CS269539B1 - A method of preparing a chroaolytic substrate to determine the activity of the endo-beta-1,4-glucan glucanohydrolase enzyme - Google Patents
A method of preparing a chroaolytic substrate to determine the activity of the endo-beta-1,4-glucan glucanohydrolase enzyme Download PDFInfo
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Abstract
Riešenie sa týká přípravy chroaolytického substrátu na stanovenie celulázovtj C aktivity /endo-beta-1,4-glukan glukaňohydroláza, EC 3.2.1.4/ postu?oa, kedy vodorozpustná hydroxyetyIcelulóza sa najprv sletuje 2,2 *-bis/oxiranýlaetyl/ éteroa alebo 2-chlóroaetyloxiránoa v reakčnej zaesi zloženej popři hydroxyetylceluloze /1 haotnostný diet/ z 6 až 12,5 haotnostných dielov roztoku, připraveného z 5 až 9 haotnostných dielov metanolu alebo etanolu, jednoho až 5 haotnostných dielov destilovanej vody, 0,01 až 0,07 haotnostných dielov hydroxydu sodného alebo draselného a 0,03 až 0,08 haotnostného dielu sietovadla, při teplote 20 až 40 °C po dobu 2 až 8 hodin, na ío vymytý sletovaný gél sa ďalej kovalentne farbi v prostředí 9 až 15 haotnostných dielov destilovanej vody za pritoanosti 0,1 až 0,5 haotnostného dielu hydroxydu sodného alebo draselného a 0,08 až 0,12 haotnostného dielu farbiva, s výhodou dvojsodnej soli kyseliny 8-aaino-5-|-3 /sulfoetylsulfonyl/anilino,-6-antrachinonsulfonovej a ďalej sa přidá 0,3 až 0,7 haotnostných dielov tuhého chloridu alebo siřenu sodného a táto zaes sa nechá reagovat při teplote 15 až 30 °c po dobu 1 až 3 hodin a chroaolytická hydroxyetytcelulóza sa na to dokonale vyaýva od nezreagovaného farbiva a suší, Riešenie aá uplatnenie pri připraví štandardných subttrátov celulázy C vo forae tabletových aonotestov, s Oplatnenia v biotechnológiach pri přesných sériových analýzach aktivity celulázovej C zložky, pri šlachteni aikroorgani zoo v, přodukujúcieh celulázy C , při priemyselných kultlváciach producentov celuláz a při dávkováni celuláz do krmných zaesi, ako aj pri analýze enzýaového vybavenia aikroflory bachorových átaiv prežúvavcov.The solution concerns the preparation of a chlorolytic substrate for the determination of cellulase C activity /endo-beta-1,4-glucan glucanohydrolase, EC 3.2.1.4/ post?oa, when water-soluble hydroxyethyl cellulose is first fused with 2,2 *-bis/oxiranylethyl/ chloroa or 2-aroaethyloxiranoa in a reaction sequence composed of hydroxyethyl cellulose /1 by weight diet/ from 6 to 12.5 parts by weight of a solution prepared from 5 to 9 parts by weight of methanol or ethanol, one to 5 parts by weight of distilled water, 0.01 to 0.07 parts by weight of sodium or potassium hydroxide and 0.03 to 0.08 parts by weight of crosslinker, at a temperature of 20 to 40 °C for 2 to 8 hours, on the ío washed slated gel is continued covalently dye in an environment of 9 to 15 parts by weight of distilled water in the presence of 0.1 to 0.5 parts by weight of sodium or potassium hydroxide and 0.08 to 0.12 parts by weight of a dye, preferably the disodium salt of 8-aaino-5-|-3/sulfoethylsulfonyl/anilino,-6-anthraquinonesulfonic acid, and further add 0.3 to 0.7 parts of solid sodium chloride or sodium sulfate, and this reaction is allowed to react at a temperature of 15 to 30 °C for 1 to 3 hours, and chroaolytic hydroxyethyl cellulose reacts perfectly to it from unreacted dye and dries. of the cellulase C component, during breeding micro-organisms that produce cellulase C, during industrial cultivation of cellulase producers and when dosing cellulases into fodder plants, as well as in the analysis of the enzyme equipment of microflora of the rumen and ruminants.
Description
Vynález ríeši přípravu chromolytického substrátu vhodného na stanovenle celulázovej aktivity, bets-1,4-glukan glukanohydroláze.The invention solves the preparation of a chromolytic substrate suitable for the determination of cellulase activity, bets-1,4-glucan glucanohydrolase.
