tca | caa | att |
| 449 | |||||||||||||||
| Val | Ala | His | Ala | Tyr | Ala | Gin | Ser | Val | Pro | Tyr | Gly | Val | Ser | Gin | Ile |
| 105 | 110 | 115 | |||||||||||||
| aaa | gcc | cct | gct | ctg | cac | tct | caa | ggc | tac | act | gga | tca | aat | gtt | aaa |
| 4 97 | |||||||||||||||
| Lys | Ala | Pro | Ala | Leu | His | Ser | Gin | Gly | Tyr | Thr | Gly | Ser | Asn | Val | Lys |
| 120 | 125 | 130 | |||||||||||||
| gta | gcg | gtt | atc | gac | agc | ggt | atc | gat | tct | tct | cat | cct | gat | tta | aag |
| 545 | |||||||||||||||
| Val | Ala | Val | Ile | Asp | Ser | Gly | Ile | Asp | Ser | Ser | His | Pro | Asp | Leu | Lys |
| 135 | 140 | 145 | 150 | ||||||||||||
| gta | gca | agc | gga | gcc | agc | atg | gtt | cct | tct | gaa | aca | aat | cct | ttc | caa |
| 593 | |||||||||||||||
| Val | Ala | Ser | Gly | Ala | Ser | Met | Val | Pro | Ser | Glu | Thr | Asn | Pro | Phe | Gin |
| 155 | 160 | 165 | |||||||||||||
| gac | aac | aac | tct | cac | gga | act | cac | gtt | gcc | ggc | aca | gtt | gcg | gct | ctt |
| 641 | |||||||||||||||
| Asp | Asn | Asn | Ser | His | Gly | Thr | His | Val | Ala | Gly | Thr | Val | Ala | Ala | Leu |
| 170 | 175 | 180 | |||||||||||||
| aat | aac | tca | atc | ggt | gta | tta | ggc | gtt | gcg | cca | agc | gca | tca | ctt | tac |
| 689 | |||||||||||||||
| Asn | Asn | Ser | Ile | Gly | Val | Leu | Gly | Val | Ala | Pro | Ser | Ala | Ser | Leu | Tyr |
| 185 | 190 | 195 | |||||||||||||
| gct | gta | aaa | gtt | ctc | ggt | gct | gac | ggt | tcc | ggc | caa | tac | agc | tgg | atc |
| 737 | |||||||||||||||
| Ala | Val | Lys | Val | Leu | Gly | Ala | Asp | Gly | Ser | Gly | Gin | Tyr | Ser | Trp | Ile |
| 200 | 205 | 210 | |||||||||||||
| att | aac | gga | atc | gag | tgg | gcg | atc | gca | aac | aat | atg | gac | gtt | att | aac |
| 785 | |||||||||||||||
| Ile | Asn | Gly | Ile | Glu | Trp | Ala | Ile | Ala | Asn | Asn | Met | Asp | Val | Ile | Asn |
| 215 | 220 | 225 | 230 | ||||||||||||
| atg | agc | ctc | ggc | gga | cct | tct | ggt | tct | gct | gct | tta | aaa | gcg | gca | gtt |
| 833 | |||||||||||||||
| Met | Ser | Leu | Gly | Gly | Pro | Ser | dy | Ser | Ala | Ala | Leu | Lys | Ala | Ala | Val |
PL 191 529 B1
| 235 | 240 | 245 | |
| gat aaa gcc gtt | gca tcc ggc gtc gta | gtc gtt gcg | gca gcc ggt aac |
| 881 | |||
| Asp Lys Ala Val | Ala Ser Gly Val Val | Val Val Ala | Ala Ala Gly Asn |
| 250 | 255 | 260 | |
| gaa ggc act tcc | ggc agc tca agc aca | gtg ggc tac | cct ggt aaa tac |
| 929 | |||
| Glu Gly Thr Ser | Gly Ser Ser Ser Thr | Val Gly Tyr | Pro Gly Lys Tyr |
| 265 | 270 | 275 | |
| cct tct gtc att | gca gta ggc gct gtt | gac agc agc | aac caa aga gca |
| 977 | |||
| Pro Ser Val Ile | Ala Val Gly Ala Val | Asp Ser Ser | Asn Gin Arg Ala |
| 280 | 285 | 290 | |
| tct ttc tca agc | gta gga cct gag ctt | gat gtc atg | gca cct ggc gta |
| 1025 | |||
| Ser Phe Ser Ser | Val Gly Pro Glu Leu | Asp Val Met | Ala Pro Gly Val |
| 295 | 300 | 305 | 310 |
| tct atc caa agc | acg ctt cct gga aac | aaa tac ggg | gcg tac aac ggt |
| 1073 | |||
| Ser Ile Gin Ser | Thr Leu Pro Gly Asn | Lys Tyr Gly | Ala Tyr Asn Gly |
| 315 | 320 | 325 | |
| acg tca atg gca | tct ccg cac gtt gcc | gga gcg gct | gct ttg att ctt |
| 1121 | |||
| Thr Ser Met Ala | Ser Pro His Val Ala | Gly Ala Ala | Ala Leu Ile Leu |
| 330 | 335 | 340 | |
| tct aag cac ccg | aac tgg aca aac act | caa gtc cgc | agc agt tta gaa |
| 1169 | |||
| Ser Lys His Pro | Asn Trp Thr Asn Thr | Gin Val Arg | Ser Ser Leu Glu |
| 345 | 350 | 355 | |
| aac acc act aca | aaa ctt ggt gat tct | ttg tac tat | gga aaa ggg ctg |
| 1217 | |||
| Asn Thr Thr Thr | Lys Leu Gly Asp Ser | Leu Tyr Tyr | Gly Lys Gly Leu |
| 360 | 365 | 370 | |
| atc aac gta caa | gcg gca gct cag taa | a acataaaaaa ccggccttgg | |
| 1265 | |||
| Ile Asn Val Gin | Ala Ala Ala Gin * | ||
| 375 | 380 |
ccccgccggt
1325 gatggctccc
1385 cggccaactc
1445 aacggtcggc
1497 tttttattat tctgaaaatt ctgaaacgtc ggcgttttcc ttttcttcct ttaacgagaa tcaatcgccg tgataccggg ccgcatgttc acggcgggtt cttcccggtt agacggcatt aatccgctcc gacccggctc tccggtcagc cgtaatcgga ataatcgacg agtcccgtaa tcaatgccat tc <210> 2 <211> 382 <212> PRT <213> B. amyloliąuefaciens
PL 191 529 B1 <400> 2
| Met | Arg | Gly | Lys | Lys | Val | Trp | Ile | Ser | Leu | Leu | Phe | Ala | Leu | Ala | Leu |
| 1 | 5 | 10 | 15 | ||||||||||||
| Ile | Phe | Thr | Met | Ala | Phe | Gly | Ser | Thr | Ser | Ser | Ala | Gin | Ala | Ala | Gly |
| 20 | 25 | 30 | |||||||||||||
| Lys | Ser | Asn | Gly | Glu | Lys | Lys | Tyr | Ile | Val | Gly | Phe | Lys | Gin | Thr | Met |
| 35 | 40 | 45 | |||||||||||||
| Ser | Thr | Met | Ser | Ala | Ala | Lys | Lys | Lys | Asp | Val | Ile | Ser | Glu | Lys | Gly |
| 50 | 55 | 60 | |||||||||||||
| Gly | Lys | Val | Gin | Lys | Gin | Phe | Lys | Tyr | Val | Asp | Ala | Ala | Ser | Val | Thr |
| 65 | 70 | 75 | 80 | ||||||||||||
| Leu | Asn | Glu | Lys | Ala | Val | Lys | Glu | Leu | Lys | Lys | Asp | Pro | Ser | Val | Ala |
| 85 | 90 | 95 | |||||||||||||
| Tyr | Val | Glu | Glu | Asp | His | Val | Ala | His | Ala | Tyr | Ala | Gin | Ser | Val | Pro |
| 100 | 105 | 110 | |||||||||||||
| Tyr | Gly | Val | Ser | Gin | Ile | Lys | Ala | Pro | Ala | Leu | His | Ser | Gin | Gly | Tyr |
| 115 | 120 | 125 | |||||||||||||
| Thr | Gly | Ser | Asn | Val | Lys | Val | Ala | Val | Ile | Asp | Ser | Gly | Ile | Asp | Ser |
| 130 | 135 | 140 | |||||||||||||
| Ser | His | Pro | Asp | Leu | Lys | Val | Ala | Ser | Gly | Ala | Ser | Met | Val | Pro | Ser |
| 145 | 150 | 155 | 160 | ||||||||||||
| Glu | Thr | Asn | Pro | Phe | Gin | Asp | Asn | Asn | Ser | His | Gly | Thr | His | Val | Ala |
| 165 | 170 | 175 | |||||||||||||
| Gly | Thr | Val | Ala | Ala | Leu | Asn | Asn | Ser | Ile | Gly | Val | Leu | Gly | Val | Ala |
| 180 | 185 | 190 | |||||||||||||
| Pro | Ser | Ala | Ser | Leu | Tyr | Ala | Val | Lys | Val | Leu | Gly | Ala | Asp | Gly | Ser |
| 195 | 200 | 205 | |||||||||||||
| Gly | Gin | Tyr | Ser | Trp | Ile | Ile | Asn | Gly | Ile | Glu | Trp | Ala | Ile | Ala | Asn |
| 210 | 215 | 220 | |||||||||||||
| Asn | Met | Asp | Val | Ile | Asn | Met | Ser | Leu | Gly | Gly | Pro | Ser | Gly | Ser | Ala |
| 225 | 230 | 235 | 240 | ||||||||||||
| Ala | Leu | Lys | Ala | Ala | Val | Asp | Lys | Ala | Val | Ala | Ser | Gly | Val | Val | Val |
| 245 | 250 | 255 | |||||||||||||
| Val | Ala | Ala | Ala | Gly | Asn | Glu | Gly | Thr | Ser | Gly | Ser | Ser | Ser | Thr | Val |
| 260 | 265 | 270 | |||||||||||||
