JP5911825B2 - セリンプロテアーゼ変異体を含む組成物及び方法 - Google Patents
セリンプロテアーゼ変異体を含む組成物及び方法 Download PDFInfo
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- JP5911825B2 JP5911825B2 JP2013120408A JP2013120408A JP5911825B2 JP 5911825 B2 JP5911825 B2 JP 5911825B2 JP 2013120408 A JP2013120408 A JP 2013120408A JP 2013120408 A JP2013120408 A JP 2013120408A JP 5911825 B2 JP5911825 B2 JP 5911825B2
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
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- C11D3/38—Products with no well-defined composition, e.g. natural products
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- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
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- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
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- C12N15/09—Recombinant DNA-technology
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
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- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21062—Subtilisin (3.4.21.62)
Description
質変異体は、少なくとも1の洗浄組成物の成分である。いくつかの好ましい実施形態では、粉末又は液体洗剤として処方され、pHが約5から約12.0の洗剤組成物を用いて洗浄性能を試験する。
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X181D, X181E, X181G, X181H, X181L, X181M, X181N, X182A, X182D, X182E, X182F, X182G, X182H, X182I, X182K, X182L, X182M, X182N, X182P, X182Q, X182R, X182S, X182T, X182V, X182W, X182Y, X183A, X183D, X183F, X183G, X183H, X183I, X183K, X183L, X183M, X183N, X183P, X183Q, X183R, X183S, X183T, X183V, X183W, X183Y, X184A, X184C, X184D, X184E, X184F, X184G, X184H, X184L, X184M, X184N, X184S, X184T, X184W, X184Y, X185A, X185C, X185E, X185F, X185G, X185H, X185I, X185K, X185L, X185M, X185N, X185Q, X185R, X185S, X185T, X185V, X185Y, X186A, X186C, X186G, X186H, X186I, X186K, X186L, X186M, X186N, X186P, X186Q, X186R, X186S, X186T, X186W, X187A, X187C, X187D, X187E, X187F, X187G, X187H, X187I, X187L, X187M, X187N, X187P, X187Q, X187S, X187T, X187V, X187W, X187Y, X188A, X188D, X188E, X188F, X188G, X188H, X188I, X188K, X188L, X188P, X188Q, X188R, X188S, X188T, X188V, X188W, X188Y, X189A, X189C, X189E, X189F, X189G, X189H, X189K, X189L, X189M, X189N, X189P, X189Q, X189R, X189S, X189T, X189V, X189Y, X190A, X190D, X190E, X190F, X190G, X190H, X190I, X190K, X190L, X190M, X190N, X190P, X190Q, X190R, X190S, X190V, X190W, X190Y, X191A, X191D, X191E, X191F, X191H, X191I, X191K, X191L, X191P, X191Q, X191R, X191S, X191T, X191V, X191W, X191Y, X192C, X192D, X192E, X192G, X192H, X192I, X192K, X192L, X192M, X192N, X192P, X192Q, X192R, X192S, X192T, X192V, X192W, X192Y, X193A, X193D, X193E, X193F, X193G, X193H, X193I, X193K, X193L, X193R, X193S, X193T, X193V, X193W, X193Y, X194A, X194C, X194D, X194E, X194F, X194G, X194H, X194I, X194L, X194M, X194P, X194Q, X194R, X194S, X194T, X194V, X194W, X194Y, X195A, X195C, X195D, X195E, X195F, X195G, X195I, X195K, X195L, X195Q, X195R, X195S, X195T, X195V, X195W, X195Y, X196A, X196D, X196E, X196F, X196I, X196L, X196M, X196P, X196Q, X196T, X196V, X196Y, X197A, X197C, X197D, X197E, X197F, X197G, X197H, X197I, X197L, X197M, X197N, X197Q, X197R, X197S, X197T, X197V, X197W, X197Y, X198A, X198D, X198E, X198F, X198G, X198H, X198I, X198L, X198M, X198N, X198Q, X198R, X198S, X198T, X198W, X198Y, X199A, X199C, X199D, X199E, X199F, X199G, X199H, X199I, X199L, X199M, X199Q, X199S, X199T, X199V, X199W, X200A, X200C, X200G, X200H, X200I, X200S, X201C, X201G, X201P, X201S, X201T, X201V, X202F, X202G, X203A, X203C, X203E, X203F, X203G, X203H, X203I, X203K, X203L, X203N, X203R, X203S, X203T, X203V, X203W, X203Y, X204A, X204C, X204E, X204F, X204G, X204I, X204K, X204L, X204N, X204P, X204R, X204S, X204T, X204W, X204Y, X205A, X205F, X205G, X205I, X205L, X205M, X205Q, X205T, X205V, X206A, X206C, X206D, X206E, X206F, X206G, X206H, X206I, X206K, X206L, X206N, X206P, X206Q, X206R, X206S, X206T, X206V, X206W, X206Y, X207A, X207G, X207H, X207S, X208A, X208C, X208L, X208N, X208P, X208S, X208T, X208V, X209A, X209C, X209D, X209E, X209F, X209G, X209H, X209I, X209K, X209L, X209M, X209N, X209R, X209S, X209T, X209V, X209W, X209Y, X210A, X210C, X210D, X210E, X210F, X210G, X210H, X210I, X210L, X210M, X210N, X210P, X210Q, X210R, X210S, X210V, X210W, X210Y, X211A, X211C, X211E, X211F, X211G, X211H, X211I, X211L, X211M, X211P, X211Q, X211R, X211T, X211V, X211W, X211Y, X212C, X212F, X212G, X212H, X212I, X212M, X212N, X212P, X212R, X212S, X212T, X212V, X212Y, X213A, X213C, X213D, X213E, X213F, X213G, X213I, X213K, X213L, X213M, X213N, X213P, X213Q, X213R, X213S, X213T, X213V, X213W, X213Y, X214A, X214C, X214E, X214F, X214G, X214H, X214I, X214K, X214L, X214M, X214N, X214P, X214Q, X214R, X214S, X214T, X214V, X214W, X214Y, X215A, X215C, X215D, X215E, X215F, X215G, X215H, X215I, X215K, X215M, X215N, X215P, X215R, X215S, X215T, X215V, X215W, X215Y, X216A, X216C, X216D, X216E, X216F, X216G, X216H, X216I, X216K, X216L, X216M, X216N, X216P, X216Q, X216R, X216S, X216V, X216W, X216Y, X217A, X217C, X217D, X217E, X217F, X217G, X217I, X217K, X217L, X217M, X217N, X217Q, X217S, X217T, X217V, X217Y, X218C, X218D, X218E, X218F, X218G, X218H, X218I, X218L, X218M, X218N, X218P, X218Q, X218R, X218S, X218T, X218V, X218W, X218Y, X219A, X219G, X219S, X220A, X220C, X220G, X220H, X220N, X220S, X220T, X220V, X221G, X221S, X221T, X222A, X222C, X222E, X222F, X222G, X222I, X222K, X222L, X222M, X222N, X222P, X222Q, X222R, X222S, X222T, X222V, X222W, X223A, X223C, X223G, X223M, X223S, X223T, X224A, X224D, X224E, X224G, X224I, X2
24L, X224M, X224N, X224P, X224S, X224T, X225A, X225C, X225G, X225I, X225N, X225P, X225S, X225T, X225V, X226C, X226F, X226G, X226H, X226I, X226L, X226M, X226N, X226R, X226S, X226T, X226V, X226Y, X227A, X227C, X227F, X227G, X227I, X227L, X227M, X227P, X227Q, X227S, X227T, X227V, X227Y, X228A, X228C, X228G, X228I, X228L, X228M, X228P, X228S, X228V, X229A, X229G, X229P, X229S, X230A, X230D, X230E, X230F, X230G, X230H, X230I, X230L, X230N, X230P, X230Q, X230S, X230T, X230V, X230W, X230Y, X231A, X231C, X231F, X231G, X231H, X231I, X231L, X231S, X231T, X231W, X231Y, X232A, X232G, X232H, X232K, X232L, X232M, X232P, X232S, X232V, X233A, X233C, X233E, X233F, X233G, X233I, X233L, X233M, X233N, X233P, X233Q, X233R, X233S, X233T, X233V, X233Y, X234D, X234F, X234G, X234H, X234L, X234M, X234N, X234P, X234Q, X234S, X234T, X234V, X234Y, X235C, X235D, X235E, X235F, X235G, X235H, X235I, X235K, X235L, X235M, X235N, X235Q, X235R, X235S, X235V, X235W, X235Y, X236A, X236C, X236E, X236F, X236G, X236H, X236K, X236L, X236N, X236P, X236Q, X236R, X236S, X236T, X236V, X236W, X236Y, X237A, X237C, X237F, X237G, X237H, X237I, X237K, X237L, X237M, X237P, X237Q, X237R, X237S, X237T, X237V, X237W, X237Y, X238C, X238D, X238E, X238F, X238G, X238H, X238I, X238K, X238L, X238M, X238N, X238Q, X238R, X238S, X238T, X238V, X238Y, X239C, X239D, X239F, X239G, X239H, X239I, X239K, X239L, X239M, X239N, X239P, X239Q, X239R, X239S, X239T, X239V, X239W, X239Y, X240A, X240C, X240E, X240F, X240I, X240K, X240L, X240M, X240N, X240Q, X240R, X240S, X240T, X240W, X240Y, X241A, X241C, X241D, X241E, X241F, X241G, X241H, X241I, X241K, X241L, X241M, X241N, X241P, X241Q, X241R, X241S, X241T, X241V, X241W, X241Y, X242A, X242C, X242D, X242F, X242G, X242H, X242I, X242L, X242M, X242P, X242Q, X242R, X242S, X242T, X242V, X242W, X243C, X243D, X243E, X243F, X243G, X243H, X243I, X243K, X243L, X243M, X243N, X243P, X243Q, X243R, X243S, X243T, X243V, X243W, X244A, X244D, X244E, X244F, X244H, X244I, X244L, X244M, X244N, X244P, X244Q, X244R, X244S, X244T, X244V, X244W, X244Y, X245A, X245C, X245E, X245G, X245H, X245K, X245L, X245P, X245Q, X245R, X245S, X245V, X245W, X245Y, X246A, X246C, X246E, X246F, X246G, X246H, X246I, X246L, X246M, X246N, X246P, X246Q, X246R, X246S, X246T, X246V, X246W, X246Y, X247A, X247C, X247D, X247E, X247F, X247G, X247H, X247I, X247K, X247L, X247M, X247N, X247P, X247Q, X247R, X247S, X247T, X247V, X247W, X247Y, X248C, X248D, X248E, X248G, X248H, X248I, X248K, X248L, X248M, X248N, X248P, X248R, X248S, X248T, X248V, X248W, X248Y, X249A, X249D, X249E, X249F, X249G, X249H, X249I, X249K, X249L, X249M, X249N, X249Q, X249R, X249S, X249T, X249V, X249W, X249Y, X250A, X250C, X250E, X250F, X250G, X250H, X250I, X250L, X250M, X250N, X250Q, X250S, X250V, X250Y, X251A, X251D, X