V sůiasnosti je známy celý rad netód stanovení· celulázovej Εχ aktivity lbets-1,4-glukan glukanohydroláze, EC 3.2.1.41 a celý rad spósobov vyjadřovaní* celulázovej C* aktivity. Tieto aetódy sů založené na stanoveni koneentrácie uvolněných redukujůcich cukrov Iglukózy, celoblózy, celotriózy a pod.I z nativnej celulózy alebo jej derivátov. Tieto metody sů však nešpeclf1cké, nakolko nezohladňujů interferencie beta-glukanáz, posoblacich exo-aechanizoom |H.J. Bailey, K.M.H. Nevalainen, Enzyme Microb. Technol. 3, 143-159, 19811. Vlskozimetrícké a nefelometrické aetódy sú podstatné špecifickejšie ale natolko zložité, že nevyhovujů k rutinným stanovenla* |T.M. Enari, P. Markkanen, Advances in Biochemical Engineering, /T.K. Chose, A. Fiechter, H. Blakesbrough, eds/, Springer Verlag, Berlin-New fork, 1977, Vol.5, 1-14 ; M. Nuaai, P.C. Fox, M.L. Ntku-Paavola, Z.H. Tenari, Anal. Biochea. 116, 133-136, 19811. Stanovenle celulázovej C* aktivity sa zjednodušilo zavedeni* chromolytických substrátov, využivajúcich fyzíkálnych interakci! niektorých chromofórov s celulózovým retazcom alebo substrátov, obsahujúcích kovalentne vlažené farbivo na retazec aaorfnej celulózy alebo jej analogu Ij. Zeaek, Z. Pavlicová, J. Kovář, L. Kuniak, Progress in Biotechnology, Vol. 5, Enzyme Technologies /A. Blažej, J. Zenek, eds./ Elsevier Sei. Publ., Amsterdam-New York-London-Tokyo, 1988, 469-478I. Uvedené chroaolytické substráty sú viak ne i tandardné, s ohladom na velkost povrchu fázového rozhrania, neoptiaálne modifikovaní, vzhledem ne typ zavedenej funkčnej skupiny Inešpecifickí interakcie s ionogínnymf karboxylovýai skupinaail a neoptímálnom zosietení chromolytického gélu.Currently, a number of methods are known for determining the cellulase E χ activity of beta-1,4-glucan glucanohydrolase, EC 3.2.1.41, and a number of methods for expressing the cellulase C* activity. These methods are based on determining the concentration of released reducing sugars Iglucose, cellolyse, cellotriose, etc. from native cellulose or its derivatives. However, these methods are non-specific, since they do not take into account the interference of beta-glucanases, exo-enzymes |HJ Bailey, KMH Nevalainen, Enzyme Microb. Technol. 3, 143-159, 19811. Viscometric and nephelometric methods are considerably more specific but so complex that they are not suitable for routine determinations. |TM Enari, P. Markkanen, Advances in Biochemical Engineering, /TK Chose, A. Fiechter, H. Blakesbrough, eds/, Springer Verlag, Berlin-New fork, 1977, Vol.5, 1-14 ; M. Nuaai, PC Fox, ML Ntku-Paavola, ZH Tenari, Anal. Biochea. 116, 133-136, 19811. The determination of cellulase C* activity has been simplified by the introduction of chromolytic substrates, which exploit the physical interactions of some chromophores with the cellulose chain or substrates containing a dye covalently bound to the chain of amorphous cellulose or its analogue. Zeaek, Z. Pavlicová, J. Kovář, L. Kuniak, Progress in Biotechnology, Vol. 5, Enzyme Technologies /A. Blažej, J. Zenek, eds./ Elsevier Sei. Publ., Amsterdam-New York-London-Tokyo, 1988, 469-478I. The mentioned chromolytic substrates are, however, not standard, with respect to the size of the phase interface surface, non-optimally modified, with respect to the type of functional group introduced, non-specific interactions with the ionic carboxyl group and non-optimal crosslinking of the chromolytic gel.
