HUE024985T2 - Oxidoreduktázok ketonvegyületek sztereoszelektív redukálására - Google Patents
Oxidoreduktázok ketonvegyületek sztereoszelektív redukálására Download PDFInfo
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- HUE024985T2 HUE024985T2 HUE11189708A HUE11189708A HUE024985T2 HU E024985 T2 HUE024985 T2 HU E024985T2 HU E11189708 A HUE11189708 A HU E11189708A HU E11189708 A HUE11189708 A HU E11189708A HU E024985 T2 HUE024985 T2 HU E024985T2
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- 102000004316 Oxidoreductases Human genes 0.000 title claims 2
- 108090000854 Oxidoreductases Proteins 0.000 title claims 2
- 125000000468 ketone group Chemical group 0.000 title description 2
- 238000011916 stereoselective reduction Methods 0.000 title 1
- 238000000034 method Methods 0.000 claims description 18
- 239000002253 acid Substances 0.000 claims description 9
- -1 cholesterol compound Chemical class 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 229910052736 halogen Inorganic materials 0.000 claims description 5
- 150000002367 halogens Chemical class 0.000 claims description 5
- 230000009467 reduction Effects 0.000 claims description 5
- 150000002576 ketones Chemical class 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 230000002255 enzymatic effect Effects 0.000 claims description 3
- 150000002440 hydroxy compounds Chemical class 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims description 3
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- 230000036528 appetite Effects 0.000 claims 1
- 235000019789 appetite Nutrition 0.000 claims 1
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Natural products C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims 1
- 235000012000 cholesterol Nutrition 0.000 claims 1
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- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 1
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical class CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 1
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- KUNSUQLRTQLHQQ-UHFFFAOYSA-N copper tin Chemical compound [Cu].[Sn] KUNSUQLRTQLHQQ-UHFFFAOYSA-N 0.000 description 1
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- 210000005069 ears Anatomy 0.000 description 1
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- 150000002148 esters Chemical class 0.000 description 1
- ILSHEBNQHBZRAD-UHFFFAOYSA-N ethanol propane Chemical compound CCC.CCO.CCO.CCO ILSHEBNQHBZRAD-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
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- 230000002068 genetic effect Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- ZIMKJLALTRLXJO-UHFFFAOYSA-N hioc Chemical compound C12=CC(O)=CC=C2NC=C1CCNC(=O)C1CCCNC1=O ZIMKJLALTRLXJO-UHFFFAOYSA-N 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
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- 229910052742 iron Inorganic materials 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 150000003017 phosphorus Chemical class 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 150000003233 pyrroles Chemical class 0.000 description 1
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- 238000011069 regeneration method Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000003333 secondary alcohols Chemical class 0.000 description 1
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 1
- NESLWCLHZZISNB-UHFFFAOYSA-M sodium phenolate Chemical compound [Na+].[O-]C1=CC=CC=C1 NESLWCLHZZISNB-UHFFFAOYSA-M 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
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- 238000001238 wet grinding Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
ΟΧΙΟΟΚΕΟυΚΤΛϊΟΚ KCTONVKCVeUETEK SírEKEOSaEÍJEKTlV RSWIKAOSAB*
Thi ptnem fevwtfo» τ*«Λ» 10 3 &* Ö* enaniioseiecttvo cmtymatse election oi n Kero compound to the χΟον.ρνν^Ρ ^ h"iSVX;' ^5νϊΓ :V k"5'! ;YM"P" U''J " ^ oxitoitsctase. Furthermore- ^v invcmi°° rdat*: 10 nw o.<idomd«ctasfe fc* ^ »' ί|κ' eoanr^electtvc ΐ^'ΙΚίϊ .,Μ»!·!. i^'·* r·'' v sctiv* bykov <oinp'.-^rviS^· are vuluncle eruroe;; vvhh broao apptieabtiiiy tor the .,ymhe.ns o: t^mmAc^ksm a-cyoltarai chemicals sad *W*ie ioiiit»!».» Bere. en „«re*** k"h™ “J *** ^ CM ** “ "**
\ V^xV lt,M.', " ' '' ,l‘U - 0',t"-.i-S pharmaceutical preparations.
The symmetric reduction ui'prochira! koto compound:; t, a sector of sucteo,elective catalyst,. onerem ttemfce» coo,mine, a powe*d competitive technology v« chemical catalysis. The chemical aaynkneh-tc hvdtogcnasiou momm the one of h#b toxic and <mviroon;cnkdty harmful heavy natal vumhu,. of Etetne ...nd dm, «-erp> onnrwe tracts χ^-ve, „ ^ U ,-'ο v auv-u w χ* oyou "h-o* ibuthvrnere. mev ^ methods am often clramcterixed by :ode tvsebons ami mskfkdektSKf* f '
' X V Κ\Λχχ">Χ 'Λ"“ NOT V "NX '"s" ' V '' \l tUC t. V NX m»«co«s biochemical pathway.;, both in the primly metahoiism and in the »«ώτ,· metabolism, m every o:vJmxm .;,,i ,uc xuuu, ον ip mho cm M-v m .«χοη-, . oúolvt xwhxxirocttta^ wU cxi*»«ducteu». \.νντ.\. .ν.>η on.'. \ «< ''-. ν-Ί' - v '" X b<s bo ο»' 'v' ! ,UM v! 'K w"" ''' '"' 'V' ];vdttov',oo;pounds wee notedly demonstrated In the puut on the beet, of model system,. wh«*in hetit
Sx xx Sx k N'X.NCS ' '< - " S^''f 5S!'f ' (' * K ^ SV >C biesatalpk: approach femet*m to be advantageous gsse:níl#y with reused to mila reckon condtpony isek cl x'Oxx ; nooix se ^ ^ ikU , 'to 'χχχχ .u oí e i *' ',. J........... ..x, -XV — xx·. s.-.......-- «**»*, pKKtou» «= twwte» * *«» “ «8^10 "· «l!,li,'“ M<TO"»' ** m of ,>,«e«si* "μ W p*a>te for ««Ϊ chemiaf »p.»y, finer ;spt|lfc Wl>r.* a*0 know fursv- i:· reqijirub kier-olbr. t'kc-mtk n h sx íven VOktik' to demon,ttak »it.M the oc o; »vi ned c\id-Mxduct<x,xo v .tpoxV-ns .ugxcx. ....... xvn. "S-rn-ix f ο Λ in thx des.-5'Cxi sx,tcni, the U'to Cx-tvtvund *o iv rviOxCd. ««ah nsud ' biV-s sohsfsfe ht-water: m thieovdsc fertds^^the brpmirpbdi» tb^ihervyh :: y f Γ, X c V X" ox . ' ' x V *-'x>„. x . x w X t X xx*n x X Ui' "in!' "* v' '' mdttoediPb Si fcm ]s in case, tkh'ieved by the enneurrent oxidation of secondary alcohols, tor which, Ik most cases, the inexpea,Tve water-misdhk 2-propanol s, n,eo. : x.nnpk* y'cui b' b .w. - s^ífv CxKioKdcxiav- and dcVevuv.;,- o: hrch ennu .ν^Λχχ . >^<·'^ * ............... ........ ...... ...,,-.,, ,·.;, .·..,· ..ο,,·.,-.,.-·.·.......... s >'- x.' '·." x>- 5,763-234 fínvymeMicsfe '1 vchnoi. 1:993 Nuvcanber; 35(Tfe-fr:!O-ffi and. frkfefe cepsuteus (DE = 933/6544), Cöffefev/ mótcAoe írom R!m«imoa:m eryibvpoL· (liECR) {ü.S. P;«. No. 5,523.223), Awrawfe? ./few; (Biosei, .Biolcchnoi. 93 i * 7! (Bfe};. ;>p. ! 721-4'Β9), (Appi Microbiol Bioiechnoi. 2003 September; 62(4):380-5. Epub 3003 Apr. 2b). ami BMöcécims wber(S Org, €hem, 2Ö03 lan. 24; 68(2):402-6.).