251E, X251F, X251G, X251K, X251L, X251M, X251P, X251Q, X251R, X251S, X251T, X251V, X251Y, X252A, X252C, X252D, X252F, X252G, X252H, X252I, X252K, X252L, X252M, X252N, X252P, X252R, X252S, X252V, X252W, X252Y, X253A, X253D, X253E, X253F, X253G, X253H, X253I, X253K, X253M, X253R, X253S, X253T, X253V, X253W, X254A, X254C, X254D, X254E, X254G, X254N, X254P, X254S, X254T, X254V, X255A, X255C, X255D, X255E, X255F, X255H, X255I, X255L, X255N, X255P, X255Q, X255R, X255S, X255T, X255V, X255W, X255Y, X256A, X256C, X256D, X256E, X256G, X256H, X256I, X256K, X256L, X256M, X256N, X256P, X256R, X256S, X256T, X256V, X256W, X256Y, X257A, X257C, X257E, X257F, X257G, X257H, X257I, X257K, X257L, X257M, X257P, X257S, X257T, X257V, X257W, X257Y, X258A, X258C, X258D, X258E, X258F, X258G, X258H, X258I, X258L, X258M, X258P, X258Q, X258R, X258S, X258T, X258V, X258W, X258Y, X259A, X259C, X259E, X259G, X259I, X259L, X259M, X259P, X259Q, X259R, X259S, X259T, X259V, X260A, X260D, X260E, X260F, X260H, X260I, X260L, X260M, X260N, X260P, X260R, X260S, X260T, X260V, X260Y, X261A, X261C, X261E, X261F, X261G, X261I, X261K, X261L, X261N, X261P, X261Q, X261R, X261S, X261T, X261V, X261W, X261Y, X262A, X262C, X262D, X262F, X262G, X262H, X262I, X262K, X262L, X262M, X262P, X262Q, X262R, X262S, X262T, X262V, X262W, X262Y, X263A, X263C, X263D, X263F, X263G, X263H, X263I, X263K, X263L, X263M, X263N, X263P, X263Q, X263R, X263W, X263Y, X264A, X264C, X264E, X264F, X264G, X264H, X264I, X264L, X264P, X264Q, X264S, X264T, X264V, X264Y, X265A, X265C, X265D, X265F, X265G, X265H, X265I, X265K, X265L, X265M, X265N, X265P, X265Q, X265R, X265S, X265T, X265V, X265W, X265Y, X266G, X266W, X266Y, X267A, X267C, X267D, X267E, X267F, X267G, X267H, X267I, X267K, X267L, X267M, X267N, X267S, X267T, X267V, X267Y, X268A, X268D, X268E, X268G, X268H, X268K, X268L, X268M, X268N, X268P, X268Q, X268R, X268S, X268V, X268W, X268Y, X269C, X269D, X269F, X269G, X269H, X269I, X269L, X269M, X269N, X269Q, X269R, X269S, X269T, X269V, X270A, X270C, X270D, X270E, X270F, X270G, X270H, X270I, X270L, X270M, X270N, X270P, X270Q, X270S, X270T, X270V, X271A, X271C, X271E, X271F, X271G, X271H, X271I, X271K, X271L, X271M, X271N, X271P, X271T, X271V, X271Y, X272A, X272C, X272D, X272E, X272F, X272G, X272H, X272K, X272L, X272M, X272N, X272P, X272R, X272S, X272T, X272W, X272Y, X273A, X273C, X273D, X273E, X273F, X273G, X273H, X273I, X273K, X273L, X273R, X273S, X273T, X273V, X273W, X273Y, X274A, X274C, X274D, X274E, X274G, X274H, X274K, X274L, X274M, X274N, X274P, X274Q, X274R, X274S, X274T, X274W, X275A, X275C, X275D, X275E, X275F, X275G, X275H, X275K, X275L, X275M, X275P, X275Q, X275R, X275V, 及び X275W。
R, X106S, X106T, X106V, X106W, X107A, X107C, X107E, X107F, X107H, X107I, X107L, X107M, X107Q, X107S, X107T, X107V, X107W, X107Y, X108A, X108C, X108F, X108G, X108I, X108L, X108M, X108Q, X108S, X108T, X108V, X109A, X109C, X109E, X109F, X109G, X109H, X109I, X109K, X109L, X109M, X109N, X109P, X109Q, X109R, X109S, X109T, X109W, X109Y, X110A, X110G, X110S, X111A, X111C, X111E, X111F, X111I, X111L, X111M, X111P, X111T, X111V, X111Y, X112A, X112C, X112D, X112E, X112F, X112G, X112I, X112L, X112M, X112N, X112Q, X112S, X112T, X112V, X112W, X112Y, X113A, X113C, X113D, X113E, X113L, X113M, X113N, X113S, X113V, X113W, X114A, X114C, X114G, X114T, X115C, X115E, X115F, X115G, X115H, X115I, X115K, X115L, X115M, X115N, X115P, X115Q, X115R, X115S, X115T, X115V, X115W, X115Y, X116A, X116C, X116D, X116F, X116G, X116I, X116K, X116L, X116M, X116N, X116Q, X116S, X116T, X116V, X116W, X117A, X117C, X117D, X117F, X117G, X117I, X117N, X117Q, X117R, X117T, X117Y, X118A, X118C, X118D, X118E, X118F, X118G, X118I, X118K, X118L, X118M, X118N, X118P, X118R, X118S, X118T, X118V, X118W, X119A, X119C, X119E, X119F, X119G, X119H, X119K, X119M, X119N, X119P, X119Q, X119T, X119W, X120A, X120C, X120E, X120F, X120G, X120H, X120I, X120L, X120M, X120N, X120R, X120S, X120T, X120W, X121A, X121C, X121E, X121F, X121G, X121I, X121K, X121L, X121M, X121Q, X121S, X121T, X121V, X121Y, X122A, X122C, X122G, X122I, X122L, X122S, X122T, X122V, X123E, X123G, X123I, X123L, X123M, X123N, X123P, X123S, X123V, X124G, X124H, X124L, X124N, X124Q, X124S, X124T, X124Y, X125A, X125C, X125G, X125P, X125S, X125T, X126A, X126C, X126E, X126F, X126G, X126H, X126I, X126K, X126L, X126M, X126N, X126Q, X126S, X126T, X126V, X126W, X126Y, X127D, X127F, X127G, X127I, X127L, X127Q, X127R, X127S, X127T, X127V, X128A, X128C, X128D, X128F, X128G, X128H, X128I, X128K, X128L, X128M, X128N, X128P, X128Q, X128R, X128S, X128T, X128W, X128Y, X129A, X129E, X129F, X129G, X129I, X129L, X129M, X129N, X129P, X129R, X129S, X129T, X129V, X129W, X129Y, X130C, X130K, X130L, X130N, X130P, X130Q, X130R, X130S, X130V, X130W, X130Y, X131A, X131D, X131E, X131F, X131G, X131I, X131K, X131L, X131M, X131P, X131Q, X131R, X131V, X132A, X132E, X132F, X132H, X132I, X132L, X132M, X132N, X132Q, X132R, X132S, X132T, X132W, X133A, X133F, X133K, X133L, X133N, X133P, X133Q, X133S, X133T, X133V, X133Y, X134A, X134F, X134I, X134L, X134M, X134P, X134S, X134T, X134V, X135A, X135C, X135E, X135F, X135L, X135M, X135Q, X135T, X135W, X136A, X136D, X136E, X136F, X136G, X136K, X136M, X136N, X136Q, X136R, X136S, X136V, X136W, X136Y, X137A, X137C, X137E, X137G, X137H, X137K, X137L, X137M, X137Q, X137R, X137S, X137V, X137W, X138A, X138C, X138E, X138G, X138H, X138L, X138M, X138Q, X138R, X138V, X139A, X139C, X139E, X139F, X139G, X139I, X139M, X139Q, X139S, X139T, X139V, X139Y, X140A, X140C, X140D, X140E, X140F, X140G, X140I, X140K, X140L, X140M, X140N, X140Q, X140R, X140S, X140T, X140V, X140Y, X141D, X141E, X141G, X141H, X141I, X141K, X141L, X141N, X141Q, X141R, X141S, X141V, X141Y, X142A, X142C, X142E, X142G, X142I, X142L, X142M, X142N, X142Q, X142S, X142T, X142V, X142Y, X143C, X143D, X143F, X143G, X143H, X143I, X143K, X143L, X143M, X143N, X143R, X143S, X143T, X143V, X143W, X143Y, X144A, X144C, X144D, X144G, X144H, X144I, X144L, X144M, X144N, X144Q, X144R, X144S, X144T, X144V, X144W, X144Y, X145A, X145C, X145D, X145E, X145F, X145G, X145K, X145L, X145M, X145N, X145Q, X145R, X145S, X145T, X145W, X145Y, X146A, X146C, X146D, X146E, X146F, X146G, X146I, X146K, X146L, X146M, X146Q, X146R, X146S, X146T, X146W, X146Y, X147F, X147G, X147I, X147L, X147M, X147P, X147Q, X147T, X147V, X148A, X148C, X148E, X148F, X148G, X148H, X148I, X148L, X148M, X148N, X148S, X148T, X148V, X148W, X148Y, X149A, X149C, X149F, X149G, X149H, X149I, X149L, X149M, X149P, X149Q, X149S, X149T, X149V, X150A, X150E, X150F, X150G, X150H, X150L, X150P, X150Q, X150S, X150T, X150V, X151A, X151G, X151M, X151S, X151T, X151V, X152A, X152C, X152P, X152R, X152S, X152T, X152V, X153A, X153E, X153F, X153G, X153I, X153M, X153N, X153Q, X153S, X153V, X153Y, X154A, X154C, X154G, X154N, X154Q, X154S, X155A, X155D, X155E, X155F, X155I, X155L, X155N, X155P, X155Q, X155R, X155S, X155T, X155V, X155Y, X156A, X156C, X156D, X156E, X156F, X156G, X156I, X156K, X156L, X156M, X156N, X156P, X156Q, X156R, X156S, X156T, X156V, X156Y, X157A, X157C, X157D, X157G, X157K, X157L, X157Q, X157R, X157S, X157V, X158A, X158C, X158E, X158F, X158H, X158K, X158L, X158M, X158P, X158Q, X158R, X158S, X158T, X158V, X158W, X159A, X159C, X159E, X159G, X159H, X159L, X159M, X159P, X159Q, X159R, X159S, X159V, X159W, X159Y, X160A, X160C, X160D, X160F, X160G, X160I, X160L, X160M, X160N, X160Q, X160R, X160S, X160T, X160V, X160Y, X165A, X165C, X165D, X165E, X165F, X165G, X165H, X165I, X165K, X165L, X165M, X165P, X165R, X165S, X165T, X165V, X165W, X165Y, X166A, X166C, X166D, X166E, X166F, X166H, X166I, X166L, X166M, X166N, X166P, X166R, X166S, X166T, X166V, X166W, X166Y, X167A, X167C, X167D, X167E, X167F, X167G, X167H, X167I, X167K, X167L, X167M, X167N, X167P, X167Q, X167R, X167S, X167T, X167V, X167W, X167Y, X168A, X168C, X168F, X168I, X168M, X168N, X168P, X168S, X168T, X168V, X169A, X169C, X169G, X169I, X169L, X169S, X170A, X170D, X170E, X170G, X170H, X170K, X170L, X170N, X170P, X170Q, X170R, X170S, X170V, X170W, X170Y, X171A, X171C, X171D, X171E, X171F, X171G, X171H, X171I, X171L, X171M, X171N, X171Q, X171S, X171T, X171V, X171W, X171Y, X172A, X172C, X172D, X172F, X172G, X172I, X172K, X172L, X172M, X172P, X172Q, X172R, X172S, X172T, X172V, X172W, X172Y, X173A, X173C, X173D, X173E, X173F, X173G, X173H, X173I, X173K, X173L, X173M, X173N, X173P, X173Q, X173R, X173T, X173V, X173W, X173Y, X174A, X174G, X174I, X174N, X174P, X174S, X174T, X174V, X175A, X175C, X175E, X175G, X175H, X175I, X175K, X175L, X175M, X175Q, X175S, X175T, X175V, X175W, X175Y, X176A, X176C, X176E, X176G, X176I, X176K, X176P, X176S, X176T, X176V, X177A, X177C, X177I, X177T, X177V, X178A, X178G, X178S, X178T, X179A, X179C, X179G, X179H, X179I, X180C, X180G, X180H, X180I, X180L, X180N, X180Q, X180S, X180T, X180V, X181A, X181D, X181E, X181G, X181H, X181L, X181M, X181N, X182A, X182D, X182E, X182F, X182G, X182H, X182I, X182K, X182L, X182M, X182N, X182P, X182Q, X182R, X182S, X182T, X182V, X182W, X182Y, X183A, X183D, X183F, X183G, X183H, X183I, X183K, X183L, X183M, X183N, X183P, X183Q, X183R, X183S, X183T, X183V, X183W, X183Y, X184A, X184C, X184D, X184E, X184F, X184G, X184H, X184L, X184M, X184N, X184S, X184T, X184W, X184Y, X185A, X185C, X185E, X185F, X185G, X185H, X185I, X185K, X185L, X185M, X185N, X185Q, X185R, X185S, X185T, X185V, X185Y, X186A, X186C, X186G, X186H, X186I, X186K, X186L, X186M, X186N, X186P, X186Q, X186R, X186S, X186T, X186W, X187A, X187C, X187D, X187E, X187F, X187G, X187H, X187I, X187L, X187M, X187N, X187P, X187Q, X187S, X187T, X187V, X187W, X187Y, X188A, X188D, X188E, X188F, X188G, X188H, X188I, X188K, X188L, X188P, X188Q, X188R, X188S, X188T, X188V, X188W, X188Y, X189A, X189C, X189E, X189F, X189G, X189H, X189K, X189L, X189M, X189N, X189P, X189Q, X189R, X189S, X189T, X189V, X189Y, X190A, X190D, X190E, X190F, X190G, X190H, X190I, X190K, X190L, X190M, X190N, X190P, X190Q, X190R, X190S, X190V, X190W, X190Y, X191A, X191D, X191E, X191F, X191H, X191I, X191K, X191L, X191P, X191Q, X191R, X191S, X191T, X191V, X191W, X191Y, X192C, X192D, X192E, X192G, X192H, X192I, X192K, X192L, X192M, X192N, X192P, X192Q, X192R, X192S, X192T, X192V, X192W, X192Y, X193A, X193D, X193E, X193F, X193G, X193H, X193I, X193K, X193L, X193R, X193S, X193T, X193V, X193W, X193Y, X194A, X194C, X194D, X194E, X194F, X194G, X194H, X194I, X194L, X194M, X194P, X194Q, X194R, X194S, X194T, X194V, X194W, X194Y, X195A, X195C, X195D, X195E, X195F, X195G, X195I, X195K, X195L, X195Q, X195R, X195S, X195T, X195V, X195W, X195Y, X196A, X196D, X196E, X196F, X196I, X196L, X196M, X196P, X196Q, X196T, X196V, X196Y, X197A, X197C, X197D, X197E, X197F, X197G, X197H, X197I, X197L, X197M, X197N, X197Q, X197R, X197S, X197T, X197V, X197W, X197Y, X198A, X198D, X198E, X198F, X198G, X198H, X198I, X198L, X198M, X198N, X198Q, X198R, X198S, X198T, X198W, X198Y, X199A, X199C, X199D, X199E, X199F, X199G, X199H, X199I, X199L, X199M, X199Q, X199S, X199T, X199V, X199W, X200A, X200C, X200G, X200H, X200I, X200S, X201C, X201G, X201P, X201S, X201T, X201V, X202F, X202G, X203A, X203C, X203E, X203F, X203G, X203H, X203I, X203K, X203L, X203N, X203R, X203S, X203T, X203V, X203W, X203Y, X204A, X204C, X204E, X204F, X204G, X204I, X204K, X204L, X204N, X204P, X204R, X204S, X204T, X204W, X204Y, X205A, X205F, X205G, X205I, X205L, X205M, X205Q, X205T, X205V, X206A, X206C, X206D, X206E, X206F, X206G, X206H, X206I, X206K, X206L, X206N, X206P, X206Q, X206R, X206S, X206T, X206V, X206W, X206Y, X207A, X207G, X207H, X207S, X208A, X208C, X208L, X208N, X208P, X208S, X208T, X208V, X209A, X209C, X209D, X209E, X209F, X209G, X209H, X209I, X209K, X209L, X209M, X209N, X209R, X209S, X209T, X209V, X209W, X209Y, X210A, X210C, X210D, X210E, X210F, X210G, X210H, X210I, X210L, X210M, X210N, X210P, X210Q, X210R, X210S, X210V, X210W, X210Y, X211A, X211C, X211E, X211F, X211G, X211H, X211I, X211L, X211M, X211P, X211Q, X211R, X211T, X211V, X211W, X211Y, X212C, X212F, X212G, X212H, X212I, X212M, X212N, X212P, X212R, X212S, X212T, X212V, X212Y, X213A, X213C, X213D, X213E, X213F, X213G, X213I, X213K, X213L, X213M, X213N, X213P, X213Q, X213R, X213S, X213T, X213V, X213W, X213Y, X214A, X214C, X214E, X214F, X214G, X214H, X214I, X214K, X214L, X214M, X214N, X214P, X214Q, X214R, X214S, X214T, X214V, X214W, X214Y, X215A, X215C, X215D, X215E, X215F, X215G, X215H, X215I, X215K, X215M, X215N, X215P, X215R, X215S, X215T, X215V, X215W, X215Y, X216A, X216C, X216D, X216E, X216F, X216G, X216H, X216I, X216K, X216L, X216M, X216N, X216P, X216Q, X216R, X216S, X216V, X216W, X216Y, X217A, X217C, X217D, X217E, X217F, X217G, X217I, X217K, X217L, X217M, X217N, X217Q, X217S, X217T, X217V, X217Y, X218C, X218D, X218E, X218F, X218G, X218H, X218I, X218L, X218M, X218N, X218P, X218Q, X218R, X218S, X218T, X218V, X218W, X218Y, X219A, X219G, X219S, X220A, X220C, X220G, X220H, X220N, X220S, X220T, X220V, X221G, X221S, X221T, X222A, X222C, X222E, X222F, X222G, X222I, X222K, X222L, X222M, X222N, X222P, X222Q, X222R, X222S, X222T, X222V, X222W, X223A, X223
C, X223G, X223M, X223S, X223T, X224A, X224D, X224E, X224G, X224I, X224L, X224M, X224N, X224P, X224S, X224T, X225A, X225C, X225G, X225I, X225N, X225P, X225S, X225T, X225V, X226C, X226F, X226G, X226H, X226I, X226L, X226M, X226N, X226R, X226S, X226T, X226V, X226Y, X227A, X227C, X227F, X227G, X227I, X227L, X227M, X227P, X227Q, X227S, X227T, X227V, X227Y, X228A, X228C, X228G, X228I, X228L, X228M, X228P, X228S, X228V, X229A, X229G, X229P, X229S, X230A, X230D, X230E, X230F, X230G, X230H, X230I, X230L, X230N, X230P, X230Q, X230S, X230T, X230V, X230W, X230Y, X231A, X231C, X231F, X231G, X231H, X231I, X231L, X231S, X231T, X231W, X231Y, X232A, X232G, X232H, X232K, X232L, X232M, X232P, X232S, X232V, X233A, X233C, X233E, X233F, X233G, X233I, X233L, X233M, X233N, X233P, X233Q, X233R, X233S, X233T, X233V, X233Y, X234D, X234F, X234G, X234H, X234L, X234M, X234N, X234P, X234Q, X234S, X234T, X234V, X234Y, X235C, X235D, X235E, X235F, X235G, X235H, X235I, X235K, X235L, X235M, X235N, X235Q, X235R, X235S, X235V, X235W, X235Y, X236A, X236C, X236E, X236F, X236G, X236H, X236K, X236L, X236N, X236P, X236Q, X236R, X236S, X236T, X236V, X236W, X236Y, X237A, X237C, X237F, X237G, X237H, X237I, X237K, X237L, X237M, X237P, X237Q, X237R, X237S, X237T, X237V, X237W, X237Y, X238C, X238D, X238E, X238F, X238G, X238H, X238I, X238K, X238L, X238M, X238N, X238Q, X238R, X238S, X238T, X238V, X238Y, X239C, X239D, X239F, X239G, X239H, X239I, X239K, X239L, X239M, X239N, X239P, X239Q, X239R, X239S, X239T, X239V, X239W, X239Y, X240A, X240C, X240E, X240F, X240I, X240K, X240L, X240M, X240N, X240Q, X240R, X240S, X240T, X240W, X240Y, X241A, X241C, X241D, X241E, X241F, X241G, X241H, X241I, X241K, X241L, X241M, X241N, X241P, X241Q, X241R, X241S, X241T, X241V, X241W, X241Y, X242A, X242C, X242D, X242F, X242G, X242H, X242I, X242L, X242M, X242P, X242Q, X242R, X242S, X242T, X242V, X242W, X243C, X243D, X243E, X243F, X243G, X243H, X243I, X243K, X243L, X243M, X243N, X243P, X243Q, X243R, X243S, X243T, X243V, X243W, X244A, X244D, X244E, X244F, X244H, X244I, X244L, X244M, X244N, X244P, X244Q, X244R, X244S, X244T, X244V, X244W, X244Y, X245A, X245C, X245E, X245G, X245H, X245K, X245L, X245P, X245Q, X245R, X245S, X245V, X245W, X245Y, X246A, X246C, X246E, X246F, X246G, X246H, X246I, X246L, X246M, X246N, X246P, X246Q, X246R, X246S, X246T, X246V, X246W, X246Y, X247A, X247C, X247D, X247E, X247F, X247G, X247H, X247I, X247K, X247L, X247M, X247N, X247P, X247Q, X247R, X247S, X247T, X247V, X247W, X247Y, X248C, X248D, X248E, X248G, X248H, X248I, X248K, X248L, X248M, X248N, X248P, X248R, X248S, X248T, X248V, X248W, X248Y, X249A, X249D, X249E, X249F, X249G, X249H, X249I, X249K, X249L, X249M, X249N, X249Q, X249R, X249S, X249T, X249V, X249W, X249Y, X250A, X250C, X250E, X250F, X250G, X250H, X250I, X250L, X250M, X250N, X250Q, X250S, X250V, X250Y, X251A, X251D, X251E, X251F, X251G, X251K, X251L, X251M, X251P, X251Q, X251R, X251S, X251T, X251V, X251Y, X252A, X252C, X252D, X252F, X252G, X252H, X252I, X252K, X252L, X252M, X252N, X252P, X252R, X252S, X252V, X252W, X252Y, X253A, X253D, X253E, X253F, X253G, X253H, X253I, X253K, X253M, X253R, X253S, X253T, X253V, X253W, X254A, X254C, X254D, X254E, X254G, X254N, X254P, X254S, X254T, X254V, X255A, X255C, X255D, X255E, X255F, X255H, X255I, X255L, X255N, X255P, X255Q, X255R, X255S, X255T, X255V, X255W, X255Y, X256A, X256C, X256D, X256E, X256G, X256H, X256I, X256K, X256L, X256M, X256N, X256P, X256R, X256S, X256T, X256V, X256W, X256Y, X257A, X257C, X257E, X257F, X257G, X257H, X257I, X257K, X257L, X257M, X257P, X257S, X257