Uvedené nedostatky odstraňuje postup podlá vynálezu, ktorého podstata spočívá v tom, že v prvnom stupni sa jeden hmotnostný diel hydroxyetylcelulózy sletuje v 6 až 12,5 hmotnostných díeloeh roztoku vztiahnuté na hmotnost hydroxyetylcelulózy, připraveného z pletich ež deviatich hmotnostných dielov metanolu alebo etanolu, jednoho až piatich hmotnostných dielov destilovanej vody, 0,01 až 0,07 hmotnostního dielu hydroxidu sodného alebo draselního a 0,03 až 0,08 hmotnostního dielu sietovadla, s výhodou 2,2'-bis/oxiranýlaetyl/éteru alebo 2-chlóronetyloxiránu pri teplote 20 až 40 °C po dobu 2 až 8 hodin, na čo sa v druhou stupni získaný gél sietovanej hydroxyetylcelulózy neutralizuje a premyje vodou a v tretom stupni sa sletovaný gél hydroxyetylcelulózy suspenduje v 9 až 15 hmotnostných dieloch destilovanej vody na hmotnost východiskovej hydroxyetyleelulózy a přidá sa jeden až dva hmotnostní dlely vodního roztoku, obsahujůceho 0,1 až 0,5 hmotnostního dielu hydroxidu sodného alebo draselného a ďalej jeden hmotnostný diel vodného roztoku 0,08 až 0,12 hmotnostního dielu farbiva s výhodou dvojsodnej soli kyseliny 8-aaino-5-1-3/sulfoetylsulfonyl/ani lino I-6-antrachinonsulfónovej a ďalej sa přidá 0,3 až 0,7 hmotnostných dielov tuhího chloridu sodního alebo síranu sodního a táto zmes sa nechá reagovat pri teplote 15 až 30 °C po dobu 1 až 3 hodin a na to sa v itvrtom stupni reakčná zmes zneutrali zuje a chromolytická hydroxyetylcelulóza sa dokonale vymyje od nezreagovaniho Farbiva najprv destilovanou vodou a potom zriedeným až koncentrovaným etanolom alebo acetónom a vysuší volné na vzduchu alebo vákuovo pri 60 až 70 °C.The above-mentioned shortcomings are eliminated by the process according to the invention, the essence of which lies in the fact that in the first stage, one part by weight of hydroxyethyl cellulose is suspended in 6 to 12.5 parts by weight of a solution based on the weight of hydroxyethyl cellulose, prepared from not more than nine parts by weight of methanol or ethanol, one to five parts by weight of distilled water, 0.01 to 0.07 parts by weight of sodium or potassium hydroxide and 0.03 to 0.08 parts by weight of a crosslinker, preferably 2,2'-bis(oxiranylethyl)ether or 2-chloromethyloxirane at a temperature of 20 to 40 °C for 2 to 8 hours, after which in the second stage the gel of crosslinked hydroxyethyl cellulose obtained is neutralized and washed with water, and in the third stage the suspended gel of hydroxyethyl cellulose is suspended in 9 to 15 parts by weight of distilled water per weight of the starting material. of hydroxyethyl cellulose and one to two parts by weight of an aqueous solution containing 0.1 to 0.5 parts by weight of sodium or potassium hydroxide and then one part by weight of an aqueous solution of 0.08 to 0.12 parts by weight of a dye, preferably the disodium salt of 8-amino-5-1-3/sulfoethylsulfonyl/anilino I-6-anthraquinonesulfonic acid, and then 0.3 to 0.7 parts by weight of solid sodium chloride or sodium sulfate are added and this mixture is allowed to react at a temperature of 15 to 30 °C for 1 to 3 hours, and then in the fourth stage the reaction mixture is neutralized and the chromolytic hydroxyethyl cellulose is thoroughly washed from the unreacted dye, first with distilled water and then with diluted to concentrated ethanol or acetone and dried in the open air or in a vacuum at 60 to 70 °C.
Připravený gél chromolytickej sietovanej hydroxyetylcelulózy sa frakcionuje na sitách na 0,25 až 0,35 mesh. Obsah viazaniho farbiva na hydroxyetylcelulózovom gíli je 6 až 9 hmotnostných X. Napučací objem sietovaního chromolytickího gélu je 10 až 16 ml.g1.The prepared chromolytic crosslinked hydroxyethylcellulose gel is fractionated on 0.25 to 0.35 mesh sieves. The content of bound dye on the hydroxyethylcellulose gel is 6 to 9% by weight. The swelling volume of the crosslinked chromolytic gel is 10 to 16 ml.g 1 .
Výhodou uvedeného postupu je příprava substrátu ďalej aplikovatelného v enzýmovej analýze vo forme tabletového monotestu, ίο zabezpečuje ítandard izovatetnost a unifiko2 CS 269539 Bl vatetnost analytického postupu. Výhodou j· vysoké citlivost připraveného ehromolytlckého substrátu a ipecifieita pro ůčlnok celulázy Cx a rozsiahle pásmo linearity, ktoré uvedený substrát poskytuje v závislosti ne aktivitách použitých celuláz C*.The advantage of the above procedure is the preparation of a substrate that can be further applied in enzyme analysis in the form of a tablet monotest, which ensures the standardization and uniformity of the analytical procedure. The advantage is the high sensitivity of the prepared enzyme-based substrate and the specificity for the action of cellulase C x and the wide linearity range that the above substrate provides depending on the activities of the used cellulases C*.