Rvspeeíns secosfenry ak-oae- ocnyopsgupgggg frpsp organisms ni the genas LacioPaciisus (Lastofeaetiks: betir (ITS. Pat. Acs, 5-200-335)- kttaotwodius breyfe (DE t0i)0924 ;A]):(Acta sfrystaifegr P Bsol Ctystsllogr. 2000 December; 56 ft :2: Hfeo-h), Lm<<éad:iys mimΨ 70130119234) or PMudomonox (U.S. Opt. No. 05:385J3J}iAppi Mkrohksi Bioisdmo!. 2002 August; 59(4-5):403-7. Epub 2002 Jan, 2b, J. Org. Cfesr. 1092, 5A 1532)
However, theknown Nűay are not. nearly suifkieni lor exploiting the estire market potential of stcrcose leaks tedoctkiOs of aetp co-npoonck, On tbe one hand,. this ears fee sxplafried fey the lset that the individual enzymes different properties tyife fsapeet to substrate spectrum, pit m well as iornperaittre: and; solvent slabtlittes,: widen otlen :: apple men t esc h other. Therefore, even relatively ssmbstr feoitnologsas snpyotss may exfeleis s oomptetely dttlemnt conversion behaviour with regard to particular salwtestc. CM site other hand ,, not neany ad of the enzymes described are ekated and ovet&spresstbl® fe n srrfltofent «xtent, wheels means that these enzymes arc not available: frw indostrim uss1. For exploiting the yvPthesic: potential of the enzymatic asymmetrse: hydrogenation as extensively as possible, it Is tkeretnze oeensferv to be in possess sop ;oi 4 portiutm or fefferen t tntlasirls Ily asdesslblé: ohfebmdnetases winch is as broad ,gg nossffele, which hKidbtednctases: at® siuthcrioere suitable ..far use In apaeousfergat-ie two-phase systema Wilts organic soKcaw. 'Bte sabred matter of the present : invention Is: now a sutnber of trove!, enastiesfeecive: 4- and S-spcettso exlderednctasss by psofr stability ip aptíboasíöi'gbnic two-phase systetvts as well as by good eyfecsHbility to Esensflekfe edit (> 599 units/g B- coll wet ibomass), ps well ss a process tor the esafrtioseleefryc enzymatic; fedltetkk of a koto cdetgooM to she corrsspetfefeg; chiral hydroxy cdmponttd.
The ofederebucíasás accowtmg to the invention are characterized is that they have an ammo scso-sequence which is identleat to the ammo acM sequence SB') 113 NO:6 by at least 70%. I he ixriypeptlde according to SB) O'.) No A can be obtained from bacteria of the etas:; Acimafesctena.,, ns psrtlcidsr from bacteria of the class Norboma, In partlcniaf Íróin öoriloaia rnbriperttsretst 13SM2 dye d). A pother safe|«pt snattcr of the invotstisM? ia a aaiiiele held aetpreoce SEC; ):D Ns> id-Avtnes codes lor the: porem tiasiog the arnino asdd segecirce SBQ: )D No: 4, 7¼ pollypopdii? is very suitable tor the reduction: ol a-osuipone <;i- y. fi-ix nn-ol a:xi, of the swo cscsolionters of 2-octnitol.. prdboibly oxidizes S-2-octaneh it u: arm very autao-c sy,.- =he reassetitn: <sf 4-hakxsceoxKxhate esicss !o B-d-hitld-A-bydrnxybutyj'ic acid esters-
The oxidoreductase 43579 Is a homoowr having a inoiecuiar weight ikdermined in the kOS-yd of 41 i ) ( Da fisc pH opbtsttm for the redaction tcectioir rastges irons o.) to ;o r0r said oxidorcsNctase, ::ansl die ph optlrmttri Br the ockiado·; reactsoss ranges front ?:-5: to >·:> t oe ysidomductase front Gosdossia oferlpatlncta DS.M2 43570 exhibits good tc=nperatow. pH and solvent statnnnes and shows only minor setivity losscv in the: »H rartA" t)f froth 4,5-..19 nod st tpinperaonos or op m ,,-. v,, wltlf Iseohotloo perityss :>f several hours.
Fttrltemam, fee Invention relates to fw&8 ^ c^“rfesá k ^ ^xlöösóduéfeScS having the hfeitto acid sebnenee tfe No 0 w. borpofegw«& u>v,tv.v,, v.„,,v„ <at, .-.v. ,ΝΜ ». .„«her 4"Λ ''“Γ'"Τ.Τ 'W^* umd «ο» mi* (rím «*» ι>ΜΛη:!«““ *» ««ο'*'»®11*· «f**1,1 "** *,Λ,*ν , . ' . .ν .. ,-¾ ... ^«ísadio:Hind to oxklofodaciases aoootdng **
Untomé the mH-ntk>o relates to anttocxndt »*h* , . v.. : : : ^, * ,T. > ...K...... *«tfhodÍOS: i$ pSXtmmá aoCOWsng H> SMi-S>^». ' <.K'V ο v n x T'C^'1*"'' ' , :: , . . „ ,. ,..,¾ woeetx oí she aTiíilxKises·. Ís«. arnunXi.te·· nwíhods by immmimhn t>! appropriate mammass *«> M.W,*- si;mbsMmiXíktml· w polygon**!.:; d'sdd , .; . . <., . ,, . i. «.., ,>rt«<fcx'!«d:a őse sittemet *f» prolim i.»«w»s«v ....... ........ . >, . ,...v . .„i,,.,......í-H "&&:. fcMS.L. InsoBattk <3.u, u»«Rs, *»v
Aid, a*. eo„ SUlS^RdP Hí *« ^ s;' 5)^Λ "- f ASTA«®swtram er-the* BtsAS i 'pro#*»». . . . ~ . . , .¾ .,...,.* jsí ihb Sd.ART i5lpön:í:nm ^easic .í,ocu> la «.kibis so. the opwnel ahgmnent ts etetenmneo by ..... v , . , <tx «,*« ^ «i“ *: "t:; ” >Ά!ν«β marb is »*d'w «««kik»* <<* ewiloiaiitg tilt sequel** '
At. ,-... 1 c :·.·..* * it··'*' -1-1- * * ·'*·*-·'·*- 1« ..stitvtq::* 1 '-'** -;-**;' > ... .. t .' i.· · ·' · ·-' '' -·: “:'i '' ' ''"' ....... . ..1 „· . . C , ** ' "1 S'® !!·' > ' S " ' ......... ............ .ÍV :«"*<«·. Ί·"-"" ’ * '" A f„.,« Stíl,.el uiiiatt t. 11*1 i»*«lii« .1 * i«ro»„l «*'>'>< *»>*«*»«-* ***» ««!*» * ««* .Ki* ^ MÍ.XW .SÍ·-' 111 Η» « N-rvrWh *.....* 1K- .««1 -A-* IMK.KAVVVP ÍS!« » >1> C«m*m m** <m of *· i™-»·««"“ ”****· IS, ,.· Λ ... SS, *1·' (SiM'Sl ·>.*Κ> lf> *>'s®i 1*1 St·!».»'5 i*1 *'i *0 , J„ \ Ui \»> '' Íin. r,u<SR«. w(SB! 111 No ΩΧ MTUWKiA(SW iD No«.>)*<»n«V0M.U«-<««> ® >* f tintomot, the ίη.«««> *>** » U.«h* vector coiisonsÍitü o« or sever,1 r.qcioc e« «isin, fe fc cetovl Rttaceises 3«»«lo.g to SBQ lO No 6. of lsqiWegii«|p»t !«<*»»«.I|K ««Wise, 1* , cloisio* vector «híd.. » Méiám to fc cqrteyl íehsictei*:, ítfixles *«»« »»»>« » rege,miit,, of NAÖ(Pi 'MW1 s*· *tl'8ie»i"’B*S ΟΓ **“* ^shyyiraiiss’íaiSe, /;:-iy. ..'::-:.i... ::y .. ;Faitóiemiös%; Öi« : 1:¾¾í«i ΐΐ^ e>i:pr&ssiO;f« vector locked s?· a b3a<sa«a «5^ p«^ u> otaaaadiao cd! cad c^mpmibg a oacidc add vhicb c-dsc: for tbc canx^-i *td«ctas« c.ccordiag to KKQ iD Nli .c <w ϊκ.,νοΚιίΧΚ^ thereof aad is isokeü so a* approptdu- ,¾ to sui cxpreeaioa ctmtro! ccqacaet:. H^Nu^ P«c ,nuo™> vdatoa a- . « ^ ^ ^ ^ ^ a'oo.otdi.*,. Cd! cod has been tturs.brswi c i'^cidxb v5^> «» ^M'^'ssda xecser e, -te!! k- , ixtxhartioa process for oNatnibg a carbonyl r^oeta» h^xi on do cnlUvasbo of *«oh a recomhlnan! hoM edl.