T, X257V, X257W, X257Y, X258A, X258C, X258D, X258E, X258F, X258G, X258H, X258I, X258L, X258M, X258P, X258Q, X258R, X258S, X258T, X258V, X258W, X258Y, X259A, X259C, X259E, X259G, X259I, X259L, X259M, X259P, X259Q, X259R, X259S, X259T, X259V, X260A, X260D, X260E, X260F, X260H, X260I, X260L, X260M, X260N, X260P, X260R, X260S, X260T, X260V, X260Y, X261A, X261C, X261E, X261F, X261G, X261I, X261K, X261L, X261N, X261P, X261Q, X261R, X261S, X261T, X261V, X261W, X261Y, X262A, X262C, X262D, X262F, X262G, X262H, X262I, X262K, X262L, X262M, X262P, X262Q, X262R, X262S, X262T, X262V, X262W, X262Y, X263A, X263C, X263D, X263F, X263G, X263H, X263I, X263K, X263L, X263M, X263N, X263P, X263Q, X263R, X263W, X263Y, X264A, X264C, X264E, X264F, X264G, X264H, X264I, X264L, X264P, X264Q, X264S, X264T, X264V, X264Y, X265A, X265C, X265D, X265F, X265G, X265H, X265I, X265K, X265L, X265M, X265N, X265P, X265Q, X265R, X265S, X265T, X265V, X265W, X265Y, X266G, X266W, X266Y, X267A, X267C, X267D, X267E, X267F, X267G, X267H, X267I, X267K, X267L, X267M, X267N, X267S, X267T, X267V, X267Y, X268A, X268D, X268E, X268G, X268H, X268K, X268L, X268M, X268N, X268P, X268Q, X268R, X268S, X268V, X268W, X268Y, X269C, X269D, X269F, X269G, X269H, X269I, X269L, X269M, X269N, X269Q, X269R, X269S, X269T, X269V, X270A, X270C, X270D, X270E, X270F, X270G, X270H, X270I, X270L, X270M, X270N, X270P, X270Q, X270S, X270T, X270V, X271A, X271C, X271E, X271F, X271G, X271H, X271I, X271K, X271L, X271M, X271N, X271P, X271T, X271V, X271Y, X272A, X272C, X272D, X272E, X272F, X272G, X272H, X272K, X272L, X272M, X272N, X272P, X272R, X272S, X272T, X272W, X272Y, X273A, X273C, X273D, X273E, X273F, X273G, X273H, X273I, X273K, X273L, X273R, X273S, X273T, X273V, X273W, X273Y, X274A, X274C, X274D, X274E, X274G, X274H, X274K, X274L, X274M, X274N, X274P, X274Q, X274R, X274S, X274T, X274W, X275A, X275C, X275D, X275E, X275F, X275G, X275H, X275K, X275L, X275M, X275P, X275Q, X275R, X275V, 及び X275W。ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される。いくつかの実施形態では、このスブチリシン変異体には、4, 5, 6, 7, 8, 9, 10, 11, 12 箇所、またはそれ以上の上記の位置が含まれる。
, X139A, X139C, X139I, X139M, X139T, X139V, X140A, X140C, X140D, X140E, X140F, X140G, X140I, X140L, X140M, X140N, X140Q, X140R, X140S, X140T, X140V, X140Y, X141D, X141E, X141G, X141H, X141I, X141K, X141L, X141N, X141Q, X141R, X141S, X141V, X141Y, X142A, X142C, X142L, X142T, X142V, X142Y, X143C, X143D, X143F, X143G, X143H, X143I, X143K, X143L, X143M, X143N, X143S, X143T, X143V, X143W, X143Y, X144A, X144C, X144D, X144G, X144H, X144I, X144L, X144M, X144N, X144Q, X144R, X144S, X144T, X144V, X144W, X144Y, X145A, X145C, X145D, X145E, X145F, X145G, X145K, X145L, X145M, X145N, X145Q, X145R, X145S, X145T, X145W, X145Y, X146A, X146C, X146D, X146E, X146G, X146K, X146Q, X147I, X147L, X147M, X147T, X147V, X148A, X148C, X148E, X148F, X148H, X148I, X148L, X148M, X148N, X148S, X148T, X148V, X148Y, X149A, X149C, X149I, X149L, X149M, X149P, X149S, X149T, X149V, X150A, X150F, X150L, X150T, X150V, X151A, X151G, X151S, X152A, X152S, X153A, X153G, X153S, X153V, X156A, X156C, X156D, X156E, X156F, X156L, X156M, X156N, X156S, X156T, X158A, X158C, X158E, X158F, X158H, X158K, X158L, X158M, X158Q, X158R, X158S, X158T, X158V, X158W, X159A, X159C, X159E, X159G, X159H, X159M, X159P, X159Q, X159R, X159S, X159W, X160A, X160C, X160D, X160F, X160G, X160I, X160L, X160M, X160N, X160Q, X160R, X160S, X160T, X160V, X160Y, X165I, X165L, X165T, X165V, X166A, X166C, X166D, X166E, X166H, X166M, X166N, X166S, X167C, X167D, X167E, X167F, X167P, X167V, X167W, X167Y, X169A, X169G, X169S, X170A, X170D, X170E, X170G, X170H, X170K, X170L, X170P, X170Q, X170R, X170S, X170V, X170W, X170Y, X171C, X171F, X171L, X171N, X171Y, X172A, X172C, X172D, X172F, X172G, X172I, X172K, X172L, X172M, X172P, X172Q, X172R, X172S, X172T, X172V, X172Y, X173A, X173C, X173D, X173E, X173F, X173G, X173H, X173K, X173L, X173M, X173N, X173Q, X173R, X173T, X173V, X173W, X174A, X174G, X174S, X174T, X174V, X175A, X175C, X175I, X175L, X175M, X175Q, X175T, X175V, X175Y, X176A, X176G, X176S, X177A, X177C, X177I, X177T, X177V, X178A, X178G, X179A, X179G, X180C, X180I, X180L, X180S, X180T, X180V, X181D, X181N, X182A, X182D, X182E, X182F, X182G, X182H, X182I, X182K, X182L, X182M, X182N, X182P, X182Q, X182R, X182S, X182T, X182V, X182W, X182Y, X183A, X183D, X183F, X183G, X183H, X183I, X183K, X183L, X183M, X183N, X183Q, X183R, X183S, X183T, X183V, X183W, X183Y, X184D, X184N, X185A, X185C, X185E, X185F, X185G, X185H, X185I, X185K, X185L, X185M, X185N, X185Q, X185R, X185S, X185T, X185V, X185Y, X186H, X186I, X186K, X186L, X186R, X187A, X187P, X187W, X188A, X188D, X188E, X188F, X188G, X188H, X188I, X188K, X188L, X188P, X188Q, X188R, X188S, X188T, X188V, X188W, X188Y, X191D, X191Q, X192W, X192Y, X194A, X194C, X194D, X194E, X194F, X194G, X194H, X194I, X194L, X194M, X194P, X194Q, X194R, X194S, X194T, X194V, X194W, X194Y, X195A, X195C, X195D, X195E, X195G, X195Q, X195Y, X196L, X196M, X197A, X197C, X197D, X197E, X197G, X197N, X197Q, X197S, X197T, X198A, X198F, X198G, X198H, X198I, X198L, X198M, X198N, X198R, X198S, X198T, X198Y, X199A, X199C, X199I, X199M, X199S, X199T, X199V, X200A, X200C, X200G, X200S, X203A, X203C, X203E, X203I, X203L, X203S, X203T, X203V, X204A, X204C, X204E, X204F, X204G, X204I, X204K, X204L, X204N, X204R, X204S, X204T, X204W, X204Y, X205I, X205T, X205V, X206A, X206C, X206D, X206E, X206G, X206H, X206I, X206K, X206L, X206N, X206P, X206Q, X206R, X206S, X206T, X206V, X206W, X206Y, X207A, X207S, X208C, X208L, X208S, X208T, X208V, X209A, X209C, X209F, X209G, X209H, X209I, X209K, X209L, X209M, X209N, X209R, X209S, X209V, X209W, X209Y, X210A, X210C, X210E, X210G, X210H, X210I, X210L, X210M, X210N, X210P, X210R, X210S, X210V, X210Y, X211A, X211C, X211E, X211F, X211G, X211H, X211I, X211L, X211M, X211P, X211Q, X211R, X211T, X211V, X211W, X211Y, X212C, X212F, X212G, X212H, X212I, X212M, X212N, X212P, X212R, X212S, X212T, X212V, X212Y, X213A, X213C, X213D, X213E, X213F, X213G, X213I, X213K, X213L, X213M, X213N, X213Q, X213R, X213S, X213T, X213V, X213W, X213Y, X214F, X214L, X214W, X214Y, X215A, X215C, X215D, X215E, X215F, X215G, X215H, X215I, X215K, X215M, X215N, X215P, X215R, X215S, X215T, X215V, X215W, X215Y, X216A, X216C, X216D, X216E, X216F, X216G, X216H, X216I, X216K, X216L, X216M, X216N, X216P, X216Q, X216R, X216S, X216V, X216W, X216Y, X217A, X217C, X217E, X217F, X217G, X217I, X217K, X217L, X217M, X217Q, X218C, X218D, X218E, X218F, X218G, X218H, X218I, X218M, X218N, X218Q, X218R, X218S, X218T, X218V, X218Y, X220S, X220T, X222A, X222M, X222Q, X223A, X223G, X223S, X224A, X224G, X224L, X224N, X224S, X224T, X225C, X225P, X226C, X226F, X226H, X226M, X226V, X227A, X227C, X227G, X227I, X227L, X227M, X227S, X227T, X227V, X228A, X228C, X228G, X228I, X228S, X228V, X229A, X229G, X229P, X229S, X230A, X230D, X230E, X230G, X230H, X230I, X230L, X230N, X230Q, X230S, X230T, X230V, X231A, X231C, X231F, X231G, X231H, X231I, X231L, X231S, X231T, X231Y, X232A, X232G, X232H, X232L, X232M, X232S, X232V, X233A, X233C, X233E, X233F, X233G, X233I, X233L, X233M, X233N, X233P, X233Q, X233S, X233T, X233V, X233Y, X234D, X234F, X234G, X234H, X234L, X234M, X234N, X234Q, X234S, X234T, X234V, X234Y, X235C, X235D, X235E, X235F, X235G, X235H, X235I, X235K, X235L, X235M, X235N, X235Q, X235R, X235S, X235V, X235W, X235Y, X236A, X236C, X236E, X236F, X236H, X236K, X236N, X236P, X236Q, X236R, X236S, X236T, X236V, X236W, X236Y, X237A, X237C, X237F, X237G, X237H, X237I, X237K, X237L, X237M, X237P, X237Q, X237R, X237S, X237T, X237V, X237W, X237Y, X238C, X238D, X238E, X238F, X238G, X238H, X238I, X238K, X238L, X238M, X238N, X238Q, X238R, X238S, X238T, X238V, X238Y, X239C, X239D, X239F, X239G, X239H, X239I, X239K, X239L, X239M, X239N, X239P, X239Q, X239R, X239S, X239T, X239V, X239W, X239Y, X240A, X240C, X240E, X240F, X240I, X240K, X240L, X240M, X240N, X240Q, X240R, X240S, X240T, X240W, X240Y, X241A, X241C, X241D, X241E, X241F, X241G, X241H, X241I, X241K, X241L, X241M, X241N, X241P, X241Q, X241R, X241S, X241T, X241V, X241W, X241Y, X242A, X242C, X242D, X242F, X242G, X242H, X242I, X242L, X242M, X242P, X242Q, X242R, X242S, X242T, X242V, X242W, X243C, X243D, X243E, X243F, X243G, X243H, X243I, X243K, X243L, X243M, X243N, X243P, X243Q, X243R, X243S, X243T, X243