| Gly | Tyr | Pro | Gly | Lys | Tyr | Pro | Ser | Val | Ile | Ala | Val | Gly | Ala | Val | Asp |
| 275 | 280 | 285 | |||||||||||||
| Ser | Ser | Asn | Gin | Arg | Ala | Ser | Phe | Ser | Ser | Val | Gly | Pro | Glu | Leu | Asp |
| 290 | 295 | 300 | |||||||||||||
| Val | Met | Ala | Pro | Gly | Val | Ser | Ile | Gin | Ser | Thr | Leu | Pro | Gly | Asn | Lys |
| 305 | 310 | 315 | 320 | ||||||||||||
| Tyr | Gly | Ala | Tyr | Asn | Gly | Thr | Ser | Met | Ala | Ser | Pro | His | Val | Ala | Gly |
| 325 | 330 | 335 | |||||||||||||
| Ala | Ala | Ala | Leu | Ile | Leu | Ser | Lys | His | Pro | Asn | Trp | Thr | Asn | Thr | Gin |
| 340 | 345 | 350 | |||||||||||||
| Val | Arg | Ser | Ser | Leu | Glu | Asn | Thr | Thr | Thr | Lys | Leu | Gly | Asp | Ser | Leu |
| 355 | 360 | 365 | |||||||||||||
| Tyr | Tyr | Gly | Lys | Gly | Leu | Ile | Asn | Val | Gin | Ala | Ala | Ala | Gin | ||
| 370 | 375 | 380 | |||||||||||||
| <210> | 3 | ||||||||||||||
| <211> | 275 | ||||||||||||||
| <212> | PRT | ||||||||||||||
| <213> | B. amyloliąuefaciens | ||||||||||||||
| <400> | 3 | ||||||||||||||
| Ala | Gin | Ser | Val | Pro | Tyr | Gly | Val | Ser | Gin | Ile | Lys | Ala | Pro | Ala | Leu |
| 1 | 5 | 10 | 15 | ||||||||||||
| His | Ser | Gin | Gly | Tyr | Thr | Gly | Ser | Asn | Val | Lys | Val | Ala | Val | Ile | Asp |
| 20 | 25. | 30 |
PL 191 529 B1
| Ser | Gly | Ile Asp 35 | Ser | Ser His | Pro Asp Leu Lys Val Ala | Gly | Gly | Ala | |||||||
| 40 | 45 | ||||||||||||||
| Ser | Met | Val | Pro | Ser | Glu | Thr | Asn | Pro | Phe | Gin | Asp | Asn | Asn | Ser | His |
| 50 | 55 | 60 | |||||||||||||
| Gly | Thr | His | Val | Ala | Gly | Thr | Val | Ala | Ala | Leu | Asn | Asn | Ser | Ile | Gly |
| 65 | 70 | 75 | 80 | ||||||||||||
| Val | Leu | Gly | Val | Ala | Pro | Ser | Ala | Ser | Leu | Tyr | Ala | val | Lys | Val | Leu |
| 85 | 90 | 95 | |||||||||||||
| Gly | Ala | Asp | Gly | Ser | Gly | Gin | Tyr | Ser | Trp | Ile | Ile | Asn | Gly | Ile | Glu |
| 100 | 105 | 110 | |||||||||||||
| Trp | Ala | Ile | Ala | Asn | Asn | Met | Asp | Val | Ile | Asn | Met | Ser | Leu | Gly | Gly |
| 115 | 120 | 125 | |||||||||||||
| Pro | Ser | Gly | Ser | Ala | Ala | Leu | Lys | Ala | Ala | Val | Asp | Lys | Ala | Val | Ala |
| 130 | 135 | 140 | |||||||||||||
| Ser | Gly | Val | Val | Val | Val | Ala | Ala | Ala | Gly | Asn | Glu | Gly | Thr | Ser | Gly |
| 145 | 150 | 155 | 160 | ||||||||||||
| Ser | Ser | Ser | Thr | Val | Gly | Tyr | Pro | Gly | Lys | Tyr | Pro | Ser | Val | Ile | Ala |
| 165 | 170 | 175 | |||||||||||||
| Val | Gly | Ala | Val | Asp | Ser | Ser | Asn | Gin | Arg | Ala | Ser | Phe | Ser | Ser | Val |
| 180 | 185 | 190 | |||||||||||||
| Gly | Pro | Glu | Leu | Asp | Val | Met | Ala | Pro | Gly | Val | Ser | Ile | Gin | Ser | Thr |
| 195 | 200 | 205 | |||||||||||||
| Leu | Pro | Gly | Asn | Lys | Tyr | Gly | Ala | Tyr | Asn | Gly | Thr | Ser | Met | Ala | Ser |
| 210 | 215 | 220 | |||||||||||||
| Pro | His | Val | Ala | Gly | Ala | Ala | Ala | Leu | Ile | Leu | Ser | Lys | His | Pro | Asn |
| 225 | 230 | 235 | 240 | ||||||||||||
| Trp | Thr | Asn | Thr | Gin | Val | Arg | Ser | Ser | Leu | Glu | Asn | Thr | Thr | Thr | Lys |
| 245 | 250 | 255 | |||||||||||||
| Leu | Gly | Asp | Ser | Phe | Tyr | Tyr | Gly | Lys | Gly | Leu | Ile | Asn | Val | Gin | Ala |
| 260 | 265 | 270 | |||||||||||||
| Ala | Ala | Gin | |||||||||||||
| 275 | |||||||||||||||
| <210> | 4 | ||||||||||||||
| <211> | 275 | ||||||||||||||
| <212> | PRT | ||||||||||||||
| <213> | B. subtilis | ||||||||||||||
| <400> | 4 | ||||||||||||||
| Ala | Gin | Ser | Val | Pro | Tyr | Gly | Ile | Ser | Gin | Ile | Lys | Ala | Pro | Ala | Leu |
| 1 | 5 | 10 | 15 | ||||||||||||
| His | Ser | Gin | Gly | Tyr | Thr | Gly | Ser | Asn | Val | Lys | Val | Ala | Val | Ile | Asp |
| 20 | 25 | 30 | |||||||||||||
| Ser | Gly | Ile | Asp | Ser | Ser | His | Pro | Asp | Leu | Asn | Val | Arg | Gly | Gly | Ala |
| 35 | 40 | 45 | |||||||||||||
| Ser | Phe | val | Pro | Ser | Glu | Thr | Asn | Pro | Tyr | Gin | Asp | Gly | Ser | Ser | His |
| 50 | 55 | 60 | |||||||||||||
| Gly | Thr | His | Val | Ala | Gly | Thr | Ile | Ala | Ala | Leu | Asn | Asn | Ser | Ile | Gly |
| 65 | 70 | 75 | 80 | ||||||||||||
| Val | Leu | Gly | Val | Ser | Pro | Ser | Ala | Ser | Leu | Tyr | Ala | Val | Lys | val | Leu |
| 85 | 90 | 95 | |||||||||||||
| Asp | Ser | Thr | Gly | Ser | Gly | Gin | Tyr | Ser | Trp | Ile | Ile | Asn | Gly | Ile | Glu |
| 100 | 105 | 110 | |||||||||||||
| Trp | Ala | Ile | Ser | Asn | Asn | Met | Asp | Val | ile | Asn | Met | Ser | Leu | Gly | Gly |
| 115 | 120 | 125 | |||||||||||||
| Pro | Thr | Gly | Ser | Thr | Ala | Leu | Lys | Thr | Val | Val | Asp | Lys | Ala | Val | Ser |
| 130 | 135 | 140 | |||||||||||||
| Ser | Gly | Ile | Val | Val | Ala | Ala | Ala | Ala | Gly | Asn | Glu | Gly | Ser | Ser | Gly |
| 145 | 150 | 155 | 160 | ||||||||||||
| Ser | Thr | Ser | Thr | Val | Gly | Tyr | Pro | Ala | Lys | Tyr | Pro | Ser | Thr | Ile | Ala |
PL 191 529 B1
| 165 | 170 | 175 | |
| Val | Gly Ala Val Asn Ser Ser Asn 180 | Gin Arg Ala Ser Phe Ser 185 190 | Ser Ala |
| Gly | Ser Glu Leu Asp Val Met Ala 195 200 | Pro Gly Val Ser Ile Gin 205 | Ser Thr |
| Leu | Pro Gly Gly Thr Tyr Gly Ala 210 215 | Tyr Asn Gly Thr Ser Met 220 | Ala Thr |
| Pro 225 | His Val Ala Gly Ala Ala Ala 230 | Leu Ile Leu Ser Lys His 235 | Pro Thr 240 |
| Trp | Thr Asn Ala Gin Val Arg Asp 245 | Arg Leu Glu Ser Thr Ala 250 | Thr Tyr 255 |
| Leu Ala | Gly Asn Ser Phe Tyr Tyr Gly 260 Ala Gin 275 <210> 5 <211> 274 <212> PRT <213> B. licheniformis <400> 5 | Lys Gly Leu Ile Asn Val 265 270 | Gin Ala |
| Ala 1 | Gin Thr Val Pro Tyr Gly Ile 5 | Pro Leu Ile Lys Ala Asp 10 | Lys Val 15 |
| Gin | Ala Gin Gly Phe Lys Gly Ala 20 | Asn Val Lys Val Ala Val 25 30 | Leu Asp |
| Thr | Gly Ile Gin Ala Ser His Pro 35 40 | Asp Leu Asn Val Val Gly 45 | Gly Ala |
| Ser | Phe Val Ala Gly Glu Ala Tyr 50 55 | Asn Thr Asp Gly Asn Gly 60 | His Gly |
| Thr 65 | His Val Ala Gly Thr Val Ala 70 | Ala Leu Asp Asn Thr Thr 75 | Gly Val 80 |
| Leu | Gly Val Ala Pro Ser Val Ser 85 | Leu Tyr Ala Val Lys Val 90 | Leu Asn 95 |
| Ser | Ser Gly Ser Gly Ser Tyr Ser 100 | Gly Ile Val Ser Gly Ile 105 110 | Glu Trp |
| Ala | Thr Thr Asn Gly Met Asp Val 115 120 | Ile Asn Met Ser Leu Gly 125 | Gly Ala |
| Ser | Gly Ser Thr Ala Met Lys Gin 130 135 | Ala Val Asp Asn Ala Tyr 140 | Ala Arg |
| Gly 145 | Val Val Val Val Ala Ala Ala 150 | Gly Asn Ser Gly Asn Ser 155 | Gly Ser 160 |
| Thr | Asn Thr Ile Gly Tyr Pro Ala 165 | Lys Tyr Asp Ser Val Ile 170 | Ala Val 175 |