Suitable doibox xecsos^ are. for «ample. ppCR-Scnpt. pCMV-Scripk pBinesctipt {SbxDgexeK PPdve domng Vect^ (gus^n. Hsid«v Germany). pS B;ae. per 8Hse. ρΒΊ I iC-veesosx (Noe^n. Madison, USA) ond i Λ -FOR edndc c c* nx* i bn. .sc«^ n. da> b?dv. t ketsvav* b
X \''c\'esN' ws x si* V» s' .«.psc ρΚκ*"' ' s ' * :"s ' t pd'px\"^s'v V pgupl'i, I'lidR, ppts.. peers. i'HMi pRMP. p1r\! as. pAbicpedcx,pKIAt.;x pd\.
Stable exnresüson cor-troi ae«xioxces arc., for «>.ample itpAt* (t«cVpmmoter. trp-lac (trc)'pronsokr. Isc-pic-ni-Xcr, rb-proroosef or J^L'pnxrxrdr. ::;;c: the ovHk'tc'JUvi.xcN .xs'sxdi. e to tide IP \o o *-r χοχκ'Κ^οον shereot van V odmxsi «« sock x manner that the ahovehneatiosed recombinant b. cob veils are cultivated, the espresslon of -he respective oxldoreduetase is induced and snbseguettdy, after shoot ft) ft; Id hours (hr the ceils ary digested by sdtrasotbe -reata-em. by seel grinding with glass beads in a globe ntill putsch, GmbH, llaan Germany Ml min, i;4 Ms) or using a mgb-prcssure honsegenlaer, The cell extract obtained can either be used directly or purified further, For tlfts purpose, the celt extract is, e..g., eeotdfnged arid the sapernataxg obtained k stsbfeeteh to son exchange chromahwmphy, Ihr exempts, by loo exchange chromatography on QPepbarose FastFlow# (Fbannacla).
Furthermore, the invention relates to a process for the enantloselcotlve enaymadc redaction of a Kelo contpomtd to the eormspomisng chiral hydroxy compound, wherein the keto cotnpound is reduced: with an oxkmredaohiSe in she presence of a cofactor, characterised fit drat an ox idoredectase Is used which has ass amino add seqaeitee which is Identical to the ssnlno acid sequence SEQ ID NO:6 by at least 70% A further preferred embodiment of the process according to tbc inveotioo consists In that the keto eoropotind has the general ibrrnttla I. %·€{0)··Ι% (If wherein % stassds for ossa of the moieties I) --(0,-0;.;)-uikyh wherein alkyl is llnear-ehttln or branched, 2; {C: oF;,:>-alkenyL wherein alkenyl is bnear-cbsm or branched and optionally contains up to four double bonds, 3} ::-tC:yC';,:;:>-alkynyl, wbarcu; Gkynyl Is ilnear-ehais or branched and optionally cPhtsans tip to tear triple bonds. 4) -/C-Cp-wyl, 5) t(l :A;Deilkyf<C;>-C: igaryk 6} {Ov-O p-hetenx ycle which is onsdbshtated nr suhstihstsd one, two or three times by OH, halogen,..-NO·; and/or- Nib , or ?> (CrCigeydoalkyi; wherein the moieties mentioned strove under I) to ?) are ansabstituted or sabsiknSed one, two or three tortes.
Independently of each other, by -OH, habgsrg --%€h and/or-htrk, and K-> stands for one of-he moieties k) ---tCrC6}'-alkyk wherein alkyl is bacar-chato or branched, 9) -tCrCvbalkenyk wherein alkettyl Is liaesr-'Chain or branched and optionally contains: no to three double bonds, 10) tCv-C,.)'-..;lkynyi. therein alkynyl Is linear-cham m- branched and optionally etwitans two triple bonds, or II) --(CrCiipmlxyi--€(0>''0''{Cr€<,}>-alkyl, wherein alkyl Is linear or hmtehed and Is wosabstianed or substituted one, two or three tiroes by Old, halogen ---19(¾ and/or -Mix, svheretn she moieties tnantioned abewe trnder b) to 11) arc ansabstitated or substituted one, two or three times, ittdepesdeody of each other, by- Old, halogen, -19:0:5 and-'or - blf i.
Ferthemwre, the invention relates to a process for the eoantloseleetiye enzymatic reduction of a keto cootptarnd to the corresponding chiral hydroxy compound, wherein the keto compound Is reduced with an exidosedoetase in the pmsence of a cofactor, which process is characterized in that an oxidoredactase is tssed which UO is eaecoad by .nucleic acid seqncttce S.EQ ID No 14, or ν·,·ι·.;ί:ρ (b) ::: k; ?»**** by o nucleic acid &spBS«e the complementary strand of which hybridizes with the nucleic V \i!i ·>:«!«)' N >fl, % the terns i:bryr, aroma-2: carbon moieties comprismg ;> *i>: 54 c«rb;.n morns within the ring arc mtimrni, ^ m* m immm «.g., !*«*$&· &μιΛΜ. dfmdbylyl, .&§,' l-feiptrenylyb 3-bipleayiyt Iipd 4-fophtmylyt, .^ry| B^henyly! m>kamt. rnm-yi wife and ip pardepiar p®mi misíim m f^kmú mi few&sr Mf iU t&m-*hateg«r, an mrnmm® m %% %»«<4 íoáfe© i3: ^ & hyűmwftmi meim id mfoérsthód the carbon chain of which fe imnor-chain or bmmMimd wmpm«s li to 20 earhen atoms, for esarnplo, methyl ethyl, propyl, isopropyl, butyl, toristry :fo*5yl, persiyi:, hesyh: hepiyl. octyl, rtooonylor tfesnyl. By the term w.-Go-AyP, a covalent Ixmd is tmderspmd'.