V, X243W, X244A, X244D, X244E, X244F, X244H, X244I, X244L, X244M, X244N, X244P, X244Q, X244R, X244S, X244T, X244V, X244W, X244Y, X245A, X245C, X245E, X245G, X245H, X245K, X245L, X245P, X245Q, X245R, X245S, X245V, X245W, X245Y, X246A, X246C, X246E, X246F, X246G, X246H, X246I, X246L, X246M, X246N, X246Q, X246S, X246T, X246V, X246W, X246Y, X247A, X247C, X247D, X247E, X247F, X247G, X247H, X247I, X247K, X247L, X247M, X247N, X247P, X247Q, X247R, X247S, X247T, X247V, X247W, X247Y, X248C, X248D, X248E, X248G, X248H, X248I, X248K, X248L, X248N, X248P, X248R, X248S, X248T, X248V, X248W, X248Y, X249A, X249D, X249E, X249F, X249G, X249H, X249I, X249K, X249L, X249M, X249N, X249Q, X249R, X249S, X249T, X249V, X249W, X249Y, X250A, X250C, X250F, X250I, X250L, X250M, X250Q, X250S, X250V, X251A, X251D, X251E, X251F, X251G, X251K, X251L, X251M, X251Q, X251R, X251T, X251V, X251Y, X252A, X252C, X252D, X252F, X252G, X252H, X252I, X252K, X252L, X252M, X252N, X252R, X252S, X252V, X252W, X252Y, X253A, X253D, X253E, X253F, X253G, X253H, X253I, X253K, X253M, X253R, X253S, X253T, X253V, X253W, X254A, X254C, X254G, X254S, X254T, X255A, X255C, X255D, X255E, X255F, X255H, X255I, X255L, X255N, X255P, X255Q, X255R, X255S, X255T, X255V, X255W, X255Y, X256A, X256C, X256D, X256E, X256G, X256H, X256I, X256K, X256L, X256M, X256N, X256P, X256R, X256S, X256T, X256V, X256W, X256Y, X257C, X257F, X257I, X257K, X257L, X257M, X257V, X258A, X258C, X258D, X258E, X258F, X258G, X258H, X258I, X258L, X258M, X258P, X258Q, X258R, X258S, X258T, X258V, X258W, X258Y, X259A, X259C, X259E, X259G, X259I, X259L, X259M, X259P, X259Q, X259R, X259S, X259T, X259V, X260A, X260D, X260E, X260F, X260H, X260I, X260L, X260M, X260N, X260P, X260R, X260S, X260T, X260V, X260Y, X261A, X261C, X261E, X261F, X261G, X261I, X261K, X261L, X261N, X261P, X261Q, X261R, X261S, X261T, X261V, X261W, X261Y, X262A, X262C, X262D, X262F, X262G, X262H, X262I, X262K, X262L, X262M, X262Q, X262R, X262S, X262T, X262V, X262W, X262Y, X263C, X263F, X263L, X263Y, X264A, X264G, X265A, X265C, X265D, X265F, X265G, X265H, X265K, X265M, X265Q, X265R, X265S, X265T, X265W, X265Y, X267G, X267I, X267L, X267M, X267N, X267V, X268A, X268G, X268H, X268L, X268M, X268N, X268P, X268Q, X268S, X268V, X269C, X269D, X269F, X269G, X269H, X269I, X269L, X269M, X269N, X269Q, X269R, X269S, X269T, X269V, X270A, X270C, X270D, X270G, X270I, X270L, X270M, X270N, X270S, X270T, X270V, X271A, X271C, X271E, X271F, X271G, X271H, X271I, X271K, X271L, X271M, X271N, X271P, X271T, X271V, X271Y, X272A, X272C, X272D, X272E, X272F, X272G, X272H, X272K, X272L, X272M, X272N, X272P, X272R, X272S, X272T, X272W, X272Y, X273A, X273C, X273D, X273E, X273F, X273G, X273H, X273I, X273L, X273S, X273T, X273V, X274A, X274C, X274D, X274E, X274G, X274H, X274L, X274M, X274N, X274P, X274Q, X274R, X274S, X274T, X274W, X275A, X275C, X275D, X275E, X275F, X275G, X275H, X275K, X275L, X275M, X275P, X275Q, X275R, X275V, 及びX275W。ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される。いくつかの実施形態では、このスブチリシン変異体には、2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 箇所、またはそれ以上の上記の位置が含まれる。
, X241M, X241N, X241Q, X241R, X241S, X241T, X241V, X241W, X241Y, X242A, X242C, X242D, X242G, X242H, X242I, X242L, X242M, X242P, X242Q, X242S, X242T, X242V, X243C, X243D, X243E, X243F, X243G, X243H, X243I, X243L, X243M, X243N, X243P, X243Q, X243S, X243T, X243V, X243W, X244D, X244E, X244F, X244H, X244I, X244L, X244M, X244N, X244Q, X244S, X244T, X244V, X244W, X245A, X245C, X245E, X245G, X245H, X245K, X245L, X245P, X245Q, X245S, X245V, X245W, X245Y, X246A, X246C, X246I, X246L, X246M, X246T, X246V, X247A, X247C, X247F, X247G, X247H, X247K, X247L, X247M, X247N, X247R, X247S, X247T, X247V, X247W, X247Y, X248C, X248D, X248E, X248G, X248H, X248I, X248L, X248N, X248P, X248R, X248S, X248T, X248V, X248Y, X249A, X249D, X249E, X249F, X249G, X249H, X249I, X249K, X249L, X249M, X249N, X249Q, X249S, X249T, X249W, X249Y, X250C, X250I, X250L, X250M, X250V, X251A, X251D, X251E, X251F, X251G, X251K, X251L, X251M, X251Q, X251R, X251T, X251V, X251Y, X252A, X252C, X252D, X252F, X252G, X252H, X252I, X252K, X252L, X252M, X252N, X252R, X252S, X252V, X252W, X253A, X253E, X253H, X253M, X253S, X253T, X253W, X255A, X255C, X255D, X255E, X255I, X255L, X255N, X255Q, X255T, X255V, X255Y, X256A, X256C, X256D, X256E, X256G, X256H, X256I, X256K, X256L, X256M, X256N, X256P, X256S, X256V, X256W, X256Y, X258D, X258G, X259A, X259C, X259E, X259P, X259Q, X259S, X260A, X260D, X260E, X260H, X260I, X260N, X260P, X260S, X260T, X260V, X261A, X261C, X261E, X261F, X261I, X261K, X261L, X261N, X261P, X261Q, X261S, X261T, X261V, X261W, X261Y, X262A, X262C, X262D, X262F, X262H, X262I, X262L, X262M, X262Q, X262T, X262V, X262Y, X265A, X265D, X265S, X267I, X267L, X268A, X268L, X268V, X269D, X269H, X269N, X269Q, X269S, X270A, X270C, X270G, X270I, X270L, X270N, X270S, X270T, X270V, X271C, X271E, X272A, X272C, X272D, X272E, X272F, X272G, X272H, X272L, X272M, X272N, X272P, X272S, X27, 2T, X272W, X272Y, X273A, X273C, X273G, X273S, X274A, X274C, X274G, X274L, X274M, X274N, X274S, X274T, X275D, X275E, X275F, X275H, X275K, X275L, X275M, X275P, X275Q, 及び X275R。ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される。いくつかの実施形態では、このスブチリシン変異体には、2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 、またはそれ以上の上記の位置が含まれる。
X241N, X241Q, X241R, X241S, X241T, X241V, X241W, X241Y, X242A, X242C, X242D, X242G, X242H, X242I, X242L, X242M, X242P, X242Q, X242S, X242T, X242V, X243C, X243D, X243E, X243F, X243G, X243H, X243I, X243L, X243M, X243N, X243P, X243Q, X243S, X243T, X243V, X243W, X244D, X244E, X244F, X244H, X244I, X244L, X244M, X244N, X244Q, X244S, X244T, X244V, X244W, X245A, X245C, X245E, X245G, X245H, X245K, X245L, X245P, X245Q, X245S, X245V, X245W, X245Y, X246A, X246C, X246I, X246L, X246M, X246T, X246V, X247A, X247C, X247F, X247G, X247H, X247K, X247L, X247M, X247N, X247R, X247S, X247T, X247V, X247W, X247Y, X248C, X248D, X248E, X248G, X248H, X248I, X248L, X248N, X248P, X248R, X248S, X248T, X248V, X248Y, X249A, X249D, X249E, X249F, X249G, X249H, X249I, X249K, X249L, X249M, X249N, X249Q, X249S, X249T, X249W, X249Y, X250C, X250I, X250L, X250M, X250V, X251A, X251D, X251E, X251F, X251G, X251K, X251L, X251M, X251Q, X251R, X251T, X251V, X251Y, X252A, X252C, X252D, X252F, X252G, X252H, X252I, X252K, X252L, X252M, X252N, X252R, X252S, X252V, X252W, X253A, X253E, X253H, X253M, X253S, X253T, X253W, X255A, X255C, X255D, X255E, X255I, X255L, X255N, X255Q, X255T, X255V, X255Y, X256A, X256C, X256D, X256E, X256G, X256H, X256I, X256K, X256L, X256M, X256N, X256P, X256S, X256V, X256W, X256Y, X258D, X258G, X259A, X259C, X259E, X259P, X259Q, X259S, X260A, X260D, X260E, X260H, X260I, X260N, X260P, X260S, X260T, X260V, X261A, X261C, X261E, X261F, X261I, X261K, X261L, X261N, X261P, X261Q, X261S, X261T, X261V, X261W, X261Y, X262A, X262C, X262D, X262F, X262H, X262I, X262L, X262M, X262Q, X262T, X262V, X262Y, X265A, X265D, X265S, X267I, X267L, X268A, X268L, X268V, X269D, X269H, X269N, X269Q, X269S, X270A, X270C, X270G, X270I, X270L, X270N, X270S, X270T, X270V, X271C, X271E, X272A, X272C, X272D, X272E, X272F, X272G, X272H, X272L, X272M, X272N, X272P, X272S, X272T, X272W, X272Y, X273A, X273C, X273G, X273S, X274A, X274C, X274G, X274L, X274M, X274N, X274S, X274T, X275D, X275E, X275F, X275H, X275K, X275L, X275M, X275P, X275Q, 及び X275R。ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される。いくつかの実施形態では、このスブチリシン変異体には、2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 箇所、またはそれ以上の上記の位置が含まれる。
S87N/Q109Q/G118D/S128L/P129Q/S130A/S188S/T213E/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213R/N248D,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213T/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213E/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213T/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213T/N248R,
S87R/Q109R/G118V/S128L/P129Q/S130A/S188D/T213E/N248R,
S87R/Q109Q/G118R/S128L/P129Q/S130A/S188D/T213T/N248R,
S87R/Q109R/G118R/S128L/P129Q/S130A/S188D/T213T/N248R,
S87N/Q109Q/G118R/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213R/N248R,
S87R/Q109Q/G118R/S128L/P129Q/S130A/S188S/T213E/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213T/N248N,
S87R/Q109Q/G118R/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213E/N248R,