| Gly | Ala Val Asp Ser Asn Ser Asn 180 | Arg Ala Ser Phe Ser Ser 185 190 | Val Gly |
| Ala | Glu Leu Glu Val Met Ala Pro 195 200 | Gly Ala Gly Val Tyr Ser 205 | Thr Tyr |
| Pro | Thr Asn Thr Tyr Ala Thr Leu 210 215 | Asn Gly Thr Ser Met Ala 220 | Ser Pro |
| His 225 | Val Ala Gly Ala Ala Ala Leu 230 | Ile Leu Ser Lys His Pro 235 | Asn Leu 240 |
| Ser | Ala Ser Gin Val Arg Asn Arg 245 | Leu Ser Ser Thr Ala Thr 250 | Tyr Leu 255 |
| Gly Ala | Ser Ser Phe Tyr Tyr Gly Lys 260 Gin <210> 6 <211> 269 <212> PRT | Gly Leu Ile Asn Val Glu 265 270 | Ala Ala |
PL 191 529 B1 <213> Β. lentus <400> 6
| Ala | Gin | Ser | Val | Pro | Trp | Gly | Ile | Ser | Arg | Val | Gin | Ala | Pro | Ala | Ala |
| 1 | 5 | 10 | 15 | ||||||||||||
| His | Asn | Arg | Gly | Leu | Thr | Gly | Ser | Gly | Val | Lys | Val | Ala | Val | Leu | Asp |
| 20 | 25 | 30 | |||||||||||||
| Thr | Gly | Ile | Ser | Thr | His | Pro | Asp | Leu | Asn | Ile | Arg | Gly | Gly | Ala | Ser |
| 35 | 40 | 45 | |||||||||||||
| Phe | Val | Pro | Gly | Glu | Pro | Ser | Thr | Gin | Asp | Gly | Asn | Gly | His | Gly | Thr |
| 50 | 55 | 60 | |||||||||||||
| His | Val | Ala | Gly | Thr | Ile | Ala | Ala | Leu | Asn | Asn | Ser | Ile | Gly | Val | Leu |
| 65 | 70 | 75 | 80 | ||||||||||||
| Gly | Val | Ala | Pro | Ser | Ala | Glu | Leu | Tyr | Ala | Val | Lys | Val | Leu | Gly | Ala |
| 85 | 90 | 95 | |||||||||||||
| Ser | Gly | Ser | Gly | Ser | Val | Ser | Ser | Ile | Ala | Gin | Gly | Leu | Glu | Trp | Ala |
| 100 | 105 | 110 | |||||||||||||
| Gly | Asn | Asn | Gly | Met | His | Val | Ala | Asn | Leu | Ser | Leu | Gly | Ser | Pro | Ser |
| 115 | 120 | 125 | |||||||||||||
| Pro | Ser | Ala | Thr | Leu | Glu | Gin | Ala | Val | Asn | Ser | Ala | Thr | Ser | Arg | Gly |
| 130 | 135 | 140 | |||||||||||||
| Val | Leu | Val | Val | Ala | Ala | Ser | Gly | Asn | Ser | Gly | Ala | Gly | Ser | Ile | Ser |
| 145 | 150 | 155 | 160 | ||||||||||||
| Tyr | Pro | Ala | Arg | Tyr | Ala | Asn | Ala | Met | Ala | Val | Gly | Ala | Thr | Asp | Gin |
| 165 | 170 | 175 | |||||||||||||
| Asn | Asn | Asn | Arg | Ala | Ser | Phe | Ser | Gin | Tyr | Gly | Ala | Gly | Leu | Asp | Ile |
| 180 | 185 | 190 | |||||||||||||
| Val | Ala | Pro | Gly | Val | Asn | Val | Gin | Ser | Thr | Tyr | Pro | Gly | Ser | Thr | Tyr |
| 195 | 200 | 205 | |||||||||||||
| Ala | Ser | Leu | Asn | Gly | Thr | Ser | Met | Ala | Thr | Pro | His | Val | Ala | Gly | Ala |
| 210 | 215 | 220 | |||||||||||||
| Ala | Ala | Leu | Val | Lys | Gin | Lys | Asn | Pro | Ser | Trp | Ser | Asn | Val | Gin | Ile |
| 225 | 230 | 235 | 240 | ||||||||||||
| Arg | Asn | His | Leu | Lys | Asn | Thr | Ala | Thr | Ser | Leu | Gly | Ser | Thr | Asn | Leu |
| 245 | 250 | 255 | |||||||||||||
| Tyr | Gly | Ser | Gly | Leu | Val | Asn | Ala | Glu | Ala | Ala | Thr | Arg | |||
| 260 | 265 |
Zastrzeżenia patentowe
Claims (16)
1.Odmiana proteazy, znamienna tym, że zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33,
37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142,
146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198,
203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232,
237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259,
260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274.
2.Odmiana proteazy według zastrz. 1, znamienna tym, że zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 i 236 oraz w przynajmniej jednej z pozycji: 1, 9, 12,
61, 62, 68, 76, 97, 98, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270 i 275.
3.Odmiana proteazy według zastrz. 1, znamienna tym, że zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 i 245 oraz w przynajmniej jednej z pozycji: 1, 9, 12,
PL 191 529 B1
61, 62, 68, 76, 97, 98, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270 i 275.
4. Odmiana proteazy według zastrz. 1, znamienna tym, że zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103, 236 i 245 oraz w przynajmniej jednej z pozycji: 1, 9, 12, 61, 62, 68, 76, 97, 98, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270 i 275.
5. Odmiana proteazy według zastrz. 1, albo 2, albo 3, albo 4, znamienna tym, że zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 159.
6. Odmiana proteazy według zastrz. 1, znamienna tym, że pochodzi od subtylizyny z Bacillus.
7. Odmiana proteazy według zastrz. 6, znamienna tym, że pochodzi od subtylizyny z Bacillus lentus.
8. Cząsteczka DNA, znamienna tym, że zawiera kodony kodujące odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274.
9. Wektor ekspresyjny, znamienny tym, że obejmuje sekwencje regulatorowe funkcjonalnie połączone z sekwencją DNA kodującą odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie sekwencja regulatorowa obejmuje promotor, sekwencje operatora, sekwencję kodującą miejsce wiązania rybosomu z mRNA oraz sekwencje regulujące terminację transkrypcji i translacji.
10. Komórka gospodarza, znamienna tym, że jest transforomowana wektorem ekspresyjnym obejmującym sekwencje regulatorowe funcjonalnie połączone z sekwencją DNA kodującą odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204,
PL 191 529 B1
206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie szczep Bacillus BG2036, Bacillus subtilis 1168 oraz B. licheniformis oraz B. lentus.
11. Kompozycja do czyszczenia, znamienna tym, że jako środek aktywny zawiera odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie w ilości od 0,01% do 5% wagowych w proszkowych lub ciekłych środkach czyszczących.
12. Karma dla zwierząt, znamienna tym, że zawiera odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie w pozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie w ilości od 0,01% do 5% wagowych.
13. Kompozycja do obróbki tekstyliów, znamienna tym, że zawiera odmianę proteazy, która zawiera podstawienie aminowasowe w pozycji reszty odpowiadającej pozycji 103 oraz przynajmniej jednej z pozycji 236 i 245 w subtylizynie z Bacillus amyloliquefaciens o sekwencji nr 1 oraz podstawienie przynajmniej jednego aminokwasu w pozycji reszty odpowiadającej następującym pozycjom w tej subtylizynie: 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177,181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 oraz 275, przy czym jeśli odmiana obejmuje podstawienie jednocześnie wpozycjach 103 i 76, to występuje również podstawienie w przynajmniej jednej z pozycji innych niż odpowiadające następującym pozycjom 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 lub 274, korzystnie w ilości od 0,01% do 5% wagowych.