By the itmi; cyclic hyéwtMb^ as cyefeigmgyl, eydohtttyk cyclopergyl syelotexytor cyclofesptyi are ttndersfood. the term "v''le-a-c;(;-sBitetereeyeie stands tor ;t nxmmtydfo ,ös· ineyclk S-merobcred to fo-sstospbered betsroeyeifo *fog which is parthtlty:orveornpietely sweated, hi, 0493 s arc examples of leforomdm Meletfes derived this pyrrole, fefofo foiPfftm pymttofo, :4oxtpoi«, thlsaote, isofoiaqdtey fetnwofc, ;,2,χ5ο^ο·ίί;3ίΙΡζο1ί··2···)Λθ^. tcsaifolorses, oxadisabifoios,: isoxamsfooes, ommfetmhdinediones. trisroks SuhstithtCil by h: --CM Cm Or 5-<»:<«" 1,2,41h5Jid pyridipS; jiVNipibíl pypmidipe, mdole, komdoto, todaxbfo.; tththshjyisPv ρΡίοί^ΡΡ, isopiuiPolipe, qtmtopslipe, •psioasohrie, tm-nthim:, phvbohpe «nd heoK-srnellsteO, cyciopenta-, cydohesa- nr cydohcpta-asHpiateii deriva-tvet. of said betctocycks are examples os the terms '-kCsyCBí^téimatyele^.; The moieties 2- # 3-twtyosyn. phehylgyoyly 1 sucf; as m or >phenYi-2--pYsroiyL· 2-foryl, 2-foicny), 4-hnidazolyi, meShyitn-idstsoly;. e.m t-mbfoye2o -No or -S-hsidatfolyl i,>'thia«>KByl. l-pyfidyi, >-pyridyl 4-pyndy!, 2-. 3· or 4myrkivl--N-oxsrici. 3-pyro.aissyL 2», 4- or S-pyrimmittyl 2-. 3 - öt foiedoiyi, sshstipped&$,. l -meiitylo fosnethyl·-, y.memnry-, N-bcstxyioxy-, .s-chioro- or 4,5~dhnetbyf-2- Indc-fyl. 1 -bcpi;yi-2-· or D-iinddyh, 4,:Svo<7”tetrahysfeo-2" lodolyj. eyetehepta:lhi|y5-pytrosyi, 2», a- t>r d-qaittoly;, K S^-or 44söe«í?toiyi, 1 -o;xo-l,2-dÍhyífo;-3~is<tqtiinöiyf, 0 -inó'oüóböyl. o.hcngoíPraKyi 2-he.8atoíhse^i 2-henaoxs2<;ty:| *» heogofhisígölyl or diht'dropyddiityk t's-í'-didtnyí, e $, 2- or 3 -fBosseiPyipyrriSlkiinyli, pipersrdrsyt, ritorphohttyp thbmorphoidyk iotrithydroPtiestyl g-s-tOrred csnopoouds oí Pxrada I are, for example, gspykdotdoroaeemaeeiste, o;e;hydec-oacesate, y i g -t-ilem-P-oxoOihanosc ac;d, dóyt-3•oxtwssterlsie. ddsydrstsy-2-hutanorie. sisyi-2-oxooaDriaie, ethyid-V?^ ::! ^„ylhntyriö: a«& sthvf ethyl iPsortyl ^yoxykde, 1 -ptetyi-B-propanoríC,: 2-cíist!rs>-f--f3- _ _ ^y0«jti:r:s:,d-oae. acetophehorie,: 2-Od:anonev 3<od:tineoe, 3-butaooae, 143,5- " '',Uí ^ ^pp^Bpiípovfsemaa^i -one, 2di-ltöXíiísedio!;c, f^áfebkTO^-kíteoae, seetexyaeefotte, phertacyf
Vx',!" ' , to the mventiom Ihe öxlíiorednehwcs ca-r be used either irt a cositpletely porlied v^-diy ourincd state or the po>eess can ha ppribrrped witit cella epoBttítiPg tbc osidorednaaset; °! !:i '' Jhv,.:5íson. in doing siti P5e cells used can be neod-ied io a osfove, hessriestblliaed:or lysed satut «ceo.n-fo ^ J ,íí( f^uc5a..cs acCoroiog to SEC) ID No δ, or honsologaes thereof are preferably used. ^ ^ ^ ^y c^idíareííoeiase are osed per ;k« of eoirt|xa:t!id 5sf fotírst-ía I to :be: eppvertetl NiO: upper limb). the ettsynte xsnií t ü correspottds to the ettayme amount svhich is repaired íor conversing i pmu) öfshs compound oí formula 1 per minute (min)·.
Fisé enzymatic redotdoo use;;'proceeds under ródd conditions sc· hsat die alcohols produced udi not feaes tardier Π.·::' {e-ncosse-s according to she iovcittior, exhibit 3 high residence rime and an enaodosoeiic purity of norms! fy roore than 95 % ot the einral alcohols produced and « isigh yield, relative to the amount or Feio compounds that is entpioyed. ;;·: the process according to toe Dhventton, the carbonyl compound is used is so amount of iron? s κ> SO %, based oo iseioisi volume, preferably i-om 5 % to· # %, to particulariron* Id |οϋ%· : fuslhesnoore, a preistred esnbodistteit:? of the invention is characterized so shea she NAD or NALtP tortnsd dorios- the reduction is cooonuousiy reduced to hi ADD or NAOPH, respectively, with s cosubaöate .in dotoa so, primary sod ^cecodasy afeohots suets as. ethanol, 2-propanol* 2-otnsnol, 2~pentanol* 2~ pentánok 4-soedwh2“pooisnoi, A-beptsool, S-octassol or cyeiohexsmol are preferably used as the cositbstrate.
Said eosubsttates ate reacted to directs encoding aldehydes or ketones ana NADH or NAPFfs, "••'Specdveh··, with the aid of an oxkk#«h*ctase anti NAD or NA0t\ respectively. fha results m & regeneration ;>t'>h,e NADU or NADPH. respectively. The pnsponion of she cosubssrate for the regeoenauon to .tots case ranges FA·· I p> PS: %|sv; vohhKth Msed óh the total vcltsde. ., ;:::e I or .¾. rcerPeiuticu ct die ,v»art* os addmumd akoliul dotty dre^nav.' can be ~ddvd Sunobk N ADtl· deoendent nkohol dehydrogenases are obtainable. for example, ltom bsker'a yeast, from Candida botdino. Candida narapstbsis or Plchia cnpsul&ta. furthermore, suitable NAOFH-depersdem alcohol dettyomgenases ate nreuen; in Lactobacillus brevis (DE 196 10 984 AD, hactohaciUus minor CDS 10i 19 274), íVadomonastü.S p*,' ^ giiys or in Thermoanaerobkim hiOC\H. Suitable costtosh’ates tor tttese alcouol oenyoiogeoases. nie the already mentioned scsxmdary alcohols such as ethanol propane! (hsopropanoli. 2-bmauoi, Z^mol :: 4-snethvt· 2-peotanoi. 2-ottanol or cyciohexattol cohusor regenerate can also be effected, for example, using NAD- or NADP-dcpeodent ,**’ ί^<·ΐ ί· “** “*» f*®1 - «,«»„ ,fSB,a,]®S NAD «ί NA» sptóflc Vem** «Ί*"***»· S“st* «·“%#“ to tM.-nr.le. Mis of to* mM «* « ?"«»«<-· >to»M '*
JlJg J’fte ,>«««, wmmttoftc ».vo»«k*»»r«K*!»ly cented«, »»««t»d, *eddideeel dehcletKocae. i.e, ®iatE-<.K-coe|>k-<l coeor.-t»' regincratios tsk»» gittee.
Se ececo.. »««« of «» «Λ» t.-.IMmc I» «Wo» Λ. ««.vomtie rejertloc fcoceedc p.eierehly e «·»ο««®»·· invHCI w******** IJEtffe. }»»iosa pH «1» of ft»w S to „ pH'„lee of tom * » 9. M «MU» >1* «« “'"f’*11 :<’,!S ^ «***» “ «·***« meeozapet-fktMM.tpfOfopoie.iOrBPiPosli.towos. „V,.,,. ,.,>1,ρΡκ**Β0Ρ»0«..90ί1ο«·'''''»'· 1 00 0 9 ^ ' -"1.Λ· Π.Κ., ,· . fteisevol If' t ·.'·“< 8-.-:--.90-0tot ,- I, «elwilmad of iteptotoci.eeiiog I» the p-vee™.,#* eaz,™t,c mmtiw* E :; „ tB-, Jé, d, JJ»C- of »-·! omooit «1« '*W. b »t« »«*>< »«> ««f Of «9 «ter «ly :» e , J'I c-seRi « «i-« -f. «r «»1*. - «-««*:“ . ar^-claa
Ji ,lk.„„ca«&« or » ™«a»*Wo ,tolat fh« if cintelertoefy r?«S«,.ihg X, J;t'íefi í S, J ,.S«,. n*.» 9., for .-.-ft-lr. ‘-9-A MM »«0 1 Uh,-. diisoprop\] etix-t diKn't csSter, tus^l .Ketnte, sscptene, hexane. Nocuno.. 2-nept.e)oh l-nH'O'sss-^'s'?'!''S:>'·' v" ,,.. v x v c ' j., \\ \c!' , 5 , ' ' s„' ' '<x c'x o svat ivi. v vO «etce \\,e r 5, v>' if w^-ef-insoiubk* solvents and eosubstrates, respect sve;y, are used the sxtacüun batch cons-sstu o: <«. nqueoas and an organic phase. i'he substrate Is dtstributoxi between the o qua me and aqueous phases aocv-; nn'n to sis suluhlky. The organic phase geaer&ily Eas-& pmpmtkm of ;l ks 9 d bit, preferably; frond *:^ baaed os the total reaction volume. The n»o l«P«d Passes are psefcrsssly mixed mechanically 5h3i 5 surface is produced between them. Also >u mss: «.isstbotitTncnl, the N.A.te or NA.DP, respectively, suo'-v ' '•'•Jvl.y the e’vun.ms reJuehon ont bn tuksred bank to N\Oi; or KVPPH. tvssvcttv.'K, using ,, lO^'w-av. *: depaskerf akwe,
'1 be concent nuion of the «factor NAi>H or NA DPH. respectively, m the aqueous obese yet-bra ;n ranges then O.t?01 n-M to t tnM, ;n particular front *1.01 tnM to O.t m.M f d!::.i:Π:if^:i^#·^síö^össííS;: aésaxirtissrrg;;to the invention,, a stabiliser of the ox idoreductaxe/dehydroip5 "**' can, ns addition, be weed. Msitnbk- siabdfeert. ere, for example, glycerol sorbiioi. !,4-Dl«dithiothreiwt {·ι>Πί or dimethyl sulfoxide (DMSO). she process according to the invention I» carried out, for esmnpic. in a closed reaction vessel mode os. yinss or metal. Far this purpose, tbc components are trtatsfenred individually Into the reaction vessel and stirred mám m 'mrnm^im öf, Tbc mwtkm feme fhoges from I bone to- ffp$i Pipire $o-24 $&#*.··......