S87D/Q109D/G118D/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213R/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213T/N248R,
S87R/Q109R/G118R/S128L/P129Q/S130A/S188D/T213E/N248R,
S87R/Q109Q/G118R/S128L/P129Q/S130A/S188D/T213E/N248N,
S87N/Q109R/G118R/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213E/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213E/N248R,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213T/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213R/N248D,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213T/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213R/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213T/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213T/N248D,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213R/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213E/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213T/N248D,
S87R/Q109D/G118D/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213R/N248R,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213R/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213R/N248N,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213E/N248N,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213E/N248N,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213R/N248R,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213T/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213R/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213T/N248N,
S87R/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109D/G118V/S128L/P129Q/S130A/S188R/T213R/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213T/N248D,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213E/N248D,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213R/N248D,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213E/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213E/N248D,
S87R/Q109D/G118R/S128L/P129Q/S130A/S188D/T213E/N248N,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213R/N248R,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213R/N248N,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213T/N248R,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213E/N248R,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213R/N248D, 及び
S87N/Q109Q/G118V/S128LP129Q/S130A/S188R/T213E/N248R。
ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される。
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213T/N248D,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213R/N248N,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213R/N248D,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213E/N248D,
S87R/Q109D/G118R/S128L/P129Q/S130A/S188S/T213E/N248R,
S87R/Q109D/G118R/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109D/G118V/S128L/P129Q/S130A/S188D/T213E/N248D, 及び
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213E/N248D。
ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される。
S87D/Q109D/G118R/S128L/P129Q/S130A/S188R/T213E/N248D,
S87N/Q109D/G118R/S128L/P129Q/S130A/S188D/T213E/N248R,
S87D/Q109Q/G118D/S128L/P129Q/S130A/S188S/T213E/N248D,
S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213E/N248D,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213E/N248D,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213T/N248D,
S87R/Q109D/G118V/S128L/P129Q/S130A/S188D/T213E/N248R,
S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213R/N248D, 及び
S87R/Q109D/G118R/S128L/P129Q/S130A/S188D/T213T/N248R
ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される。
A001C/S024V/I107L, A001I/R045T/I107R/A172T, A001K/A172L, A001K/A172W, A001K/I072C/L126F, A001K/I072C/L126F/S164Q, A001K/I072C/V104I/L126F, A001K/I072C/V104I/L126F/S164Q, A001K/I072V, A001K/I072V/L126F, A001K/I072V/L126F/S164F, A001K/I072V/V104I, A001K/I072V/V104I/L126F/S164I, A001K/I072V/V104I/S164F, A001K/I072V/V104I/S164Q, A001K/I107T/A172Q, A001K/I107V/G127Q, A001K/I107V/G127Q/A172Q, A001K/I107V/G127R/A172Q, A001K/L126F, A001K/L126F/S164F, A001K/L126F/S164I, A001K/L126F/S164Q, A001K/R045F/I072C, A001K/R045F/I072C/L126F, A001K/R045F/I072C/V104I/S164F, A001K/R045F/I072V/L126F/S164I, A001K/R045F/I072V/L126F/S164Q, A001K/R045F/I072V/S164F, A001K/R045F/I072V/V104G/S164T, A001K/R045F/I107L/A172N, A001K/R045F/I107T, A001K/R045F/I107V/G127Q, A001K/R045F/L126F/S164F, A001K/R045F/L126F/S164I, A001K/R045F/S164F, A001K/R045F/V104I/L126F/S164Q, A001K/R045K/I072C/L126F/S164Q, A001K/R045K/I072V/L126F, A001K/R045K/I072V/L126F/S164F, A001K/R045K/I072V/L126F/S164Q, A001K/R045K/I072V/S164F, A001K/R045K/I072V/S164Q, A001K/R045K/I072V/V104I/L126F/S164F, A001K/R045K/I072V/V104I/L126F/S164I, A001K/R045K/I072V/V104I/L126F/S164Q, A001K/R045K/I107A/A172T, A001K/R045K/I107R/G127A/A172N, A001K/R045K/I107T/G127R, A001K/R045K/I107T/G127R/A172Q, A001K/R045K/L126F, A001K/R045K/L126F/S164I, A001K/R045K/L126F/S164P, A001K/R045K/L126F/S164Q, A001K/R045K/S164F, A001K/R045K/V104I/S164Q, A001K/R045K/V104L/S164A, A001K/R045N/I107T/G127Q, A001K/R045S/V104C/L126Y/S164F, A001K/S024H/I107T/G127Q, A001K/S024H/I107T/G127R, A001K/S024H/I107V, A001K/S024H/R045N/I107T, A001K/S024H/R045N/I107T/G127Q/A172Q, A001K/S024L/R045A, A001K/S024N/I107V/G127T, A001K/S024N/R045K/A172Y, A001K/S024R/I107T, A001K/S024R/R045N/A172C, A001K/S024R/R045N/I107T, A001K/S024R/R045N/I107T/G127R, A001K/S024T/I107T/G127Q/A172Q, A001K/S024T/R045N/G127Q/A172Q, A001K/S024W/I107T/G127R, A001K/S024W/I107V/G127T/A172Q, A001K/S164Q, A001K/V104E, A001K/V104I/L126F, A001K/V104I/L126F/S164I, A001K/V104I/L126F/S164Q, A001L/I072C, A001L/R045Q/G127H/A172R, A001L/S024D/R045Y/I107Y, A001M/R045S/I107E/A172D, A001P/A172V, A001Q/R045K/I107T, A001R/A172G, A001R/A172Q, A001R/I107T, A001R/I107T/A172Q, A001R/I107T/G127Q/A172Q, A001R/I107V/A172Q, A001R/R045F, A001R/R045K/A172Q, A001R/R045K/G127R, A001R/R045K/I107T/G127Q, A001R/R045K/I107V/G127R, A001R/R045N/A172K, A001R/R045N/A172Q, A001R/R045N/I107T, A001R/R045N/I107T/G127Q, A001R/R045N/I107V/A172V, A001R/R045N/I107V/G127Q/A172Q, A001R/R045N/I107V/G127R, A001R/S024H/I107T, A001R/S024H/R045K/I107T, A001R/S024H/R045K/I107T/A172Q, A001R/S024H/R045N/I107T/A172Q, A001R/S024N, A001R/S024R, A001R/S024R/A172H, A001R/S024R/A172Q, A001R/S024R/I107V/A172L, A001R/S024R/I107V/G127R, A001R/S024R/R045F/I107T/G127Q, A001R/S024R/R045F/I107T/G127R, A001R/S024R/R045K/I107T/G127Q, A001R/S024T/I107T/A172Q, A001R/S024T/R045K/A172Q, A001R/S024T/R045K/I107L/G127P/A172P, A001R/S024T/R045N/I107T/A172Q, A001R/S024W/I107V/A172S, A001R/S024W/R045F/I107V/G127Q, A001R/S024W/R045F/I107V/G127R, 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S087N/G118V/S128L/P129Q/S130A/R045N/A172Q, S087N/G118V/S128L/P129Q/S130A/R045N/I107V/A172P, S087N/G118V/S128L/P129Q/S130A/R045S, S087N/G118V/S128L/P129Q/S130A/S024H, S087N/G118V/S128L/P129Q/S130A/S024H/I107V/A172S, S087N/G118V/S128L/P129Q/S130A/S024H/Q109V, S087N/G118V/S128L/P129Q/S130A/S024H/Q109V/G127Q, S087N/G118V/S128L/P129Q/S130A/S024H/R045N/I107T, S087N/G118V/S128L/P129Q/S130A/S024H/S099K/S103N, S087N/G118V/S128L/P129Q/S130A/S024H/S099K/S103N/S132N, S087N/G118V/S128L/P129Q/S130A/S024H/S099Q, S087N/G118V/S128L/P129Q/S130A/S024H/S099Q/S103P, S087N/G118V/S128L/P129Q/S130A/S024H/S099Q/S132N, S087N/G118V/S128L/P129Q/S130A/S024H/S099T/S103P, S087N/G118V/S128L/P129Q/S130A/S024H/S099T/S103P/S132H, S087N/G118V/S128L/P129Q/S130A/S024H/S099T/S103P/S132N, S087N/G118V/S128L/P129Q/S130A/S024H/S099T/S132N, S087N/G118V/S128L/P129Q/S130A/S024H/S103P/S132N, S087N/G118V/S128L/P129Q/S130A/S024L/S099K/S103P/S132N, S087N/G118V/S128L/P129Q/S130A/S024N/I107N, S087N/G118V/S128L/P129Q/S130A/S024N/I107T, S087N/G118V/S128L/P129Q/S130A/S024N/I107V, S087N/G118V/S128L/P129Q/S130A/S024N/Q109K, S087N/G118V/S128L/P129Q/S130A/S024N/Q109T/G127T, S087N/G118V/S128L/P129Q/S130A/S024R, S087N/G118V/S128L/P129Q/S130A/S024R/G127R, S087N/G118V/S128L/P129Q/S130A/S024R/I107T/A172Q, S087N/G118V/S128L/P129Q/S130A/S024R/I107V/A172S, S087N/G118V/S128L/P129Q/S130A/S024R/Q109K, S087N/G118V/S128L/P129Q/S130A/S024R/Q109L, S087N/G118V/S128L/P129Q/S130A/S024R/Q109L/G127Q, S087N/G118V/S128L/P129Q/S130A/S024R/Q109V, S087N/G118V/S128L/P129Q/S130A/S024R/R045F/I107T, S087N/G118V/S128L/P129Q/S130A/S024T, S087N/G118V/S128L/P129Q/S130A/S024T/G127T, S087