14. Odmiana proteazy według zastrz. 1, znamienna tym, że zawiera następujący zestaw podstawień wybrany z grupy obejmującej pozycje reszt odpowiadających pozycjom subtylizyny z Bacillus amyloliquefaciens:
PL 191 529 B1
76,103,104,222; 76,98,103,104; 76,78,103,104; 76,103,104,107; 4,76,103,104;
76,103,104,246; 76,77,103,104; 76,103,104,183,218; 16,76,103,104,248;
1,76,103,104; 76,103,104,261; 76,103,104,160; 76,103,104,216; 17,76,103,104;
37,76,103,104; 76,77,103,104,174; 38,76,103,104; 38,76,103,104,237;
8,76,103,104; 76,103,104,183; 19,76,103,104; 13,76,103,104; 19,76,103,104;
76,103,104,184; 76,103,104,252; 76,103,104,259; 76,103,104,251; 76,86,103,104;
72,76,103,104,185; 76,103,104,237,274; 76,103,104,160; 76,103,104,228;
55,76,103,104,240; 76,103,104,254; 76,103,104,204; 76,103,104,204;
43,76,103,104; 76,103,104,159; 10,76,103,104,177; 58,76,103,104;
76,103,104,270; 76,103,104,185; 27,76,103,104; 76,103,104,262; 76,78,103,104;
24,76,103,104; 76,103,104,166,236,251; 17,76,103,104,237; 76,103,104,130;
76,103,104,109; 76,99,103,104,204; 76,103,104,181; 12,76,103,104;
76,103,104,212,271; 76.103,104,252,261; 76,103,104,242; 76,103,104,271;
12,76,103,104,242:43,76,103,104,116,183: 76,103,104,258:76,103,104,271
PL 191 529 B1
61,76,103,104; 38,76,103,104,182,263; 76,103,104,182,272; 76,103,104,109,246;
76,87,103,104,206,249,265; 76,103,104,137,238,271; 103,104,228,;
76,103,104,182,198; 21,76,103,104,182; 76,103,104,119,137; 76,103,104,137,248; 13,76,103,104,206; 76,103,104,206; 76,103,104,212,258; 58,76,103,104,271;
76,103,104,206,261; 4,76,103,104,206; 76,77,103,104,206; 76,103,104,158;
76,103,104,206; 4,76,103,104,159,217,251; 4,76,103,104,159,217,252;
76,77,103,104,133,185,251; 76,103,104,159,206,244; 4,76,103,104,188;
4,76,103,104,158; 76,77,103,104,185; 76,103,104,206,251; 48,76,103,104,111,159; 68,76,103,104,159,236; 42,76,103,104,159; 12,62,76,103,104,159;
42,76,103,104,159; 76,103,104,146,159; 76,103,104,159,238; 76,103,104,159,224; 76,103,104,212,268,271; 76,89,103,104; 76,87,103,104,212,271;
76,103,104,212,245,271; 76,103,104,134,141,212,271;
76,103,104,212,236,243,271; 76,103,104,109,245; 76,103,104,109,210;
20,62,76,103,104; 68,76,103,104,236; 68,76,103,104,159,236,271;
68,76,103,104,159,236,245; 68,76,103,104,159,217,236,271; 17,68,76,103,104;
68,76,103,104; 68,76,103,104,159,236; 68,75,76,103,104,159,236;
68,76,76,103,114,121,159,236,245; 12,68,76,103,104,159,236;
68,76,103,104,159,209,236,253; 68,76,103,104,117,159,184,236;
68,76,103,104,159,236,243; 68,76,103,104,159,236,245; 68,76,103,104,142,159;
68,76,103,104,123,159,236,249; 68,76,103,104,159,236,249; 76,103,104,222,245;
12,76,103,104,222,249; 76,103,104,173,222; 76,103,104,222,263;
21,76,103,104,222,237,263; 76,103,104,109,222; 76,103,104,109,222,271;
61,76,103,104,222; 76,103,104,137,222; 76,103,104,109,222,248;
76,103,104,222,249:68,76,103,104,159,236,245,261;
68,76,103,104,141,159,236,245,255; 68,76,103,104,159,236,245,247;
PL 191 529 B1
68,76,103,104,159,174,204,236,245; 68,76,103,104,159,204,236,245;
68,76,103,104,133,159,218,236,245; 68,76,103,104,159,232,236,245; 68,76,103,104,159,194,203,236,245; 12,76,103,104,222,245; 76,103,104,232,245; 24,68,76,103,104,159,232,236,245; 68,103,104,159,232,236,245,252; 68,76,103,104,159,213,232,236,245,260; 12,76,103,104,222,244,245; 12,76,103,222,210,245; 12,76,103,104,130,222,245; 22,68,76,103,104;
68,76,103,104,184; 68,103,104,159,232,236,245,248,252; 68,103,104,159,232,236,245; 68,103,104,140,159,232,236,245,252; 43,68,103,104,159,232,236,245,252;43,68,103,104,159,232,236,245; 43,68,103,104,159,232,236,245,252; 68,87,103,104,159,232,236,245,252,275; 12,76,103,104,130,222,245,248,262; 12,76,103,104,130,215,222,245; 12,76,103,104,130,222,227,245,262; 12,76,103,104,130,222,245,261; 76,103,104,130,222,245; 12,76,103,104,130,218,222,245,262,269; 12,57,76,103,104,130,222,245,251; 12,76,103,104,130,170,185,222,243,245; 12,76,103,104,130,222,245,268; 12,76,103,104,130,222,210,245;
68,103,104,159,232,236,245,257; 68,103,104,116,159,232,236,245;
68,103,104,159,232,236,245,248; 10,68,103,104,159,232,236,245; 68,103,104,159,203,232,236,245; 68,103,104,159,232,236,237,245; 68,76,79,103,104,159,232,236,245; 68,103,104,159,183,232,236,245;
68,103,104,159,174,206,232,236,245; 68,103,104,159,188,232,236,245;
68,103,104,159,230,232,236,245; 68,98,103,104,159,232,236,245; 68,103,104,159,215,232,236,245; 68,103,104,159,232,236,245,248;
68,76,103,104,159,232,236,245; 68,76,103,104,159,210,232,236,245;
68,76,103,104,159,232,236,245,257; 76,103,104,232,236,245,257;
68,103,104,159,232,236,245,257,275; 76,103,104,257,275;
PL 191 529 B1
68,103,104,159,224,232,236,245,257; 76,103,104,159,232,236,245,257;
68,76,103,104,159,209,232,236,245; 68,76,103,104,159,211,232,236,245;
12,68,76,103,104,159,214,232,236,245; 68,76,103,104,159,215,232,236,245;
12,68,76,103,104,159,232,236,245:20,68,76,103,104,159,232,236,245,259; 68,87,76,103,104,159,232,236,245,260; 68,76,103,104,169,232,236,245,261; 76,103,104,232,236,242,245; 68,76,103,104,159,210,232,236,245;
12,48,68,76,103,104,159,232,236,245; 76,103,104,232,236,245;
76,103,104,159,192,232,236,245; 76,103,104,147,159,232,236,245,248,251;
12,68,76,103,104,159,232,236,245,272; 68,76,103,104,159,183,206,232,236,245;
.68,76,103,104,159,232,236,245,256; 68,76,103,104,159,206,232,236,245;
27,68,76,103,104,159,232,236,245; 68,76,103,104,116,159,170,185,232,236,245;
61,68,103,104,159,232,236,245,248,252; 43,68,103,104,159,232,236,245,248,252;
68,103,104,159,212,232,236,245,248,252;
68,103,104,99,159,184,232,236,245,248,252; 103,104,159,232,236,245,248,252;
68,103,104,159,209,232,236,245,248,252;
68,103,104,109,159,232,236,245,248,252;
20,68,103,104,159,232,236,245,248,252;
68,103,104,159,309,232,236,245,248,252;
68,103,104,159,232,236,245,248,252,261;
68,103,104,159,185,232,236,245,248,252;
68,103,104,159,210,232,236,245,248,252;
68,103,104,159,185,210,232,236,245,248,252;
68,103,104,159,212,232,236,245,248,252;
68,103,104,159,213,232,236,245,248,252; 68,103,104,213,232,236,245,248,252;
68,103,104,159,215,232,236,245,248,252;
PL 191 529 B1
68,103,104,159,216,232,236,245,248,252;
20,68,103,104,159,232,236,245,248,252;
68,103,104,159,173,232,236,245,248,252;
68,103,104,159,232,236,245,248,251,252;
68,103,104,159,206,232,236,245,248,252; 68,103,104,159,232,236,245,248,252; 55,68,103,104,159,232,236,245,248,252;
68,103,104,159,232,236,245,248,252,255;
68,103,104,159,232,236,245,248,252,256;
68,103,104,159,232,236,245,248,252,260;
68,103,104,159,232,236,245,248,252,257;
68,103,104,159,232,236,245,248,252,258;
8,68,103,104,159,232,236,245,248,252,269;
68,103,104,116,159,232,236,245,248,252,260;
68,103,104,159,232,236,245,248,252,261;
68,103,104,159,232,236,245,248,252,261;
68,76,103,104,159,232,236,245,248,252; 68,103,104,232,236,245,248,252;
103,104,159,232,236,245,248,252; 68,103,104,159,232,236,245,248,252; 18,68,103,104,159,232,236,245,248,252; 68,103,104,159,232,236,245,248,252; 68,76,101,103,104,159,213,218,232,236,245,260;
68,103,104,159,228,232,236,245,248,252;
33,68,76,103,104,159,232,236,245,248,252;
68,76,89,103,104,159,210,213,232,236,245,260;
61,68,76,103,104,159,232,236,245,248,252; 103,104,159,205,210,232,236,245;
61,68,103,104,130,159,232,236,245,248,252;
61,68,103,104,133,137,159,232,236,245,248,252;
PL 191 529 B1
61,103,104,133,159,232,236,245,248,252; 68,103,104,159,232,236,245,248,252; 68,103,104,159,218,232,236,245,248,252;
61,68,103,104,159,160,232,236,245,248,252;
3,61,68,76,103,104,232,236,245,248,252;
61,68,103,104,159,167,232,236,245,248,252;