Subsequently, the reaction rondure * processed. For Ihts purpose, the aqueous phase is separatee, me organic phase is filtered. The aqueous phase can optionally be extracted once «tore and cat) lx-, processed further tkes the orpstk: phase. Theteupsts, die solvent ss optson&Uy evaporated hum- the filteren orgetne prsea··
Kieshet more, tbc im centes; «felles to a process for obtaining chiral hydroxy compounds of S*mus& I!
Rhctom-Hd an wherein HIand R2.areas rkttnedabove, which ts dtaraetetieso to that a? a mixture containing tbs racemic compound of forsrtma is is incubated i.o toe patsence o.t onU>»..0".:. oxidoreduemes of Site invention according to S£0 U> Fio 6. or homologate thereof. NAD* **) tmd wafer, whcsvapoB as) enantiomer of the hydroxy compound of formula 11 is converted into the cotresponamg keío:coíópt)ís»d aáiFaÁÍ)(Pi:fl, askl ; : ; ο; b) the semainlng efs&alioiner of she hydroxy cotnpound ot lortneda tl ;s -solatcxt. ...... It· she csolxaiyt: mhjetiss<)s aeeotd-ing to SEt| tid No 6 arc uaexi, too eorresponthng «>«<»> N ~ cdrnpíhttsdsíare preferably öbfahsed. y the sx-actlot) conditktns me basically dte setae us it) she aboveunenfbnvd process for the enuntiospx'Oltc reduction of the keto compound of Ibrtnuia !. llosvevet, intend of an enantiosclective reduction of the Kefe· coo?pound of forntuia I he!), tbc s.'.Ovove stji'lutv ot th, t eoqx und ot Oi’nit ) U. ο Ή O; e es ) -Ji's \ · ·. t th, S W OV w 5Γ X Ski < f t ) f '* ' s V S ' V * S ' . S , .x ' t!,!lh U' t sp<' < ' 'y h „ m' s s the onpx'Sitc enanftotner of tbs hxdroxy cotnpoutsd of sorosok; 11 resrunsss tst-d can he isohaco. s atihesraote. instead of the alcohol;: used .,s cosubstraiet:, stseb as efissrsos, d-proparsoi írsopropttnol). d -ssufa.ool, ..•.-prudanoi °; d'-octanol, she c;strespos>vbng ketones thereof such us acetone arc used is; the: process for the regeneration ol she MAD. Pur example, she acetone and NAD(iAH are convmed into NAD and tsopropxnol by meuny of the oxidorcductxv) according to the mxentmn or an addnionat quhycsrpgenase.
Ore lerced embodiments «r the invention. are illustrated is fOrther detail by means &f the MhvMsg ess síple X.
Exunfpk /:
Ci-íüví:!;o>! ísfOrgimmn* andScrennmg finCarhamd.EeAiHuse Acdvm
The strain Oíírdotda nshripertiocia, DSM2I 43570 was cultivated on a medium of yeast extract (4} gladose (4). malt extract (10} and (sC(E (2)at 3?:> C and with 140 revolutions per minute trpmi
Subsequsmtly, 125 mg of cells wore resnaixmded with 8Ö0 pi of a digestion bollir (100 mM triethasekmios (TEA), pH-T-O)» mixed with i g -of glass beads and digested the 10 mkastes (min) at 4!> C is the globe mtli (Reiach), The supernatant (lysate) obtained aRer 2 min of emixiRtgalion· .at 12,000 ypn?: was. used in the Rdsowhig activity scrcexdxsg sod lor ddmoining the suaotknnsrfc· «sees» (ee-vaise), Different Ketones such as 2-1500:0080,. 2'öctsnene, etbHM-ehbroaceroaeetat®, acetophenone or ethyM-oxoH-phenyisntyrie acid wem used as substrates.
Batch tor activity screening:
MO ;.d 0.1 M K?f.P(h'K;dT): pH * 7,0 I mM MgCE 20 pi HA DEM or HA OH (; 0 mM) 20 pi lysate 1:00 pi substrate: (100 -oM.s 1 be reaction was pursued for I no:? at 340 om.
Bate!: tor the émműmtlm of the ee-vaioe: 20 μ i Hsam 100)0 ΜΑΒΗ or HAW! (SO utM)..................................................................................................................... 00 pi sobsirate (100 mM)
Alter 24 boars (h), the batches lor ee-detenoinaborr were extracted, e.g. with chloroform and the eoastkioterk: excess seas dHersnhtsd via gas chromatography ((IC), Ebe estasítiooteríc excess is eaienisted as fpHows: e«(5ó)'0(R-;ileöhoÍ- S-akeluH}/(RMc«ho! ί BmleohmíH löO.
Tabic I:
DEMZ stands tor Herasshe Sammiong inr hSikxxaxrgatdsmea and ZeOinihoxeo, Mascharoder Weg fh, 30)24 Bmsosehweig. Ddlnhioo of enayrne boils: 1 U; corresponds to the coxywe amount weleb Is feouhed tor cortvorthtg I stool stf substrate per mux /I.: ampin 2: keimim md Purdleidimifi'NADfidH^mandmiMIcmbsnliMdn^emmmes bs ostler to isolate the HlMXPfH-dependest tnkrohlal oxkleredítctases, the· sBlerosrrgaalsms were cultivated as describe!) under Example I, Upon reaching the stationary phase, -he ceils were: harvested and separated from the medium by eeatrifapBott, The enzyme release was efleeted by wet grinding usmg glass heads bot o;ay also be achieved bt odtet digestion method-.:. tor mss purpose. sor e.vampw. ;G> g m wet ceo ;;mx: were suspended wkh <H)o taí of u digestion buffer (UK.· trM ír;eih.inoíu;vníie; ? roM MgOk . pH==7.<A and V * Λ,\' Ovc S .1 > SV. the erode extract obtained after eerurdugation í 70<H^ rpm> was ínén puriheo tyt»he· via FPs-t νν~-> pmteht liquid chromatography).
Ab osidoreduciases according to the invention coo id ne punfteu by mfíeretu contohusuon.-: oi :Oe exchange chminaimgr&phy... e.e. on QSepharotie Fast Fiow (Pea· mac sas nr unó Q t hsur.-.d. h-nmich. Uvuneov;. hydrophobic interaction chmmntography. e.g. on OetyFbwpharoce t act r low or BtnvnSepharoxe east mow «Pharmacia). ceramic hydroxyiapatue thromaiugraphv and g*d permem ton.
Exsmpie i ' r Deroi-mbm/mo of the m-Terrnm &φο«ίΑ Ο, ,n r ' ot i \ « ,0 it x. m a «w SNM txv,v'exv.v. t -»o- *'
Fxtnople 2 were separated and tmotidorred onto a polyvmyhocnc ontuonue mentát asu U* VDx-membtam:;.
The conspmnoe, band was subject*! to N-terndnai sequenennp via bdtnan depredation (Frocke 4<G (P£- iTiqSpteihx)).