N/G118V/S128L/P129Q/S130A/S024T/I107T/A172Q, S087N/G118V/S128L/P129Q/S130A/S024T/Q109V/G127T, S087N/G118V/S128L/P129Q/S130A/S024T/R045N/I107V/A172S, S087N/G118V/S128L/P129Q/S130A/S024W, S087N/G118V/S128L/P129Q/S130A/S024W/G127T, S087N/G118V/S128L/P129Q/S130A/S024W/I107T, S087N/G118V/S128L/P129Q/S130A/S024W/Q109I, S087N/G118V/S128L/P129Q/S130A/S024W/Q109L, S087N/G118V/S128L/P129Q/S130A/S024W/Q109R/G127Q, S087N/G118V/S128L/P129Q/S130A/S024W/Q109T, S087N/G118V/S128L/P129Q/S130A/S024W/Q109V, S087N/G118V/S128L/P129Q/S130A/S024W/R045F/I107V/A172Q, S087N/G118V/S128L/P129Q/S130A/S024W/S099K, S087N/G118V/S128L/P129Q/S130A/S024W/S099K/S103P, S087N/G118V/S128L/P129Q/S130A/S024W/S099K/S132H, S087N/G118V/S128L/P129Q/S130A/S024W/S099Q, S087N/G118V/S128L/P129Q/S130A/S024W/S099Q/S103N/G127R/S132N, S087N/G118V/S128L/P129Q/S130A/S024W/S099Q/S103N/S132N, S087N/G118V/S128L/P129Q/S130A/S024W/S099Q/S103P, S087N/G118V/S128L/P129Q/S130A/S024W/S099Q/S132H, S087N/G118V/S128L/P129Q/S130A/S024W/S099Q/S132N, S087N/G118V/S128L/P129Q/S130A/S024W/S099T, S087N/G118V/S128L/P129Q/S130A/S024W/S099T/G127R/S132H, S087N/G118V/S128L/P129Q/S130A/S024W/S099T/S103N, S087N/G118V/S128L/P129Q/S130A/S024W/S099T/S103N/G127R/S132N, S087N/G118V/S128L/P129Q/S130A/S024W/S099T/S103N/S132N, S087N/G118V/S128L/P129Q/S130A/S024W/S099T/S103P/S132H, S087N/G118V/S128L/P129Q/S130A/S024W/S099T/S132N, S087N/G118V/S128L/P129Q/S130A/S099K, S087N/G118V/S128L/P129Q/S130A/S099K/S103N, S087N/G118V/S128L/P129Q/S130A/S099K/S103P, S087N/G118V/S128L/P129Q/S130A/S099K/S132H, S087N/G118V/S128L/P129Q/S130A/S099Q, S087N/G118V/S128L/P129Q/S130A/S099Q/S103N, S087N/G118V/S128L/P129Q/S130A/S099Q/S103P, S087N/G118V/S128L/P129Q/S130A/S099Q/S103P/S132H, S087N/G118V/S128L/P129Q/S130A/S099Q/S132H, S087N/G118V/S128L/P129Q/S130A/S099T/G127Q, S087N/G118V/S128L/P129Q/S130A/S099T/S103N, S087N/G118V/S128L/P129Q/S130A/S099T/S103N/S132H, S087N/G118V/S128L/P129Q/S130A/S099T/S103P, S087N/G118V/S128L/P129Q/S130A/S099T/S103P/S132H, S087N/G118V/S128L/P129Q/S130A/S099T/S132H, S087N/G118V/S128L/P129Q/S130A/S101E, S087N/G118V/S128L/P129Q/S130A/S101E/N248K, S087N/G118V/S128L/P129Q/S130A/S101F/N248K, S087N/G118V/S128L/P129Q/S130A/S101N/N248K, S087N/G118V/S128L/P129Q/S130A/S101R/N248K, S087N/G118V/S128L/P129Q/S130A/S101Y, S087N/G118V/S128L/P129Q/S130A/S103N/S132H, S087N/G118V/S128L/P129Q/S130A/S103P, S087N/G118V/S128L/P129Q/S130A/S144T/N185R, S087N/G118V/S128L/P129Q/S130A/S144T/N185R/S256I, S087N/G118V/S128L/P129Q/S130A/S144T/N185R/Y209W, S087N/G118V/S128L/P129Q/S130A/S144T/N185R/Y209W/S256I, S087N/G118V/S128L/P129Q/S130A/S144T/N185V/S256I, S087N/G118V/S128L/P129Q/S130A/S144T/N185V/Y209W, S087N/G118V/S128L/P129Q/S130A/S144T/S256I, S087N/G118V/S128L/P129Q/S130A/S144T/Y209W/S256I, S087N/G118V/S128L/P129Q/S130A/S144V, S087N/G118V/S128L/P129Q/S130A/S144V/N185R/S256I, S087N/G118V/S128L/P129Q/S130A/S144V/N185R/Y209W, S087N/G118V/S128L/P129Q/S130A/S144V/N185V, S087N/G118V/S128L/P129Q/S130A/S144V/N185V/S256I, S087N/G118V/S128L/P129Q/S130A/S144V/N185V/Y209W, S087N/G118V/S128L/P129Q/S130A/S144V/Y209W/S256I, S087N/G118V/S128L/P129Q/S130A/S144V/Y209W/S256T, S087N/G118V/S128L/P129Q/S130A/S144W/Y209W, S087N/G118V/S128L/P129Q/S130A/S144Y/N185R, S087N/G118V/S128L/P129Q/S130A/S144Y/N185R/Y209W/S256T, S087N/G118V/S128L/P129Q/S130A/S144Y/N185V/Y209W, S087N/G118V/S128L/P129Q/S130A/S164I, S087N/G118V/S128L/P129Q/S130A/S164Q, S087N/G118V/S128L/P129Q/S130A/V104I, S087N/G118V/S128L/P129Q/S130A/V104I/L126F/S164F, S087N/G118V/S128L/P129Q/S130A/V104I/L126F/S164I, S087N/G118V/S128L/P129Q/S130A/V104I/S164I, S087N/G118V/S128L/P129Q/S130A/V104I/S164Q, S087N/G118V/S128L/P129Q/S130A/Y209W, S087N/G118V/S128L/P129Q/S130A/Y209W/A215I/S256W, S087N/G118V/S128L/P129Q/S130A/Y209W/S256W, S099H/S103I/G118N, S099K/G118R, S099K/S103N/G118T, S099K/S103P/G118L/S132N, S099K/S103P/G127Q, S099K/S103P/S132H, S099K/S103R/G118A/S132P, S099N/S132R, S099Q/G118F/G127R, S099Q/G118T/G127Q, S099Q/S103N/G118R/S132N, S099Q/S103N/G118T, S099Q/S103N/G118T/S132H, S099Q/S103N/S132H, S099Q/S103P/G118R, S099Q/S103P/G118R/S132N, S099Q/S103P/S132H, S099Q/S132R, S099T/G118F, S099T/G118R/S132H, S099T/G118T/S132H, S099T/G118T/S132N, S099T/S103P, S099T/S103P/G118L/S132H, S101E/N117I, S101E/N117Y/N248K, S101F/N117I/N248I, S101F/N117Y/N248I, S101F/N117Y/N248K, S101F/N248K, S101R/N117I/N248K, S101R/N117Y, S101R/N248K, S101T/N117Y/N248K, S101T/N248I, S101V/N248K, S101Y/N117I/N248K, S101Y/N117Y, S101Y/N248K, S103G/G118I/G127E/S132E, S103L/S132E, S103R/S132P, S144T/N185R, S144T/N185R/Y209W/S256I, S144T/N185R/Y209W/S256T, S144T/N185V, S144V/N185R/S256I, S144V/N185R/Y209W, S144V/N185R/Y209W/S256T, S144V/S256I, S144V/Y209W, S144W/N185R/Y209W/S256T, S144W/N185V/Y209W/S256I, S144W/S256I, S144Y/Y209W/S256I, V104E/L126I/S164L。
ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される。
TS87N/G118V/S128L/P129Q/S130A/S24W/S101L/Q109K, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101L/Q109R/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/Q109R/S164I, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109R/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101Y/Q109L/N185R/A215R, S87R/G118R/S128L/P129Q/S130A/S101K/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/S24W/S101L/Q109L, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101Y/Q109R/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101L/Q109R/S164I, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109R/S188D/T213E/N248R, S87R/G118R/S128L/P129Q/S130A/I72V/S164I/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N76D, S87N/G118V/S128L/P129Q/S130A/S24W/S101Y/Q109K, S87N/G118V/S128L/P129Q/S130A/N43S/P52V/S101R/Q109R, S87N/G118V/S128L/P129Q/S130A/I72V/S101Y/Q109R/S164I, S87N/G118V/S128L/P129Q/S130A/S101L/Q109L/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/I72V/Q109R/S164I/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N76D/S101L, S87N/G118V/S128L/P129Q/S130A/S24W/S101Y/Q109L, S87N/G118V/S128L/P129Q/S130A/N76D/S164I, S87R/G118R/S128L/P129Q/S130A/N76D/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109L/S188D/N248R, S87R/G118R/S128L/P129Q/S130A/V118R/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N76D/S101M, S87N/G118V/S128L/P129Q/S130A/N18K/N76D/G97P/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101L/S164I, S87R/G118R/S128L/P129Q/S130A/S101L/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109L/S188D/T213E/N248R, S87R/G118R/S128L/P129Q/S130A/Q109L/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101L/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101Y/S164I, S87N/G118V/S128L/P129Q/S130A/S101Y/T213E/N248R, S87N/G118V/S128L/P129Q/S130A/I72V/S101L/Q109L/S164I, S87R/G118R/S128L/P129Q/S130A/S101V/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101Y/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101V/S164I, S87N/G118V/S128L/P129Q/S130A/S101L/T213E/N248R, S87N/G118V/S128L/P129Q/S130A/I72V/S101Y/Q109L/S164I, S87R/G118R/S128L/P129Q/S130A/S101H/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/Q109R/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101H/S164I, S87N/G118V/S128L/P129Q/S130A/S101L/Q109R/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101L/Q109L/N185R/A215R, S87R/G118R/S128L/P129Q/S130A/S101M/S188D/N248R, N76D/S87R/G118R/S128L/P129Q/S130A/S188D, N76D/S87R/G118R/S128L/P129Q/S130A/S188D/N248K, S87N/G118V/G127S/S128L/P129Q/S130A, S87N/S101H/G118V/S128L/P129Q/S130A, S87N/S101K/G118V/S128L/P129Q/S130A, S87N/S101V/G118V/S128L/P129Q/S130A, S87N/S101Y/G118V/S128L/P129Q/S130A, S87N/S101L/G118V/S128L/P129Q/S130A, I72V/S87N/G118V/S128L/P129Q/S130A//S164I。
ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される。
ΔΔ G = -RT ln (kvariant/kreference )
ここで、kvariantは変異体酵素の速度定数、kreferenceは参照酵素の速度定数、Rは気体定数で、Tは絶対温度である。大部分の分析は真の自由エネルギを測定できるように計画されていないため、本願では次の量を用いた;
ΔΔ G app = -RT ln ( Pvariant/Preference )
ここで、Pvariantは変異体の性能値、Preferenceは同一条件下での参照酵素の性能値である。ΔΔ G app値は、データ分散及び相加性において、ΔΔ Gと同様の挙動を示すと考えられるであろう。しかし、ΔΔ Gは参照酵素との比較において変異体が行える最大の仕事量であるため、ΔΔ G app量は通常ΔΔ Gを過小評価していることになるため、2つの追加的位置の性質の相乗効果は、それらのΔΔ G app値を足し合わせた値から予想される値寄りも大きく現れる結果となる。
特に断りのない限り、この発明を実施する際、分子生物学、タンパク質工学、微生物学、及び組換えDNAの分野において当業者が一般に用いている公知技術が用いられる。このような技術は当業者にとって公知であり、当業者によく知られた多数の教科書及び総説に開示されている。本願に引用する特許、特許出願、記事、及び刊行物はすべて参照として本願に組み込まれる。