97,103,104,159,232,236,245,248,252; 98,103,104,159,232,236,245,248,252; 99,103,104,159,232,236,245,248,252; 101,103,104,159,232,236,245,248,252; 102,103,104,159,232,236,245,248,252; 103,104,106,159,232,236,245,248,252; 103,104,109,159,232,236,245,248,252; 103,104,159,232,236,245,248,252,261; 62,103,104,159,232,236,245,248,252; 103,104,159,184,232,236,245,248,252; 103,104,159,166,232,236,245,248,252; 103,104,159,217,232,236,245,248,252; 20,62,103,104,159,213,232,236,245,248,252;
62,103,104,159,213,232,236,245,248,252;
103,104,159,206,217,232,236,245,248,252;
62,103,104,159,206,232,236,245,248,252; 103,104,130,159,232,236,245,248,252; 103,104,131,159,232,236,245,248,252; 27,103,104,159,2-32,236,245,248,252; 38,103,104,159,232,236,245,248,252; 38,76,103,104,159,213,232,236,245,260; 68,76,103,104,159,213,232,236,245,260,271;
68,76,103,104,159,209,213,232,236,245,260;
68,76,103,104,159,210,213,232,236,245,260;
68,76,103,104,159,205,213,232,236,245,260;
68,76,103,104,159,210,232,236,245,260; 68,103,104,159,213,232,236,245,260;
76,103,104,159,213,232,236,245,260; 68,103,104,159,209,232,236,245; 68,103,104,159,210,232,236,245; 68,103,104,159,230,232,236,245; 68,103,104,159,126,232,236,245; 68,103,104,159,205,232,236,245;
PL 191 529 B1
68,103,104,159,210,232,236,245; 103,104,159,230,236,245;
68,103,104,159,232,236,245,260; 103,104,159,232,236,245;
68,103,104,159,174,232,236,245,257; 68,103,104,159,194,232,236,245,257; 68,103,104,159,209,232,236,245,257; 103,104,159,232,236,245,257;
68,76,103,104,159,213,232,236,245,260,261;
68,103,104,159,232,236,245,257,261; 103,104,159,213,232,236,245,260;
103,104,159,210,232,236,245,248,252; 103,104,159,209,232,236,245,257;
68,76,103,104,159,210,213,232,236,245,260;
12,103,104,159,209,213,232,236,245,260; 103,104,209,232,236,245,257;
103,104,159,205,210,213,232,236,245,260; 103,104,159,205,209,232,236,245,260; 68,103,104,159,205,209,210,232,236,245;
103,104,159,205,209,210,232,236,245,257; 103,104,159,205,209,232,236,245,257; 68,103,104,159,205,209,210,232,236,245,260;
103,104,159,205,209,210,232,236,245; 103,104,159,209,210,232,236,245; 103,104,159,205,210,232,236,245; 68,103,104,128,159,232,236,245; 48,103,104,159,230,236,245; 48,68,103,104,169,209,232,236,245; 48,68,103,104,159,232,236,245,248,252; 48,68,103,104,159,232,236,245,257,261; 102,103,104,159,212,232,236,245,248,252;
12,102,103,104,159,212,232,236,245,248,252;
101,102,103,104,159,212,232,236,245,248,252;
98,102,103,104,159,212,232,236,245,248,252;
102,103,104,159,213,232,236,245,248,252; 103,104,131,159,232,236,245,248,252; 103,104,159,184,232,236,245,248,252; 103,104,159,232,236,244,245,248,252; 62,103,104,159,213,232,236,245,248,252,256;
12,62,103,104,159,213,232,236,245,248,252;
PL 191 529 B1
101,103,104,159,185,232,236,245,248,252;
101,103,104,159,206,232,236,245,248,252;
101,103,104,159,213,232,236,245,248,252;
98,102,103,104,159,232,236,245,248,252;
101,102,103,104,159,232,236,245,248,252;
98,102,103,104,159,212,232,236,245,248,252;
98,102,103,104,159,212,232,236,248,252;
62,103,104,109,159,213,232,236,245,248,252;
62,103,104,159,212,213,232,236,245,248,252;
62,101,103,104,159,212,213,232,236,245,248,252; 103,104,159,232,245,248,252; 103,104,159,230,245; 62,103,104,130,159,213,232,236,245,248,252;
101,103,104,130,159,232,236,245,248,252;
101,103,104,128,159,232,236,245,248,252;
62,101,103,104,159,213,232,236,245,248,252;
62,103,104,128,159,213,232,236,245,248,252;
62,103,104,128,159,213,232,236,245,248,252;
101,103,104,159,232,236,245,248,252,260;
101,103,104,131,159,232,236,245,248,252;
98,101,103,104,159,232,236,245,248,252;
99,101,103,104,159,232,236,245,248,252;
101,103,104,159,212,232,236,245,248,252;
101,103,104,159,209,232,236,245,248,252;
101,103,104,159,210,232,236,245,248,252;
101,103,104,159,205,232,236,245,248,252; 101,103,104,159,230,236,245;
101,103,104,159,194,232,236,245,248,252;
76,101,103,104,159,194,232,236,245,248,252;
101,103,104,159,230,232,236,245,248,252;
62,103,104,159,185,206,213,232,236,245,248,252,271.
PL 191 529 B1
15. Odmiana proteazy według zastrz. 14, znamienna tym, że zawiera następujący zestaw podstawień wybrany z grupy obejmującej pozycje reszt odpowiadających pozycjom subtylizyny z Bacillus amyloliquefaciens:
N76D, S103A, V104l, M222S; N76D, A98E, S103A, V104l; N76D, S78T, S103A,
Y104I; N76D, S103A, V104l, I107Y; V4E, N76D, S103A, V104I; N76D, S103A,
V104I, I246Y; N76D, N77D, S103A, V104I; N76D, S103A, V104l, N183D, N218I;
A16T, N76D, S103A, V104l, N248D; A1E, N76D, S103A, V104I; N76D, S103A,
V104l, N261D; N76D, S103A, V104l, S160T; N76D, S103A, Y104I, S216C; H17Q,
N76D, S103A, V104l; S37T, N76D, S103A, V104l; N76D, N77D, S103A, V104l,
A174V; T38S, N76D, S103A, V104I; T38S, N76D, S103A, V104l, K237Q; I8V,
N76D, S103A, V104l; N76D, S103A, V 1041, N183D; R19L, N76D, S103A, V104I;
A13V, N76D, S103A, V104I; R19C, N76D, S103A, V104l; N76D, S103A, V104l,
N184D; N76D, S103A, V104l, N252D; N76D, S103A, V104l, S259C; N76D,
S103A, V104l, K251T; N76D, P86S, S103A, V104l; I72V, N76D, S103A, V104l,
N185D; N76D, S103A, V104l, K237E, T274A; N76D, S103A, V104I, S160L;
N76D, S103A, V104l, A228Y; P55S, N76D, S103A, V104l, S240T; N76D, S103A,
V104l, A254T; N76D, S103A, I104N, N204T; N76D, S103A, V104l, N204D; N43S,
N76D, S103A, V104I; N76D, S103A, V104l, G159D; R10H, N76D, S103A, V104l,
Y177A; T58S, N76D, S103A, V104l; N76D, S103A, V104l, A270Y; N76D, S103A,
V104l, N185D; K27N, N76D, S103A, V104l; N76D, S103A, V104l, L262M; N76D,
S78P, S103A, V104l; S24P, N76D, S103A, V104l; N76D, S103A, V104l, S166G,
Q236R, K251R; H17L, N76D, S103A, V104l, K237E; N76D, S103A, V104l, S130L;
PL 191 529 B1
N76D, S103A, V104I, Q109R; N76D, S99R, S103A, V104I, N204T; N76D, S103A, V104l, D181N; Q12R, N76D, S103A, V104l; N76D, S103A, V104I, S212P, E271V; N76D, S103A, V104l, N252K, N261Y; N76D, S103A, V104I, S242T; N76D, S103A, V104l, E271Q; Q12R, N76D, S103A, V104l, S242T; N43S, N76D, S103A, V104l, N116K, N1831; N76D, S103A, V104l, G258R; N76D, S103A, V104l, E271G;
G61R, N76D, S103A, V104l; T38S, N76D, S103A, V104l, Q182R, Y263H; N76D, S103A, V104I, Q182R, A272S; N76D, S103A, V104I, Q109R, I246Y; N76D, S87G, S103A, V104l, Q206R, H249Q, S265G; N76D, S103A, V104I, Q137R, N238Y, E271V; S103A, V104l, A228T, ; N76D, S103A, V104l, Q182R, 1198 V; L21M, N76D, S103A, V104l, Q182R; N76D, S103A, V104l, M119I, Q137R; N76D, S103A, V104l, Q137R, N248S; A13T, N76D, S103A, V104I, Q206R; N76D, S103A, V104l, Q206R; N76D, S103A, Y104I, 8212P, G258R; T58S, N76D, S103A; V104l,
E271G; N76D, S103A; V104l, Q206E, N261D; V4E, N76D, S103A; V104l,
Q206E; N76D, N77D, S103A; V104l, Q206E; N76D, S103A; V104l, A158E;
N76D, S103A; V104l, Q206E; V4E, N76D, S103A; V104l, G159D, L217E, K251Q; V4E, N76D, S103A; V104l, G159D, L217E, N252D; N76D, N77D, S103A; V104l,
A1SST, N185D, K251T; N76D, S103A; V104l, G159D, Q206E, Y244A; V4E,
N76D, S103A; V104l,8188E; V4E, N76D, S103A; V104l,A158E; N76D, N77D, S103A; V104l, N185D; N76D, S103A; V104l, Q206E, K251T; A48T, N76D,
S103A; V104l, L111M, G159D; V68A, N76D, S103A; V104l, G159D, Q2S6H;
L42V, N76D, S103A; V104l, G159D; Q12H, N62H, N76D, S103A; V104l, G159D;
L421, N76D, S103A, V104I, G159D; N76D, S103A, Y104I, G146S, G159D; N76D,
S103A, V104l, G159D, N238S; N76D, S103A, Y104I, G159D, T224A; N76D, S103A, V104I, S212P, Y268F, E271Y; N76D, E89A, S103A, V104l; N76D, S87R,
S103A, V104l, S212P, E271Y; N76D, S103A, V104I, S212P, Q245L, E271V;
PL 191 529 B1
N76D, S103A, V104l, T1A4S, S141N, S212P, E271V; N76D, S103A, V104l, S212P, Q236L, N243S, E271V; N76D, S103A, V104l, Q109R, Q245R; N76D, S103A,
V104I, Q109R, P210L; G20V, N62S, N76D, S103A, V104l; V68A, N76D, S103A,