-cm:'''.:::::::1 &0$pi%, TvA
C)\ „ V ,-..UíC 0'\.'ί;Λϊ- ,V.O: f <U >.ϊ. ;Λ OC h.' W!> ' V». 'XX; .'-'..-'t '*'"**. -. 'X · CSO
Chromosomal ON Λ ia extracted accordtog kt the method described in “Molecular Ciotnne" by Mamtmik ,¾ Sambrouk The resulting ttuckdc acid serves as a twpiate for Ihe Polymerase chain reaction (PCR) with degenerate primers. Its domg so. 5’-primers are derived hmn the amino acid seaoen.ee (SEQ ID No. 6ft; 72: 80) «« n '> -peters arc dc"VM tk"" 'tv car "o x ,i ·χ·οη·'η»ν pi \* ¢0 V · h ^ st^oh up 'Pc n n coda ...... «swede: for she organism (SEQ ID Ho 68: 69: 74. 75; $2; 8s).
Amy' xcmex ..¾ eua-xd --ct m a 1VF t-ofer !'Α1 -fi-' ϊ!ΐ ! vFiJ ^ '* !fN *n'* ! ··> ·η.Λ« \'v V v ' i·. ·.. sc ocv \\ .n. d' pbosphatv cv.v x>.N ~ . a,, .v pNiOl vOvC p-.- .v- v,\. 2.S is Biolher-n Star Polysnerase (Getsec-atfs. l.-udingshttsistsi, Germany;) .Auer aenvahon of kte R'.ot itetsn St<n l\'Ktnerase (8 mis 9?° (.") and subseqttvot 45-50 cycles oi t; Í tutctn-s.-Otvst kc k, irse reaetjon x cornea o«\>u so a:> C. a«d Ihe catste FOR batc-'n is applied onto a 1 % agarose gei tor am»ty»ts, i'he epecirx ttagtneoi resakisc. front the poiynter&sC cnain reuettos ts ngatco into Use 1 A-ctonmg 'm\ k>; pc'FJ ; tlnvitrogcti. Karhrnhe. Germany) and sequenced with toe pruners ha: s rev t.StQ is) NO bn? ano M: * : usecSEO ID HfGIStywdhdbe aid ofan ABHfNA stoioenesr.; ;
The ,V- and .V-ter-nisai reghurn of ihe gene-coding se<|cfcnec are deiemu;v.a osutg n-c RAt. w utetntxi (ranict astpUhcation of eDNA ends). Ba.ed on ihe jtuvleic acid sequence m the specific fr^mcat G.$en.x..m;des t> 5 -RAGV a;v ''-RAO .m consnnued tout R\A p'-ryS'c. fton dx min- xm-m n a temofete tor the synthesis of the tust cDNA strarO using the 3'-RACb. systettt (Invitrogco. Karlsmhe, Gtrrmanv s This is foiknved b\ an amphficaoms und a ivaniphtice.tion of tne specific ctSNA wnh tne mu e-t > -; KAOi: (SKQ 0K mmh Rdbsedfpdy, it«: p Oh agarose get Τρ tor analv;ns. The speciftc irugmct.t cartytttg the rttkstng T'-ftattking sequence inforniadon is isolated, ttuated i;sio a TA-chonutg vector pCR2. i and sequenced. v. . v . i ' <U. it Ά V^v '· ‘ >· “ O. s kt s'v i'hiViiroaen). Fof this purpose. niRNA nmn; the total SNA oh^od previously is e-ndched with the aid ot Ohgo .rr..celiuk«:e (NF\B, ftcvetly< URA) and employed For she syndesis of the first cDNA strand wth she £δ5Κ“ ^ecjflc ofigonodeotides {5BO ID N'm ?0: ?*: ?v': 86; 8'Jf- The subsequent amplification and svamph wattos <}|··tlvs spéciik cDNA tvsalis ja s xragmeut which it, hgated into a pCRAs TA-cloning valor slnvitrcgei*) ^ , s - ' Kt , ... - - \ " - “ '!< ·'-d - > X»1 UN ' N"'s^ mtvdng sequence information aNn« toe > -end os me gene 1¾ ontteneu.
Based: on *b«: sequence coding ^ *« i«M»og*· gene, spéciik primers For subsequent elérttel teOsmd . DHA scerim? im rn app-te^R "^'«t am coostvued i'-Nfthn ontw. to: -'umpk * i^n$cm y ^,-0 %m^ém mm& far Ntte i -ör with a raeogififien éiíígssaee far %|J, or mé $<Φμ*Μ****λ\'* vxo ' '' x as S' *' * " ^ '' v" ' 'v in the sobsesssss?; FOR, ebraotesomal DMA Sams as titte test#!». lie 0fe see;#?'? coding hsrottc ..^eetlve otedomdxtstese is anjtfUSadl wAte fim ted m&mirnm i#: FP|«ieasse (tevhmgsrs}. Alter pufsRsatRts? - ef · v ·ν.:·Γ·ο--' «d fim resulting FA'R product is treated with appropriate DNA endonucleases and hg&teo mm loeihoPorflhs oliTaisi vector iNpv^h Msdfeop, ISA) or into fim hadteste of fim lP<P?e veetor <>mgen, Hűden, Ochmdv). mspecsivety. which backbone has been nested with fim ssme enaonocleases. -^'^ndas :hc espseasksn construct formed is taken into the eeptession stram Btmt Star grogén) or RB7<0 if.. coli genetic stock. Yale. USA), sxspcctívcíy. : :: A.CíÍÍ?P>/í:' 5.' . > . „ '\(ίΐ(Μ. ,>,. , M1.), - .-.
.-,. . \\ ^ „'Anvil), oe.hod dx >u?S.-fi r Moku a -,0-¾ wV ' . R garnbrooit. The retteg ttudek acid serves as a template tor the polymerase chain read ton tPCRi with , ; «> v.prisaets am derived from the amino add sequence (SFQ ID No 104: U~) degenerate primer*· I» - ! : , - :. T'-ortme-s ere derived From the asnmo acid sequence {SEO ID do 105: Hid. invoivmg fim genetm coon (S®S<3 306; ..... ">:VV is carried tapt, in a POi btitld fd7 mM ΎηΜΚί #11:0), JR mM (NFAASÖ,:; Π5 :mM ,;; . »*. -nvi 0 ? #4 desoxvitockoitidO: thtpbospbaie ssix (dbil Ps), 48 pMol ot cadt pnstmr turn k#CP. O.OI% TwCOO^„ ,.- , :...; , : ... „, .x..: 0. ....... -> ';'V Bv a ή Sásf BpFymet^ (OértsrcfalA ÜldmgabiDsdh dersaassy)1 After setsvaimh ofcfhe mor*®motw ,. :«« po i,«d snoscoaetfi 45-50 oyetes «As Toseti-ikiwn PCR; fiw msetiem is c«oie<i ddtvtt to :: polymerase # tfit;:i: v';' 'x/. "'" ' ...... Γ A ί% Ληϊ5Λ, pat biscit is: sppliod osm a \% agarose gei for analysis. trsamed restfififig « the polymerase chain reaciion is ligated into the TA-doning vow sr ..vk,-.ihc <km»anv>and seanenced wxth the primers Mil tc.v (SIFQ ID Kk> fids sod Ml* pCRil d itixvio-ogetfi .,..,,...,. ^gjwrtMN: aid ofanAHIDNA sequencer. tot' tbS'D tu Do >- .... „ , v .««{'.terminal regions os fi a gene Cod-ng ;.'>:..m kc .it. doietmmsM »w ng ot·' »nxc«ss ρι>οηκη.».. apf'Xt Based on the nneicie tti.vd setgtenee oF the .specific smemal fregmet-t. chain icaidiot; '*·--' '·" , , :; ., . .SÓD ID :¾ to# Ifit; 102; ;03; 10R; RN; l Ifi; FI Ϊ yllfiF i 1# Ufk i lv ore ooswiraemoy ;;::
. , s me&ss oFa reslmlion «mdmntcitKise and used in a txtitgatinrs so that tmmfic; DMA
Ueoomu: Γ.ΐΡΐΛ a:‘“•'v ' -· , . r . ... i'."h*aflois mjsttnre ss then sweti as a template .For an sPC.A ano ptinsesa-Sliv , sections can cttvui^'- «= . λ hí. sfít The POR sternal is mcreased by a sobsasque-nt nesteo K.R was? prunes »u TOO; ioi: H)g, i 1?}> · " , ,; ;:?; tg hc soc-.dfisti? specific fi'ttgmtms is Ussmcd niter dution front the t'te agarose