Přiklad 1.Example 1.
Do sklenenej 2 litrové) nádoby opatrenej oleiadloo sa přidal etanol /0,5 kg/ a destilovaná voda /0,5 kg/ obeahujúe.e 0,8 g hydroxidu sodného a 2,4 g 2,2*-b1s/ox1ranyloe~ tyl/éteru a 80 g hydroxyetylcelulózy /rozpustnej vo vodě, poekytujůcej v svojom 2 X vodnom roztoku pri 25 °C vlskozltu 5000 mPa.s/ a reakcia sietovania sa uskutečnila pri teplote 20 °C po dobu 8 hodin. Sletovaný gél sa zneutralizoval kyselinou octovou a preoyl nadbytkom vody /2 x 1000 ml/ a suspendoval v. 720 al destilováno) vody a přidalo sa 80 g roztoku destilované) vody, obsahujůceho 8 g NaOH e Sele) 80 al roztoku obsahujůceho 6,4 g dvojsodné) soli kyseliny 8-ea1no-5-l-3/sulfoetylsulfonyl/an1linol-6-antrachinonsulfonove) a Sálej sa přidalo 24 g tuhého chloridu sodného. Kovalentné farbenie sletovaného gélu hydroxyetylcelulózy sa uskutečnilo pri teplote 15 °C po dobu 3 hodin. Potoa sa reakčná zaes zneutrallzovala a preaývala postupné 3 x 1000 al destilovenej vody na frite, potoa 3 x 1000 al etanolu /50 X/ a nakoniec sa suspendoval tento gél 3 x 500 al etanolu /96 X/. Vymytý gél neobsahujůci fyzikálně zachytené farblvo sa vysuiil na rotačnej vákuovej odparke pri 70 °C, aechanicky rozaelnil a vysiloval na site s 0,35 eesh. Obsah naviazaného farbiva bol 6 X. Napučací objea chromolytického celulózového gélu bol 16 ml.g“\In a 2 liter glass vessel equipped with an oil separator, ethanol (0.5 kg) and distilled water (0.5 kg) were added, while 0.8 g of sodium hydroxide and 2.4 g of 2,2-bis(oxalyl)ethyl ether and 80 g of hydroxyethyl cellulose (water-soluble, having a viscosity of 5000 mPa.s in its 2X aqueous solution at 25 °C) were added and the crosslinking reaction was carried out at 20 °C for 8 hours. The gel was neutralized with acetic acid and diluted with excess water (2 x 1000 ml) and suspended in. 720 l of distilled water and 80 g of a solution of distilled water containing 8 g of NaOH were added. Then 80 l of a solution containing 6.4 g of disodium salt of 8-amino-5-1-3-sulfoethylsulfonyl-an1linol-6-anthraquinonesulfonic acid were added and 24 g of solid sodium chloride were added. The covalent staining of the hydroxyethylcellulose gel was carried out at a temperature of 15 °C for 3 hours. Then the reaction mixture was neutralized again and washed successively with 3 x 1000 l of distilled water on a frit, then with 3 x 1000 l of ethanol (50 X) and finally this gel was suspended in 3 x 500 l of ethanol (96 X). The washed gel, free of physically bound dye, was dried on a rotary vacuum evaporator at 70°C, mechanically disintegrated and sieved through a 0.35 mesh sieve. The bound dye content was 6%. The swelling volume of the chromolytic cellulose gel was 16 ml.g".
Přiklad 2.Example 2.