V104I, Q236H; V68A, N76D, S103A, V104l, G159D, Q236H, E271V; V68A, N76D, S103A, V104I, G159D, Q236H, Q245R; V68A, N76D, S103A, V104I, G159D,
L217I, Q236H, E271V; H17Q, V68A, N76D, S103A, V104l; V68A, N76D, S103A,
V104I; V68A, N76D, S103A, V104I, G159D, Q236R; V68A, L75R, N76D, S103A, V104l, G159D, Q236H; V68A, N76D, N76D, S103A, Α114Y, ¥1211, G159D, Q236H,Q245R; Q12R, V68A, N76D, S103A, V104l, G159D, Q236H; V68A, N76D, S103A, V104I, G159D, Y209S, Q236H, T253K; V68A, N76D, S103A, V104I,
N117K, G159D, N184S, Q236H; V68A, N76D, S103A, V104l, G159D, Ω236Η, N243I; V68A, N76D, S103A, V104l, G159D, Q236H, Q245L; V68A, N76D, S103A,
V104l, A142V, G159D; V68A, N76D, S103A, V104I, N123S, G159D, Q236H,
H249Y; V68A, N76D, S103A, V104I, G159D, Q236H, H249Q; N76D, S103A;
V104l, M222S, Q245R; N76D, S103A, V104l, Q12R, M222S, H249R; N76D,
S103A, V104l, N173R, M222S; N76D, S103A, V104l, M222S, Y263F; L21M,
N76D, S103A, V104I, M222S, K237R, Y263F; N76D, S103A, V104l, Q109R,
M222S; N76D, S103A, Y104I, Q109R, M222S, E271D; G61R, N76D, S103A,
V104I, M222S; N76D, S103A, V104l, Q137R, M222S; N76D, S103A, V104l,
Q109R, M222S, N248S; N76D, S103A, Y104I, M222S, H249R; V68A, N76D,
S103A, V104l, G159D, Q236H, Q245R, N261D; V68A, N76D, S103A, V104l,
S141N, G159D, Q236H, Q245R, T255S; V68A, N76D, S103A, V104l, G159D,
Q236H, Q245R, R247H; V68A, N76D, S103A, V104l, G159D, A174V, N204D,
Q236H, Q245R; V68A, N76D, S103A, V104l, G159D, N204D, Q236H, Q245R;
V68A, N76D, S103A, V104I, A133V, G159D, N218D, Q236H, Q245R; V68A, N76D,
PL 191 529 B1
S103A, V104Ι, G159D, A232V, Q236H, Q245R; V68A, N76D, S103A, V104l, G159D, A194I, Y203A, Q236H, Q245R; Q12R, N76D, S103A, V104l, M222S, Q245R; N76D, S103A, V104l, A232Y, Q245R; S24T, V68A, N76D, S103A, V104l, G159D, A232V, Q236H, Q245R; V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N252K; V68A, N76D, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, T260A; Q12R, N76D, S103A, I104T, M222S, Y244I, Q245R; Q12R, N76D, S103A, M222S, P210T, Q245R; Q12R, N76D, S103A, I104T, S130T, M222S, Q245R; T22K, V68A, N76D, S103A, V104l; V68A, N76D, S103A, N184D; V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, Y104I, G159D, A232Y, Q236H, Q245R; V68A, S103A, V104l, N140D, G159D, A232V, Q236H, Q245R, N252K; N43S, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N252K; N43K, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R; N43D, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N252K; V68A, S87G, S103A, V104l, G159D, A232V, Q236H, Q245R, N252K, R275S;
Q12R, N76D, S103A, I104T, S130T, M222S, Q245R, N248S, L262M; Q12R, N76D, S103A, I104T, S130T, A215Y, M222S, Q245R; Q12R, N76D, S103A, I104T,
S130T, M222S, V227A, Q245R, L262S; Q12R, N76D, S103A, I104T, S130T, A215T, M222S, Q245R; Q12R, N76D, S103A, I104T, S130T, M222S, Q245R, N261D; N76D, S103A, I104T, S130T, M222S, Q245R; Q12R, N76D, S103A,
I104T, S130T, N218D, M222S, Q245R, L262S, N269D; Q12R, S57P, N76D,
S103A, I104T, S130T, M222S, Q245R, K251Q; Q12R, N76D, S103A, I104T,
S130T, R170S, N185D, M222S, N243D, Q245R; Q12R, N76D, S103A, I104T, S130T, M222S, Q245R, Y268A; Q12R, N76D, S103A, I104T, S130T, M222S,
P210S, Q245R; V68A, S103A, V104I, G159D, A232Y, Q236H, Q245R, L257V; V68A, S103A, V104l, N116D, G159D, A232Y, Q236H, Q245R; V68A, S103A,
PL 191 529 B1
V1041, G159D, Α232Υ, Q236H, Q245R, N248D; R10C, V68A, S103A, V104l, G159D, A232Y, Q236H, Q245R; V68A, S103A, V104l, G159D, Y203E, A232Y, Ω236Η, Q245R; V68A, S103A, V104l, G159D, A232Y, Ω236Η, K237E, Q245R;
V68A, N76D, 179N, S103A, V104l, G159D, A232Y, Ω236Η, Q245R; V68A, S103A,
V104I, G159D, N183D, A232Y, Q236H, Q245R; V68A, S103A, V104I, G159D, A174Y, Q206L, A232Y, Ω236Η, Q245R; V68A, S103A, V104I, G159D, S188C, A232Y, Ω236Η, Q245R; V68A, S103A, V104l, G159D, A230T, A232Y, Q236H, Q245R; V68A, A98T, S103A, V104l, G159D, A232Y, Q236H, Q245R; V68A, S103A, V104I, G159D, A215T, A232Y, Q236H, Q245R; V68A, S103A, V104l, G159D, A232Y, Q236H, Q245R, N248S; V68A, N76D, S103A, V104l, G159D, A232Y, Q236H, Q245R; V68A, N76D, S103A, V104I, G159D, P210R, A232Y, Q236H, Q245R; V68A, N76D, S103A, V104l, G159D, A232Y, Q236H, Q245R, L257V; N76D, S103A, V104I, A232Y, Q236H, Q245R, L257V; V68A; S103A, V104l, G159D, A232Y, Q236H, Q245R, L257Y, R275H; N76D, S103A, V104l, L257Y, R275H, ; V68A, S103A, V104l, G159D, T224A, A232Y, Q236H, Q245R, L257Y; N76D, S103A, V104l, G159D, A232Y, Q236H, Q245R, L257Y; V68A,
N76D, S103A, V104l, G159D, Y209W, A232V, Q236H, Q245R; V68A, N76D,
S103A, V104I, G159D, G211R, A232V, Q236H, Q245R; V68A, N76D, S103A,
V104l, G159D, G211Y, A232V, Q236H, Q245R; Q12R, V68A, N76D, S103A,
V104l, G159D, Y214L, A232V, Q236H, Q245R; V68A, N76D, S103A, V104l,
G159D, A215R, A232V, Q236H, Q245R; Q12R, V68A, N76D, S103A, V104l,
G159D, A232V, Q236H, Q245R; G20R, V68A, N76D, S103A, V104l, G159D,
A232V, Q236H, Q245R, S259G; V68A, S87R, N76D, S103A, V104l, G159D,
A232V, Q236H, Q245R, T260Y; V68A, N76D, S103A, V104l, G159D, A232V,
Q236H, Q245R, N261G; V68A, N76D, S103A, V104I, G159D, A232Y, Q236H,
PL 191 529 B1
Q245R, N261W; N76D, S103A, V104I, A232Y, Q236H, S242P, Q245R; V68A, N76D, S103A, V104l, G159D, P210L, A232V, Q236H, Q245R; Q12R, A48Y, V68A, N76D, S103A, V104l, G159D, A232V, Q236H, Q245R; N76D, S103A, V104l, A232Y, Q236H, Q245R; N76D, S103A, V104l, G159D, Y192F, A232V, Q236H, Q245R; V68A, S103A, V104I, G159D, A232Y, Q236H, Q245R, L257Y, R275H; N76D, S103A, V104l, L257Y, R275H; V68A, S103A, V104l, G159D, T224A, A232Y,
Q236H, Q245R, L257V; N76D, S103A, V1041, G159D, A232Y, Q236H, Q245R, L257V; V68A, N76D, S103A, V104I, G159D, Y209W, A232V, Q236H, Q245R;
V68A, N76D, S103A, V104l, G159D, G211R, A232V, Q236H, Q245R; V68A, N76D,
S103A, V104I, G159D, G211Y, A232V, Q236H, Q245R; Q12R, V68A, N76D,
S103A, V104l, G159D, Y214L, A232V, Q236H, Q245R; V68A, N76D, S103A,
VfÓ4l, G159D, A215R, A232V, Q236H, Q245R; Q12R, V68A, N76D, S103A, V104l, G159D, A232V, Q236H, Q245R; G20R, V68A, N76D, S103A, V104l, G159D,
A232V, Q236H, Q245R, S259G; V68A, S87R, N76D, S103A, V104l, G159D,
A232V, Q236H, G245R, T260Y; V68A, N76D, S103A, V104l, G159D, A232V,
Q236H, Q245R, N261G; V68A, N76D, S103A, V104l, G159D, A232Y, Q236H,
Q245R, N261W; N76D, S103A, V104l, A232Y, Q236H, S242P, Q245R; V68A,
N76D, S103A, V104I, G159D, P210L, A232V, Q236H, Q245R; Q12R, A48V, V68A,
N76D, S103A, V104l, G159D, A232V, Q236H, Q245R; N76D, S103A, V104l,
A232Y, Q236H, Q245R; N76D, S103A, V104l, G159D, Y192F, A232V, Q236H,
Q245R; N76D, S103A, V104l, Y147I, G159D, A232V, Q236H, Q245R, N248S,
K251R; Q12R, V68A, N76D, S103A, V104l, G159D, A232V, Q236H, Q245R,
A272S; V68A, N76D, S103A, V104l, G159D, N183K, Q206L, A232V, Q236H,
Q245R; V68A, N76D, S103A, V104i, G159D, A232V, Q236H, Q245R, S256R;
V68A, N76D, S103A, V104I, G159D, Q206R, A232V, Q236H, Q245R; K27R, V68A,
PL 191 529 B1
N76D, S103A, V104l, G159D, A232V, Q236H, Q245R; V68A, N76D, S103A, V104l, N116T, G159D, R170S, N185S, A232V, Q236H, Q245R; G61E, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; N43D, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, S212P, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, S99N, G159D, N184D, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, G159D, A232Y, Q236H, Q245R, N248D, N252K; V68A, S103A, V1041, G159D, Y209W, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, 0109R, G159D, A232V, Q236H, Q245R, N248D, N252K; G20R, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, Y209F, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K, N261 D; V68A, S103A, V104l, G159D, N185D, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, P210R, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, P210T, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, P210S, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, N185D, P210L, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V1041, G159D, P210L, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, S212A, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, S212G, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, S212E, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, T213E, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, T213S, A232V, Q236H, Q245R, N248D, N252K; V68A, A103V, V104l, G159D, T213E, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D,