No 10): 10s, Plo ; : ^ ^ - ''':vI;:!::::. : :::' 0::0::)1 ..... , ,. ......-,CR0'i tiovisTogetst. •SO.: iOiO Pic VcCi<-> r ., , , . ., -ν,ιΚ!,,.,.., „maKm. v-t tre wetos pf R7 i -md'uorac ?Re h-iguvot - \ -κ «v i.xgsc.w
Hused on the sequence *i^«g *'«* *S»g& gene tSTO Hi No, 14}. specific prkeets ^ sub^uem ; ;: X;!i v,,i ^tj.-sa;d DK-A seeiion into anappsrogmls expression system are cornhasewd. In doing so.:5-^¾¾¾¾.,^ v N Λ vv0_ { V S..s ' V W vk . v H lb * MPg'fto' ~2 ' - "* Nn> 1 ' ' '* > n resswaivoly, Md ikpnsnes's *hh * #&#»*#**** tor H*«d Hi tSFQ No. ;20. 12\ L'X ex |^; 125; 12d; 127). : :; .pip ampiHksHos; of *4 h&b&k DMA fom gestorak: DNA, which fuH-kflgih DNA codes: ibr fe ·:=/ iM sahsessuesk restrict m& %*&» im- the expressitm vectorh performed m émáhf4 m B»Sk 2 τ;κ> essresxkm straits Bid: i dt;r powtoagest) or RB791 CH. cad generic stock. Vein, USA). i«sp&:tisx;^.%. ; psawbrmed - oh she espsvssuw v-nvnv óvnod :. ;2vomo/o 6:
Xvnrtvykw (rfXzcm&mM'Dkfyérogems&z'g^waM? m E. <v// : The Escherichia cots drsins Hi.21 Star (ksvktxsgesn Hlarismhe,· Germany.) aod HB7HI i,E, colt poods ; : rock W*' dA4 i resoechy*lv.t wideh had beets bxassfomsed1 wills the expressktts oopsmict, were cisRiyated hs 7 VV} 0<- LBondhatP (l*e fryptone. **asl 0Μπκί' N;* Π with sonp-ciido«&« ca^bomciliin < n, us mi\ respective!;,. tank an opmai den.,sit of 0 5 we.wared ,n vp- ,.,,, e,n* .wUwu'd, Ike esps^-kv v* LpipbDsnt protein was indaced W && of isopropyl fhK>g;rfa.Ptqgda| (tFHl at a concentration of Hi :; V x c Tv S' it.' 'Vs' >' N H'"''' 'i ''t's.KO 'S , " Ί" k sC's Os s . * s d .,,..0 ρ·0'..·Λ:« at -20'· C. for the activity was. ID too of ceils were mixes with 50O Ms ^ ^ TEA bailor pH r P and ?00 μ! of glass beads and wtv digested for 10 mm using a glebe ml* The iy.vse obtained was rasn esőd for the respeeiwe raeasswemoots ?« a dikhod state. Tfw activity test vww a;5 m $ ^ too mM TT:A buffer pH 7.0, Has p« HADtPiH. Η) μΙ diksiod oeil |ysMe the rcastu)!'. was sorted with the 5 a.pttots 0' ibO .d of a u«> mhi atiPskaie aolutwa so th«. v sosata oosts.oe.
Eiwk?# A f .· Chef ticwr&á&B °f Recombimftt Oxxiw-eáucíoses :v; i Ϊ 'iMiűÉmmt
Tbc bts ííerá Hstsd In Table 4 ο<;·κ·. produced, The eorweníratsosr of the respecti ve be Her cot«p<«d§ In each case aosoenSed to. .10 mbi Tabled:
Measuring bascls 00* 0>·ρΗ optimum reikictiosn :P(s μ I o f the bssifc syetessrs each merbiossed is Table 3 30 pi of NADfP)H 10 ssM 10 pi of a diluted enzy s-te incubation was performed for shoal 2 to 1 .mfo, subosgueraly 100 p! of a subssnste sblutiofr (100 hrM) were added,
Depmdmg tar the odtforednchrsc. E-bntanone or O-ocísíkíbc was used as ike xubslrase, The reset km was pursued for I .min at 340 one In order le determine ike pH-optfonan, Ike etseyníalse reaction k; the respective >a·Her listed m Table 4 was analysed, in order to detennlne the pil-optismms for Ike oxidation reaction, NAD; IE wets asted as the cefscter and d.-propastoi os' 2'oetahol was issed ;o. lise sphstmte.
Tbc result.* for the oxkforedectase* according lo the bsventlon are: compiled In Table 5. fable 5:
lÁimlStiélM
The detemiisiatioa of Ike atds vky or she reeombhrasti oxsdbredssefases seas es.anoued by storing ihens as the balder systems merstforsed 1st lakle 4. f or this purpose,· the dtfforeot bisders (SO nsM) were prepared la Ike muge of h ont pH 4 to 11, and the oxidorsdstesase prodaeed acoosxlksg to Exasngle 4 was diluted theresvpb, Aker 50. 00 gad 120 ruinates of Irseobatioa, 10 pi were taken Ifosn the batch ara! esed la the activity test according to Example I,
Ike Initial value is in this esse the nseasstted value which was obtained Immediately alter she dilution (1:30) of she enzyme In a pskassssun phosphate boiler 10 ®M p1.|~?J.. Under the given conditions, said vglnc corresponded to ssn extinction change of approx, OTIfonin and was set as s 100 Of value, and sll subsegiseat measured values were put, its .relation.to this value, hr 'fable 6, the psl ranges, with the enzymes ekbfoiifog no less fossr 50 if of the ksibal sseuvky wkb an inetskalion lasting for 120 ns is·, are complied for the oxlderedoctsses according so the mvenifoo.
Table $:
Is order to detetroiae the oplniona test lessrpex&tms; lit® enayme activity for the «xidereácstasss accitsding kt tbs invention ·«?»« measured in tbs standard measuring batch in a temperature mage of koto 15* C ks 70 ' C
The setnperatere ogtinas deiemnoed are complied so Table 7:
Table 7:
In se anstksgoov Sana ns? as beseribedl under Example 5c. the temperature stability was detemsissed for tbs rsage of from 15" C to TCP C. for this psrrpoae, a dilation of the oxidosbdoeiases aecosbwg to the invention was in each case incubated at the respective unnperaiurc for Oil ntks and 130 mm and was sunseaucully measured at 50" C with the above-aneitiioned test baieh. In Table 0. the tentperatnre ranges. with tee aoyyotes exhibiting no teas than 50 % of the inhsat acti vity with so inoxb&tksn lading ibr 130 min, a rc compiled í&r she oxMorsductases according to tbs invention.
Tabic 8:
The substrate spectrum of the oxidoredoetesec aecordhtg so the invenPoo was determined sty measuring the enoyroe activity tor redtretion ants oxkktkm with a norober of betonos and alcohols. hot this purpose, the standard nseaatnhsg baton according so Essantpie i teas tssed edfo diiferen; ssfosirales, 'fee activity wish tnethyi acetoaeesatc teas set to i 0d 33 for sil anaytoes and all the other -mbslyates wets; pot in reiatioo thereto·. fable 0-: Snbsteaie spectra redoction.........................................................................................................................