Do sklenenej 2 Utrovej nádoby opatrenej miečadlom sa přidal etanol /0,9 kg/, destilovaná voda /0,1 kg/, obsahujůca 11,2 g Na OH a 12,8 g 2-chlórometyloxi ránu a nakoniec 160 g hydroxyetylcelulózy /viskozita 1 X vodného roztoku pri 25 °C 1500 mPa.s/ a reakčná zmes sa miešala pri teplote 20 °C po dobu 8 hodin. Sletovaný gél sa zneutralizoval kyselinou chlorovodíkovou a prenyl nadbytkom vody /2 x 1000 ml/ a suspendoval v 2,4 kg destilované) vody obsahujůcej 80 g KOH a Sálej 160 ml roztoku obsahujůceho 19,2 g dvojsodné) soli kyseliny 8-amino-5-1-3/sulfoetyl sulfonyl/ani Uno I-6-antraehinonsulfónovej, pričom sa přidávalo súčasne 112 g síranu sodného. Kovalentné farbenie sletovaného gélu hydroxyetylcelulózy sa uskutečnilo pri teplote 30 °C po dobu 1 hodiny. Potom sa reakčná zmes zneutrallzovala a premývala postupné 3 x 2000 ml destilované) vody na frite, potom 3 x 1000 ml etanolu /50 X/ a nakoniec sa tento gél suspendoval v 1000 ml acetonu, čo sa opakovalo 2 krát. Vymytý gél neobsahujůci fyzikálně zachytené farblvo sa vysušil pri izbovej teplote v digestoři, mechanicky rozmělnil a vysitoval na site s 0,35 mesh. Obsah naviazeného farbiva bol 9 X hmotnostných. Napučací objem sletovaného gélu chromolytickej hydroxyetylcelulózy bol 10 ml.g~\Ethanol (0.9 kg), distilled water (0.1 kg) containing 11.2 g NaOH and 12.8 g 2-chloromethyloxirane and finally 160 g hydroxyethylcellulose (viscosity of 1X aqueous solution at 25 °C 1500 mPa.s) were added to a 2-liter glass vessel equipped with a stirrer and the reaction mixture was stirred at 20 °C for 8 hours. The fused gel was neutralized with hydrochloric acid and prenyl with excess water (2 x 1000 ml) and suspended in 2.4 kg of distilled water containing 80 g of KOH and 160 ml of a solution containing 19.2 g of disodium salt of 8-amino-5-1-3-(sulfoethyl sulfonyl)ani Uno I-6-anthraquinonesulfonic acid, while 112 g of sodium sulfate was added simultaneously. Covalent staining of the fused hydroxyethyl cellulose gel was carried out at 30 ° C for 1 hour. Then the reaction mixture was neutralized and washed successively 3 x 2000 ml of distilled water on a frit, then 3 x 1000 ml of ethanol (50 X) and finally this gel was suspended in 1000 ml of acetone, which was repeated 2 times. The washed gel, free of physically bound dye, was dried at room temperature in a fume hood, mechanically ground, and sieved through a 0.35 mesh sieve. The bound dye content was 9% by weight. The swelling volume of the fused chromolytic hydroxyethyl cellulose gel was 10 ml.g~\
Přiklad 3.Example 3.
Postupuje sa tak, ako je uvedené v přiklade '1 s tým rozdielom, že namiesto etanolu sa použil metanol. Získaný gél chromolytickej hydroxyetylcelulózy obsahoval 7,2 X hmotnostného naviazaného farbiva, napučací objem gélu bol 14,8 ml.g”1.The procedure is as described in Example '1, except that methanol was used instead of ethanol. The obtained chromolytic hydroxyethyl cellulose gel contained 7.2% by weight of bound dye, the swelling volume of the gel was 14.8 ml.g' 1 .
Přiklad 4.Example 4.
Postupuje sa tak, ako )e uvedené v příklade 2, s tým rozdielom, že k sietovaniu sa použije hydroxyetylcelulóza, ktorej 1 X vodný roztok poskytuje viskozitu 3500 mPa.s pri 25 °C. Připravený sletovaný gél chromolyticke) hydroxyetylcelulózy obsahoval 6,5 X hmotnostných naviazaného farbiva a napučací objem vo vodě mal 10,5 ml.g~\The procedure is as described in Example 2, with the difference that hydroxyethylcellulose is used for crosslinking, a 1X aqueous solution of which provides a viscosity of 3500 mPa.s at 25 °C. The prepared fused gel of chromolytic hydroxyethylcellulose contained 6.5% by weight of bound dye and the swelling volume in water was 10.5 ml.g~\
Vynález má použitie v biotechnológiach pri přesných sériových analýzach aktivity celulázovej €χ zložky. pri ilachteni mikroorganizmov produkujůcich celulázy C*. pri pr1emyselných kultiváciach mikroorganizmov, produkujůcich celulázy C* a pri dávkováni celuláz do krmných zmesi, ako aj pri analýze enzýmového vybavenia mikroflóry báchorových štiav prežůvavcov.The invention has application in biotechnology in precise serial analyses of the activity of the cellulase € χ component. in the breeding of microorganisms producing cellulases C*. in industrial cultivations of microorganisms producing cellulases C* and in the dosing of cellulases into feed mixtures, as well as in the analysis of the enzyme equipment of the microflora of rumen juices of ruminants.
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| CS881655A CS269539B1 (en) | 1988-03-14 | 1988-03-14 | A method of preparing a chroaolytic substrate to determine the activity of the endo-beta-1,4-glucan glucanohydrolase enzyme |
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