PL 191 529 B1
T213G, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104I, G159D, A215Y, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104I, G159D,
A215R, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104I, G159D, S216T, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, S216Y, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, S216C, A232V, Q236H, Q245R, N248D, N252K; G20A, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, N173D, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, K251Y, N252K; V68A, S103A, V104l, G159D, Q206R, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252F; V68A, S103A, V1041, G159D, A232V, Q236H, Q245R, N248D, N252L; P55S, V68A, S103A, V104I, G159D, A232V, Q236H, Q245R, N248D, N252F; V68A, S103A, V104l, G159D, A232V, Q236H,
Q245R, N248D, N252K, T255Y; V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K, S256N; V68A, S103A, V104I, G159D, A232V, Q236H,
Q245R, N248D, N252K, S256E; V68A, S103A, V104l, G159D, A232V, Q236H,
Q245R, N248D, N252K, S256R; V68A, S103A, V104l, G159D, A232V, Q236H,
Q245R, N248D, N252K, T260R; V68A, S103A, V104l, G159D, A232V, Q236H,
Q245R, N248D, N252K, L257R; V68A, S103A, V104l, G159D, A232V, Q236H,
Q245R, N248D, N252K, G258D; 18V, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K, N269D; V68A, S103A, V104l, N116S, G159D,
A232V, Q236H, Q245R, N248D, N252K, T260E; V68A, S103A, V104l, G159D,
A232V, Q236H, Q245R, N248D, N252K, N261R; V68A, S103A, V104l, G159D,
A232V, Q236H, Q245R, N248D, N252K, N261D; V68A, N76D, S103A, V104l,
G159D, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, A232V,
PL 191 529 B1
Q236H, Q245R, N248D, N252K; S103A, V104l, G159D, A232S, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159DD; A232Y, Q236R, Q245R, N248D, N252K; N18S, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, A232Y, Q236H, Q245V, N248D, N252K; V68A, N76D, S101T, S103A, V104l, G159D, T213R, N218S, A232V, Q236H, Q245R, T260A; V68A, S103A, V104l, G159D, A228Y, A232V, Q236H, Q245R, N248D, N252K; T33S, V68A, N76D, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; V68A, N76D, E89D, S103A, V104l, G159D, P210L, T213R, A232V, Q236H, Q245R, T260A; G61E, V68A, N76D, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, G159D, V205l, P210I, A232V, Q236H, Q245R; G61E, V68A, S103A, V104l, S130A, G159D, A232V, Q236H, Q245R, N248D, N252K; G61E, V68A, S103A, V104l, A133S, Q137R, G159D, A232V, Q236H, Q245R, N248D, N252K; G61E, S103A, V104l, A133Y, G159D, A232V, Q236H, Q245R, N248D, N252K; V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248G, N252K; V68A, S103A, V104l, G159D, N218S, A232V, Q236H, Q245R, N248D, N252K; G61E, V68A, S103A, V104l, G159D, S 160 V, A232V, Q236H, Q245R, N248D, N252K; S3L, G61E, V68A, N76D, S103A, V104l, A232V, Q236H, Q245R, N248D, N252K; G61E, V68A, S103A, V104I, G159D, S167F, A232V, Q236H, Q245R, N248D, N252K; G97E, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; A98D, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; S99E, S103A, V104l, G159D, A232V, Q236K, Q245R, N248D, N252K; S101E, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; G102A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, S106E, G159D, A232V, Q236H, Q245R,
PL 191 529 B1
N248D, Ν252Κ; S103A, V104l, Q109E, G159D, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K, N261 R; S103A, V104l, Q109R, G159D, A232V, Q236H, Q245R, N248D, N252K; N62D, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, G159D, N184D, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, G159D, S166D, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, G159D, L217E, A232V, Q236H, Q245R, N248D, N252K; G20R, N62D, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, N248D, N252K; N62D, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, G159D, Q206R, L217E, A232V, Q236H, Q245R, N248D, N252K; N62D, S103A, V104l, G159D, Q206R, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, S130G, G159D, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, P131Y, G159D, A232V, Q236H, Q245R, N248D, N252K; K27N, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; T38G, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; T38A, N76D, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, T260A; V68A, N76D, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, T260A, E271G; V68A, N76D, S103A, V104l, G159D, Y209W, T213R, A232V, Q236H, Q245R, T260A; V68A, N76D, S103A, V104l, G159D, P210I, T213R, A232V, Q236H, Q245R, T260A; V68A, N76D, S103A, V104l, G159D, Y205I, T213R, A232V, Q236H, Q245R, T260A; V68A, N76D, S103A, V104l, G159D, P210I, A232V, Q236H, Q245R, T260A; V68A, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, T260A; N76D, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, T260A; V68A, S103A, V104l, G159D, Y209W, A232V, Q236H, Q245R; V68A, S103A, V104l, G159D, P210I, A232V, Q236H, Q245R; V68A, S103A, V104l, G159D, A230Y, A232V, Q236H, Q245R; V68A, S103A,
PL 191 529 B1
V104l, G159D, L126F, A232V, Q236H, Q245R; V68A, S103A, V104l, G159D, Y205I, A232V, Q236H, Q245R; V68A, S103A, V104I, G159D, P210L, A232V, Q236H, Q245R; S103A, V104l, G159D, A230Y, Q236H, Q245R; V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, T260A; S103A, V104l, G159D, A232V, Q236H, Q245R; V68A, S103A, V104l, G159D, A174V, A232V, Q236H, Q245R, L257V; V68A, S103A, V1041, G159D, A194S, A232V, Q236H, Q245R, L257V; V68A, S103A, V1041, G159D, Y209W, A232V, Q236H, Q245R, L257V; S103A, V1041, G159D, A232V, Q236H, Q245R, L257V; V68A, N76D, S103A, V1041, G159D, T213R, A232V, Q236H, Q245R, T260A, N261W; V68A, S103A, V1041, G159D, A232V, Q236H, Q245R, L257V, N261W; S103A, V1041, G159D, T213R, A232V, Q236H, Q245R, T260A; S103A, V1041, G159D, P210I, A232V, Q236H, Q245R, N248D, N252K; S103A, Y104I, G159D, Y209W, A232V, Q236H, Q245R, L257V; V68A, N76D, S103A, V104l, G159D, P210L, T213R, A232V, Q236H, Q245R, T260A; Q12R, S103A, V1041, G159D, Y209W, T213R, A232V, Q236H, Q245R, T260A; S103A, V1041, Y209W, A232V, Q236H, Q245R, L257V, *; S103A, V1041, G159D, V205l, P210I, T213R, A232V, Q236H, Q245R, T260A; S103A, Y104I, G159D, V205l, Y209W, A232V, Q236H, Q245R, T260A; V68A, S103A, V1041, G159D, V205l, Y209W, P210I, A232V, Q236H, Q245R; S103A, V1041, G159D, V205l, Y209W, P210I, A232V, Q236H, Q245R, L257V; S103A, V1041, G159D, V205l, Y209W, A232V, Q236H, Q245R, L257V; V68A, S103A, V1041, G159D, V205l, Y209W, P210I, A232V, Q236H, Q245R, T260A; S103A, V1041, G159D, V205l, Y209W, P210I, A232V, Q236H, Q245R; S103A, V1041, G159D, Y209W, P210I, A232V, Q236H, Q245R; S103A, V104l, G159D, V205l, P210I, A232V, Q236H, Q245R; V68A, S103A, V104l, S128L, G159D, A232V, Q236H, Q245R; A48V, S103A, V104l, G159D, A230Y, Q236H, Q245R; A48V, V68A,
PL 191 529 B1