X. íVDΛί f .í «·ί;.; :l aAíOí: ítí ? ;Λ:. 1 >v:% ?;:/!áíí^.%:. ;V;;S r*: r?j 'The. stability of the novel oxidocedoctsises te. a<|o«ees/tagtshe iwo-phase systems was. exssriecd by dilating the lysates obtained m Example 6 (from a recombmant exptesstoa) ta an a<pmoos bolter suitable fat the respective oxldoredaetssc t spares. Ki units/iru bobén. Tfeen, the soon volume of an organic solves;! not misdate with water was added to the oxldoreductase diluted In the batter arid the hatch was teetrbaleh at room iedtdaralufs with constant thorough mixing (fherntcuniner at 170 spat). Alter 24 h cT ine«bation:, Hs al each mete taboo irons the aqueous, phase and oaed tor the detenttmanott of the enxyme activity in the stassdard test bsteh (potassinm phosphate bafler (KPP) ttKt utM, pH:: 7.0. 0,2 mM AAD(P}H, 10 tsM substrate). Also iu this case, the-háttal value immediately alter the dilution la the buffer was set to 100 %5 and si; further mines mere pot h; reladort thereto.
Table 9t Brtaytne aedvity alter 24 h of recohatioe in the apoeoos/orgastle twoodtasc system
Mi Bh MethylAendraiyf ether
Table 10: Substrate spectra oxidation
Claims (5)
- .................... igéayjMMtóek1, Eljárás keibnvegYület e^santiomers^efekiv eazímatite - redukálására a megtelő kíráiis hklroxivegyületté, ahol a keton vegyüleíet kofaktor jelenlétében oxidoreaukbuzai redtAéifok;: tiszidJeHémezm, hogy -t'-köv^tke^d oxMorediillázök allíítís «^értből ^íák^it @3odo reduMzt alkalmazómé: ft): SBQ IQ :^ö^í4n]öktefe^-mk¥^c<a által kódol oxM^reduMá^k vagy |b| olyan ouMeiíisav-sxekvencia Altai kódolt oxldoreduktá^ok, melynek komplementer sMla az (a) pontban megnevezett nukteinsswaJ erősen sztxmgefts körülmények között ykrlfeák f; % Az 1. kénypoot szerinti eljárás, azzal Jellemezve, hogy a keton vegyidet általános képlete a következő (1) képlet RrCCOl-Rs (!)> ahol R{ íelemese a kővetkező csoportok közül egy; .. i) eláe=«ő láncú, ; : 2} ^C.rCvo)-alkonü, amely egyenes vagy elágazó láncú és adott esetben legfeljebb négy kétfős kötést taRaltnnz,: ; : M I) -(Cs-CbohaiVinil am# egyenes vagy elágazó láncú és adott esetben legfeljebb négy hármas kötést tanatewg 4| A^A-őzbarú,; ; ö) ό(Α··€i-sbbeterocikitts, amely sztmszMuálatlaa vágy egyszeresen, kétszeresen vagy háromszorosan sznbsztitnált -OH-csoporttal, halogénad, -NOr csoporttal Ésfvagy «jHEHfc* csoporttsl, vagy ;; ; ' 7l '(Ci-Cvpeikloalkü, e érni mj}*?): pontban mm^VCZQlt csoportok szubsztituálatlanok vagy egymástól függetlenül egyszeresen* kétszeresen vagy háromszorosan szuhaxti múltak -OH-csoporttal, halogénnek - A Qr csoport tal étvágy -NHrCsoportla), és ll2 jelentése a következő csoportok közül egy 3) -CrökH&íl, amely egyenes vagy elágazó láncú, 9} -(Cb-CO-alkenil, amely egyenes vagy elágazó láncú ős adott esetben legfeljebb bárom kettős kötést tartalmaz..0) -CCrC^-alkmíl, amely egyenes vagy elágazó láncú és adott esetben két hármas kötést tartalmaz, vagy 1Π .{CrCjoVaikiKW-O-CCrCs^alkik amely egyenes vagy elágazd láncú és sz.ubszüluálatían vagy egyszeresen, kétszeresen vagy háromszorosan szahsxtituált -OH-csoporttal, halogénéi, -NOrcsoporttal énvagy -NH-r-esoporttal, ahol a 8.M I) pontom nmgimxereU esopo"to\ omdvynmiribnnok \ agy emrnumb függetlenül egysemen, kétszeresen vagy háromszorosan sxabsziituállak -OH-csopontaL halogénnek :: -é{ö>-esoportt;ú ésriogy Ι:1^η#ρρ11η1,
- 3. Klónozó vektor, amely SEQ ID N();í> szekvencia szerinti oxidoredoktáxt kódoló egy vagy :Úsbb nukleináav-székyéstetát tartalmaz., s
- 4. Expressztös vektor, amely bakteriális, rovar*, növényi vagy emlőssejtben in vitro található és SEQ ÍD NOkl szekvencia szerinti oaidorednktázt kódoló és expressziés szabályozd szekvenciához kötött nulrieinsav-azelcvenciai tartalmaz.,
- 5. Rekombi.náns gazdasejt, amely bakteriális, rovar*, növényi vagy emlőssejt, és 4. igénypont szerinti exnressziés vektorral transzformált vagy iransztektatt.
- 6. Oxidoreduktáz. I. vagy 2. igénypont sxe.ri.nd eljárásban történő at kai mazasra, őzen/ joUemezvo,. hogv a. következők alkotta csoportból választott nuxicmsav-a/.ek véneta kódoga: NtQ *D"N'(.V"a-xrekv-orn éa ímklemsav-szekvencia melynek koinnmniente- szaki SeQ fD V * lo a, i i'Unc; o é oto-i n s.",rmgoo\ kdnilmenyck kozott rakodna: 1- kijárás ketorivegyület erumnomerszeIcktsv enzimahkns redukmasara a megtelem kárába hidroxivegyületté, ahol a kotos oegyelőtől lm,',Mm n'edeh uvídmednki m redukáljuk, «zen/ /e//m?m:r;o, hogy az. alkalmazott oxidoreditktáz: aromosav-szekvenedha a SEQ !D Nőid szerinti aminosav*szekvendával legalább 70%-ban meg^y****··
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| CN101784669B (zh) | 2007-08-24 | 2015-02-18 | 科德克希思公司 | 用于(r)-3-羟基四氢噻吩的立体选择性制备的改善的酮还原酶多肽 |
| KR101586503B1 (ko) | 2007-09-13 | 2016-01-18 | 코덱시스, 인코포레이티드 | 아세토페논의 환원을 위한 케토리덕타제 폴리펩티드 |
| TWI601825B (zh) | 2007-09-27 | 2017-10-11 | Ｉｅｐ有限公司 | 對映異構選擇性酶催化還原中間產物之方法 |
| WO2009042984A1 (en) | 2007-09-28 | 2009-04-02 | Codexis, Inc. | Ketoreductase polypeptides and uses thereof |
| WO2009046153A1 (en) | 2007-10-01 | 2009-04-09 | Codexis, Inc. | Ketoreductase polypeptides for the production of azetidinone |
| US8288141B2 (en) * | 2008-08-27 | 2012-10-16 | Codexis, Inc. | Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine |
| HUE026181T2 (hu) | 2008-08-27 | 2016-05-30 | Codexis Inc | Ketoreduktáz polipeptid 3-aril-3-hidroxipropánamin elõállítására 3-aril-3-ketopropánaminból |
| CN102186972B (zh) | 2008-08-29 | 2014-08-20 | 科德克希思公司 | 用于立体选择性生产(4s)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1，3-噁唑烷-2-酮的酮还原酶多肽 |
| EP2226386A1 (de) * | 2009-03-05 | 2010-09-08 | IEP GmbH | Verfahren zur stereoselektiven enzymatischen Reduktion von Ketoverbindungen |
| WO2010150946A1 (en) | 2009-06-22 | 2010-12-29 | Sk Holdings Co., Ltd. | Method for preparation of carbamic acid (r)-1-aryl-2-tetrazolyl-ethyl ester |
| SG178456A1 (en) | 2009-08-19 | 2012-04-27 | Codexis Inc | Ketoreductase polypeptides for the preparation of phenylephrine |
| US8404461B2 (en) | 2009-10-15 | 2013-03-26 | SK Biopharmaceutical Co. Ltd. | Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester |
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| WO2011140219A1 (en) | 2010-05-04 | 2011-11-10 | Codexis, Inc. | Biocatalysts for ezetimibe synthesis |
| BR112014031474A2 (pt) | 2012-06-18 | 2017-08-01 | Laboratorio Chimico Int S P A | processo para preparar um composto, uso de uma oxidoredutase, uso de uma oxidoredutase e composto |
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