S103A, V104Ι, G159D, Y209W, A232V, Q236H, Q245R; A48V, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; A48V, V68A, S103A, V104l, G159D, A232V, Q236H, Q245R, L257Y, N261W; G102A, S103A, V1041, G159D, S212G, A232V, Q236H, Q245R, N248D, N252K; Q12R, G102A, S103A, V104l, G159D, S212G, A232V, Q236H, Q245R, N248D, N252K; S101G, G102A, S103A, V104l, G159D, S212G, A232V, Q236H, Q245R, N248D, N252K; A98L, G102A, S103A, V104I, G159D, S212G, A232V, Q236H, Q245R, N248D, N252K; G102A, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, N248D, N252K; S103A, Y104I, P131Y, G159D, A232V, Q236H, Q245R, N248D, N252K; S103A, Y104I, G159D, N184S, A232V, Q236H, Q245R, N248D, N252K; S103A, Y104I, G159D, N184G, A232V, Q236H, Q245R, N248D, N252K; S103A, V104l, G159D, A232Y, Q236H, V244T, Q245R, N248D, N252K; S103A, V104l, G159D, A232V, Q236H, Y244A, Q245R, N248D, N252K; N62D, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, N248D, N252K, S256R; Q12R, N62D, S103A, V104l, G159D, T213R, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A, V104l, G159D, N185D, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A, V104I, G159D, Q206E, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A, V104I, G159D, T213Q, A232V, Q236H, Q245R, N248D, N252K; A98L, G102A, S103A, V104l, G159D, A232V, Q236H, Q245R, N248D, N252K; S101G, G102A, S103A, V104t, G159D, A232V, Q236H, Q245R, N248D, N252K; A98L, G102A, S103A, V104l, G159D, S212G, A232V, Q236H, Q245R, N248D, N252K; A98L, G102A, S103A, V104I, G159D, S212G, A232V, Q236H, N248D, N252K; N62D, S103A, V104l, Q109R, G159D, T213R, A232V, Q236H, Q245R, N248D, N252K; N62D, S103A, V104I, G159D, S212G, T213R, A232V, Q236H, Q245R, N248D, N252K; N62D, S101G, S103A, V104l, G159D, S212G, T213R, A232V, Q236H, Q245R,
PL 191 529 B1
N248D, Ν252Κ; S103A, V104l, G159D, A232V, Q245R, N248D, Ν252Κ; S103A,
V104I, G159D, A230V, Q245R; N62D, S103A, V104l, S130G, G159D, T213R,
A232V, Q236H, Q245R, N248D, N252K; S101G, S103A, V104l, S130G, G159D,
A232V, Q236H, Q245R, N248D, N252K; S101G, S103A, V104l, S128G, G159D,
A232V, Q236H, Q245R, N248D, N252K; S101G, S103A, V104l, S128L, G159D,
A232V, Q236H, Q245R, N248D, N252K; N62D, S101G, S103A, V104l, G159D,
T213R, A232V, Q236H, Q245R, N248D, N252K; N62D, S103A, V104I, S128G,
G159D, T213R, A232V, Q236H, Q245R, N248D, N252K; N62D, S103A, V104l,
S128L, G159D, T213R, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A,
V104I, G159D, A232V, Q236H, Q245R, N248D, N252K, T260A; S101G, S103A,
V104I, P131Y, G159D, A232V, Q236H, Q245R, N248D, N252K; A98V, S101G,
S103A, V104I, G159D, A232V, Q236H, Q245R, N248D, N252K; S99G, S101G,
S103A, V104l, G159D, A232V, G236H, Q245R, N248D, N252K; S101G, S103A,
V104I, G159D, S212G, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A,
V104I, G159D, Y209W, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A,
V104I, G159D, P210I, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A,
V104l, G159D, Y205I, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A,
V104l, G159D, A230Y, 0236H, Q245R; S101G, S103A, V104l, G159, A194P,
A232V, Q236H, Q245R, N248D, N252K; N76D, S101G, S103A, V104l, G159D,
A194P, A232V, Q236H, Q245R, N248D, N252K; S101G, S103A, V104l, G159D,
A230Y, A232V, Q236H, Q245R, N248D, N252K; N62D, S103A, V104l, G159D,
N185D, 0206E, T213R, A232V, Q236H, Q245R, N248D, N252K, E271Q.
16. Odmiana proteazy według zastrz. 14, znamienna tym, że zawiera następujący zestaw podstawień wybrany z grupy obejmującej pozycje reszt odpowiadających pozycjom subtylizyny z Bacillus amyloliquefaciens przedstawionym: 76, 103, 104, 222, 245; 76, 103, 104, 222, 249; 68, 103, 104, 159, 232, 236, 245, 252; 68, 76, 103, 104, 159, 213, 232, 236, 245, 260; 22, 68, 76, 103, | 104; 68, 103, 104, 159, 232, 236, 245, 248, 252; 68, 103, 104, 159, 232, 236, 245; 68, 103, 104, 140, 159, 232, 236, 245, 252; 43, 68, 103, 104, 159, 232, 236, 245, 252; 43, 68, 103, 104, 159, 232, 236, 245; 12, 76, 103, 104, 130, 222, 245, 261; 76, 103, 104, 130, 222, 245; 68, 103, 104, 159, 232, 236, 245, 257; 68, 76, 103, 104, 159, 210, 232, 236, 245; 68, 103, 104, 159, 224, 232, 236, 245, 257; 76, 103, 10 104, 159, 232, 236, 245, 257; 68, 76, 103, 104, 159, 211, 232, 236, 245; 12, 68, 76, 103, 104, 159, 214, 232, 236, 245; 68, 76, 103, 104, 159, 215, 232, 236, 245; 12, 68, 76, 103, 104, 159, 232, 236, 245; 20, 68, 76, 103, 104, 159, 232, 236, 245, 259; 68, 76, 87, 103, 104, 159, 232, 236, 245, 260; 68, 76, 103, 104, 159, 232, 236, 245, 261; 12, 48, 68, 76, 103, 104, 159, 232, 236, 245; 76, 103, 104, 159, 15 192, 232, 236, 245; 76, 103, 104, 147, 159, 232, 236, 245, 248, 251; 12, 68, 76, 103, 104, 159, 232, 236, 245, 272; 68, 76, 103, 104, 159, 183, 206, 232, 236, 245; 68, 76, 103, 104, 159', 232, 236, 245, 256; 68, 76, 103, 104, 159, 206, 232, 236, 245; 27, 68, 76, 103, 104, 159, 232, 236, 245; 68, 103, 104, 159, 212, 232, 236, 245, 248, 252; 103, 104, 159, 232, 236, 245, 248, 252; 68, 103, 104, 159, 209, 20 232,
PL 191 529 B1
236, 245, 248, 252; 68, 103, 104, 109, 159, 232, 236, 245, 248, 252; 20, 68, 103, 104, 159, 232, 236, 245, 248, 252; 68, 103, 104, 159, 209, 232, 236, 245, 248, 252; 68, 103, 104, 159, 210, 232, 236, 245, 248, 252; 68, 103, 104, 159, 212, 232, 236, 245, 248, 252; 68, 103, 104, 159, 213, 232, 236, 245, 248, 252; 68, 103, 104, 213, 232, 236, 245, 248, 252; 68, 103, 104, 159, 215, 232, 236, 245, 248, 252; 68, 25 103, 104, 159, 216, 232, 236, 245, 248, 252; 20, 68, 103, 104, 159, 232, 236, 245, 248, 252; 68, 103, 104, 159, 232, 236, 245, 248, 252, 255; 68, 103, 104, 159, 232, 236, 245, 248, 252, 256; 68, 103, 104, 159, 232, 236, 245, 248, 252, 260; 68, 103, 104, 159, 228, 232, 236, 245, 248, 252; 68, 76, 89, 103, 104, 159, 210, 213, 232, 236, 245, 260; 68, 103, 104, 159, 218, 232, 236, 245, 248, 252.
Rysunki
PL 191 529 B1
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FIG..1B-2
PL 191 529 B1
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PORÓWNANIE SEKWENCJI SUBTILIZYN Z: B. amyloliquefaciens B. subtilis
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Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US95632397A | 1997-10-23 | 1997-10-23 | |
| US95632497A | 1997-10-23 | 1997-10-23 | |
| US95656497A | 1997-10-23 | 1997-10-23 | |
| PCT/US1998/022572 WO1999020770A2 (en) | 1997-10-23 | 1998-10-23 | Multiply-substituted protease variants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| PL340600A1 PL340600A1 (en) | 2001-02-12 |
| PL191529B1 true PL191529B1 (pl) | 2006-06-30 |
Family
ID=27420734
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PL340600A PL191529B1 (pl) | 1997-10-23 | 1998-10-23 | Nowa odmiana proteazy, cząsteczka DNA kodująca nową odmianę proteazy, wektor zawierający tę cząsteczkę oraz karma dla zwierząt i kompozycje zawierające nową odmianę proteazy |
Country Status (26)
| Country | Link |
|---|---|
| US (5) | US6482628B1 (pl) |
| EP (10) | EP1398367B1 (pl) |
| JP (7) | JP4346237B2 (pl) |
| KR (6) | KR100612981B1 (pl) |
| CN (9) | CN100558871C (pl) |
| AR (10) | AR015977A1 (pl) |
| AT (9) | ATE314478T1 (pl) |
| AU (7) | AU1276299A (pl) |
| BR (6) | BR9813260A (pl) |
| CA (9) | CA2307640C (pl) |
| CZ (6) | CZ300347B6 (pl) |
| DE (7) | DE69841226D1 (pl) |
| DK (9) | DK1624050T3 (pl) |
| EG (2) | EG22075A (pl) |
| ES (7) | ES2319401T3 (pl) |
| HK (1) | HK1039159B (pl) |
| HU (3) | HUP0104539A3 (pl) |
| ID (4) | ID25637A (pl) |
| MA (2) | MA24811A1 (pl) |
| NO (3) | NO327607B1 (pl) |
| NZ (3) | NZ519204A (pl) |
| PL (1) | PL191529B1 (pl) |
| PT (8) | PT1025239E (pl) |
| TR (8) | TR200002014T2 (pl) |
| TW (2) | TW585912B (pl) |
| WO (5) | WO1999020726A1 (pl) |
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1999
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2000
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2001
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2002
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2006
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