同伴する領域の同定法は、当業者に良く知られている(例えば、Dynan and Tijan, Nature 316:774-78 [1985]参照)。「単離されたDNA配列」を、「クローンされたDNA配列」と言うこともある。
を調節することができる。
(a)少なくとも1の要素又は置換基を失って不飽和になること;又は
(b)その化合物又は置換基中の少なくとも1の水素が(i)炭素(ii)酸素(iii)硫黄(iv)窒素又は(v)ハロゲン原子の1以上を含む基で置換されること;又は
(c)(a)及び(b)の両者。
上記(b)に記載した水素を置換する置換基であって、炭素と水素原子のみを含む置換基は炭化水素置換基であり、例えばアルキル、アルケニル、アルキニル、アルキルジエニル、シクロアルキル、フェニル、アルキルフェニル、ナフチル、アントリル、フェナントリル、フルオリル、ステロイド基、及びこれらの基の相互の組み合わせ及びアルキレン、アルキリデン、アルキリジン基のような多価の炭化水素基との組み合わせが含まれるが、これらに限定されない。上記(b)に記載した水素を置換する置換基であって、酸素原子を含む置換基にはヒドロキシル、アシル、又はケト、エーテル、エポキシ、カルボキシル、及びエステル基が含まれるが、これらに限定されない。上記(b)に記載した水素を置換する置換基であって、硫黄原子を含む置換基には硫黄含有酸及び酸エステル基、チオエーテル基、メルカプト基及びチオケト基が含まれるが、これらに限定されない。上記(b)に記載した水素を置換する置換基であって、窒素原子を含む置換基には、アミノ基、ニトロ基、アゾ基、アンモニウム基、アミド基、アジド基、イソシアネート基、シアノ基、及びニトリル基が含まれるが、これらに限定されない。上記(b)に記載した水素を置換する置換基であって、ハロゲン原子を含む置換基には塩素、臭素、フッ素、ヨウ素基、及びペンダントアルキル基又は水素がハロ基によって置換され、安定した置換部位を形成し得るいずれかの置換基が含まれる。
特に断りのない限り、本明細書に記載の成分または組成物のレベルはすべて、その成分または組成物の活性レベルに関するものであり、不純物、例えば、市販の商品に存在する残留溶媒または副生物を除いたものである。酵素成分の重量は全活性タンパク質に基づく。全てのパーセント及び比率は、特に断りのない限り重量に基づき計算される。全てのパーセント及び比率は、特に示さない限り全組成物に基づき計算される。例示した洗剤組成物においては、酵素レベルは全組成物の重量に対する純粋な酵素により表わし、特に断りのない限り、洗剤成分の量は全組成物の重量により表わす。
このような洗浄組成物には通常、原液pHが約3から約5となるように、水酸化ナトリウム、モノエタノールアミンまたは塩酸のような十分な量のpH調整剤が含有されている。この洗浄組成物には通常、1以上の酸性下で安定な酵素が含有されている。いくつかの実施形態では組成物は液体であり、別の実施形態では固体である。液体組成物のpHは原液pHとして測定する。固体組成物のpHは、溶媒に蒸留水を用いて組成物の10%固体溶液として測定する。これらの実施形態では、特に断りのない限り、全てのpH測定は20℃で行う。
<分析>
以下の実施例では、理解を容易にするために下記に示した様々な分析を用いた。下記に示した手順を変更したときは、その旨記を記載した。
BPN’(例えば参照プロテアーゼ)及びその変異体について、加湿エアレーション下で、230 rpmで振蕩しながら33℃で3-4日間培養したマイクロタイタープレートからろ過して調製した培養上清液を用いて、TCA分析を開始した。この分析には、新品の96-ウエル平底マイクロタイタープレートを用いた。最初に100μL/ウエルの0.25 N HCIをウエルに入れた。次に、50μLのろ過された培養ブロスをウエルに加えた。「ブランク」の測定地を求めるために、405nm(プレートリーダーの5秒のミキシングモードを使用)での光散乱/吸光度を測定した。測定では、15% (w/v) トリクロロ酢酸 (TCA)を100μL /ウエルでマイクロタイタープレートに加え、室温で5から30分間インキュベートした。次に、405nm(プレートリーダーの5秒のミキシングモードを使用)での光散乱/吸光度を測定した。
プロテアーゼ、及びこの発明のプロテアーゼ変異体のプロテアーゼ活性を評価するために、N-サクシニル-L-アラニル-L-アラニル-L-プロリル-L-フェニル-p-ニトロアニリド (suc-AAPF-pNA) の加水分解を測定した。使用した試薬溶液は:100 mM Tris/HCl、pH 8.6、0.005% TWEENTM-80 (Tris 希釈緩衝液)含有;100 mM Tris 緩衝液、pH 8.6、10 mM CaCl2及び 0.005% TWEENTM-80 (Tris/Ca 緩衝液)含有; 及び160 mM suc-AAPF-pNAのDMSO溶液 (suc-AAPF-pNAストック溶液) (Sigma: S-7388)であった。suc-AAPF-pNAワーキμング溶液を調製するため、1 ml のsuc-AAPF-pNA溶液を100 mlのTris/Ca緩衝液に加え、少なくとも10秒間、よく撹拌した。分析は、希釈したプロテアーゼ溶液10 μlを各ウエルに加え、直ちに1 mg/ml suc-AAPF-pNAワーキング溶液190μLを加えることにより行った。溶液を5秒間撹拌し、410 nmにおいてキネティック・モード(5分間に20回測定)での吸光度変化を、25℃のMTPリーダー中で測定した。プロテアーゼ活性はAU (活性 = ΔOD・min-1 ml-1)で表した。
プロセス変異体を68℃で1時間インキュベートした後、AAPF分析を用いてプロテアーゼ活性を分析することにより、セリンプロテアーゼの熱安定性を評価した。この分析条件下では、参照プロテアーゼ(例えば野生型 GG36 = GCI-P036)の残留活性は約50%であった。使用した装置は:平底MTP (Costar No. 9017)、Biomek FX 及び/又は Biomek FXp Robot (Beckman Coulter社)、Spectramax Plus 384 MTP リーダー (Molecular Devices社)、iEMS インキュベーター/シェーカー (振幅1 mm) (Thermo/Labsystems社), シーリングテープ (Nunc No. 236366)、及び氷浴であった。グリシン緩衝液を、960mlの水にグリシン(Merck No. 1.04201.1000) 3.75gを溶解させて調製した。このグリシン緩衝液に、1 mlの5% Tween 80 (Sigma No. P-8074)と、10 ml の1000 mM CaCl2 (Merck No. 1.02382.1000) (200 ml 中に29.4 g 溶解) ストック溶液とを加えた。4N NaOHでpHを10.5に調整し、容積を1000mlにした。グリシン、CaCl2及びTWEENTM-80の最終濃度は、それぞれ50 mM, 10 mM 及び 0.005%であった。複数のインキュベーターを、68℃(インキュベート用)及び25℃(AAPF分析用)に設定した。90μL と 190μLのグリシン緩衝液を、それぞれ空の希釈プレートとインキュベーションプレートに入れた。次に上清10μLを希釈プレートに加えた後、希釈プレートから採取した10μLをインキュベーションプレートに移した。インキュベーションプレートから採取した混合物10μLを、予熱しsuc-AAPF-pNA基質を入れたプレートに加えた。このsuc-AAPF-pNAプレートを、410 nm (t = 0測定)においてMTPリーダーで測定した。インキュベーションプレートをテープで覆い、68℃、400 rpmで1時間インキュベートした。インキュベート完了後、プレートをインキュベーターから取り出し、氷上で少なくとも5分間冷却した。インキュベーションプレートから採取した混合物10μLをsuc-AAPF-pNA基質を入れたプレートに移し、このプレートを410 nm (t=60 測定) において測定した。パーセント残留活性を:%残留活性: (t=60におけるmOD.min-1) / (t=0におけるmOD.min-1) x 100として計算した。
LAS/EDTA安定性は、被検プロテアーゼをLAS/EDTAそれぞれの存在下でインキュベートした後、AAPF分析により求めた残留活性の関数として評価した。
プロテアーゼ変異体の汚れ除去性能を、市販の洗剤を用いて評価した。市販の洗剤処方品の熱不活性化により、タンパク質成分の酵素活性が破壊され、一方、非酵素成分の性質は保持された。従ってこの方法は、この発明の酵素変異体の試験に用いるための市販の洗剤の調整に適している。
直径1/4インチの円形の小見本片をCFT (Vlaardingen, The Netherlands)から購入した。シングル小見本片又は2つの小見本片を、96ウエルMTPの各ウエルに、表面積全体が露出するようにして置いた(すなわち、ウエルの底に広げたのではない)。
直径0.25インチで、血液、牛乳及びインク(BMI)を含有する小見本片をCFT Vlaardingenから入手した。小見本片を切断する前に、織物(EMPA 116)を水で洗浄した。表面全体を暴露するために、ひとつの小見本片を96-ウエルのマイクロタイタープレートの各ウエルに垂直になるように置いた(すなわち、ウエルの底に広げたのではない)。所望の洗剤液を本願に開示する方法で調製した。Thermomixerを25℃の一定温度にした後、小見本片を入れたMTPの各ウエルに洗剤液190 μlを添加した。この混合液に希釈酵素溶液10 μlを加え、最終酵素濃度を1 μg/ml(BCA分析で測定)にした。MTPをテープでシールし、インキュベーターに取り付けて1400rpmで30分間攪拌した。適切な条件下でインキュベートした後、各ウエルから100μlの溶液を採取し、新しいMTPへ移した。各ウエルに溶液を100μl入れた新しいMTPを、MTP SpectraMax readerリーダーを用いて405nmにおいて測定した。ブランクコントロール、2つの小見本片からなるコントロール、及び酵素を含有しない洗剤も含め、評価した。
ニワトリ卵黄を用いて、96-ウエル加熱卵黄基質プレートを調製した。ニワトリ卵黄を卵白から分離し、卵黄膜を除き、Milli-Q水で20%(体積/重量)に希釈した。希釈した卵黄を、マグネティックスターラーを用いて室温で15分間攪拌した。8チャンネルピペットを用いて、5μlを注意深く96穴V底型プレート(Costar #3894)の各ウエルの中央に滴下した。プレートを90℃で1時間加熱し、室温で冷却した。加熱卵黄基質プレートを室温で保存し、調製後1週間以内に使用した。自動食器洗い機用洗剤を本明細書に記載の方法で調製し、50℃まで予熱した。8チャンネルピペット装置を用いて、洗剤液190μlを96-ウエルプレートの各ウエルに添加した。希釈した酵素10μlを、96チャンネルピペットを用いて各ウエルに添加した。このプレートを注意深く接着性ホイルシーラーで覆い、50℃で攪拌しながら30分間インキュベートした。この反応混合液120μlを新しい96-ウエル平底プレートに移し、吸光度/光散乱を405nmで測定した。405nmでの吸光度/光散乱は卵黄除去に比例する。
食器洗浄機用洗剤を、本願に開示した方法で調製した。用いた装置はNew Brunswick Innova 4230 シェーカー/インキュベーター及びSSpectraMAX (type 340) MTPリーダーであった。Costar 社製(type 9017)のMTPを用いた。Center for Test Materials (Vlaardingen, Netherlands)から入手した染料含有熟成卵黄見本(CSp-8)を用いた。 布地を水で洗浄した後、0.25インチの円形の小見本片を切り出した。を96穴マイクロタイタープレートの各ウエルに小見本片を1つ置いた。試験洗剤を50℃にした。小見本片を入れたMTPの各ウエルに、洗剤液190μlを添加した。この液に希釈酵素溶液10μlを加えた。MTPを粘着ホイルでシールし、インキュベーターで30分間攪拌した。インキュベーションに続いて、各ウエルから溶液100μlを採取し、新しいMTPへ移した。このMTPをSpectraMax MTPリーダーを用いて405nmで測定した。ブランクコントロール、及び小見本片及び洗剤を含有するが酵素は含有していないコントロールも測定した。
このタイプの小見本片は、室温において脱イオン水で20分間予備洗浄して調製した。予備洗浄ステップの後、見本片をペーパータオル上で乾燥させた。風乾した見本変を、打ち抜きパンチに取り付けた1/4インチ円形ダイで打ち抜いた。最後に、2つの小見本片を、96-ウエルMTPの各ウエルに、表面積全体が露出するようにして置いた(すなわち、ウエルの底に広げたのではない)。
北米(NA)及び西ヨーロッパ(WE)強力液体洗剤(HDL) について、予め秤量した(ガラス瓶に入れた)液体洗剤を95℃の水浴に2時間入れて熱不活性化した。実施例で用いた洗剤は、各地のスーパーマーケットで購入した市販の洗剤であった。上記のように、これらの洗剤に含まれていた酵素を加熱処理により不活性化した。北米(NA)及び日本(JPN)の顆粒状強力洗濯(HDG)洗剤の熱不活性化時間は8時間で、西ヨーロッパ(WE)のHDG洗剤の熱不活性化時間は5時間であった。NA及びWEの食器洗浄機 (ADW) 用洗剤を熱不活性化するときの加熱時間は8時間であった。非加熱及び加熱処理洗剤の両者について、不活性化のパーセンテージを性格に測定するために、洗剤を溶解させた後5分以内に評価した。酵素活性はAAPF分析により測定した。
GCI-P036のサンプル、及びその変異体のサンプルは、MTPプレートで培養した培養ブロスを濾過して調製した。使用した装置は、Biomek FX Robot (Beckman Coulter社)、SpectraMAX MTPリーダー (type 340; Molecular Devices社)、iEMSインキュベータ/シェーカー (Thermo/Labsystems社); 平底MTP (Costar社 type 9017、インキュベーション後の反応プレートの測定に使用); 及びV-底MTP (Greiner社651101、上清の予備希釈に使用) であった。この分析では、プロテアーゼが基質を加水分解し、基質から色素と不溶性部粒子が放出される。従って、濁度の増加速度が酵素活性の指標となる。
測定された吸光値をブランク値(酵素を含有しない基質)について修正し、加水分解活性を評価した。各サンプル(変異体)について性能指数を計算した。性能指数は、同一のタンパク質濃度における変異体の性能(実際の値)と、標準酵素(理論値)との比較である。さらに理論値は、標準酵素のラングミュア式のパラメータを使って計算できる。
プロテアーゼ変異体を識別するために、野生型又は標準プロテアーゼに対するプロテアーゼ変異体の対比及び順位付けを可能にするsuc-AAPF-pNAを基質として用いて、相対的比活性度を計算した。suc-AAPF-pNA基質に対する比活性度は、上記の分析を用いてタンパク質分解活性を、各サンプルのTCA-測定値で割り算することにより求めた。これらの値を用いて、相対的比活性度を求めた(変異体の比活性度/参照プロテアーゼの比活性度)。
本願に開示するように、セリンプロテアーゼの濃度及び比活性度を、阻害剤の滴定により求めた。ヒルのヒルド メディシナリス(Hirudo medicinalis)由来のエグリンCは、 スブチリシンに強く結合するタンパク質阻害剤である。 (Heinz 他、Biochemistry, 31(37): 8755-66 [1992])。従って、エグリンCを酵素濃度測定に用いることができ、同様に、特異的酵素活性を計算することができる。簡潔に言えば、いくつかの既知濃度のエグリンCにより生じる酵素阻害の量を測定する。この情報から、完全な阻害に必要とされるエグリンCの濃度を算出することができる。この値はサンプルにおける酵素濃度と同等である。
性能指数は、同一のタンパク質濃度における変異体の性能(実際の値)と、標準または参照プロテアーゼ(理論値)との対比である。また、理論値は、標準プロテアーゼのラングミュア式のパラメータを使って計算することができる。性能指数(PI)が1より大きい場合( PI>1)は、標準(例えば、野生型)と比較してより優れた変異体であるものとし、他方、PIが1である場合( PI=1)は、変異体は標準と同じように機能するものとし、及びPIが1より小さい場合( PI<1)は、変異体は標準よりも機能が低いものとする。従って、PIにより勝者と、一定の環境の下での使用に関してあまり望ましくない変異体とが特定される。
<B.スブチリス中でのGCI-P036プロテアーゼの産出>
この実施例では、B.スブチリス中でGCI-P036プロテアーゼを産出させるために行った実験について説明する。以前開示した方法(例えばWO 2002/014490参照)により、GCI-P036及びその変異体をコードする発現ベクターを、B.スブチリスの細胞中 (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::[xylR,pxylA-comK]) に形質転換した。
B.スブチリス中でGCI-P036 (本願では「バシラス・レンタス・スブチリシン」及び「GG36」とも呼ぶ)を産出する方法を例示する。発現プラスミドpAC-GG36ciを、コンセンサスaprEプロモータの制御下にある aprEシグナル配列の8番目のコドンに融合したGCI-P036コドン改善遺伝子、及び BPN’ 転写ターミネータを用いて調製した。いかに示す配列では、太字及びイタリック体はコンセンサスaprEプロモータを表し、標準フォントはシグナル配列を表し、下線部のフォントはプロ配列を表し、太字のフォントはGCI-P036成熟プロテアーゼをコードするDNAを表し、下線部のイタリック体フォントはBPN’ ターミネータを表す。KpnI 及びクローン化のためのXhoI制限酵素認識部位が、GCI-P036成熟プロテアーゼのコード領域に隣接する:
B.スブチリス中でGCI-P036参照プロテアーゼを産出する別の方法について説明する。GCI-P036プロテアーゼ前駆体をコードする合成遺伝子を、合成オリゴヌクレオチド及びPCR産品から調製した。このフラグメントを、BsmBI 及び HindIII制限酵素認識部位を用いてプラスミド骨格pHPLT (U.S. Patent No. 5,024,943) 中にクローン化した。pHPLT-GCI-P036の成熟GCI-P036プロテアーゼ(SEQ ID NO:2)のアミノ酸配列は、pAC-GG36ciと同じである。pHPLT B.スブチリス発現ベクターにはthe B. リケニフォルミス LAT プロモータ(Plat)が含まれ、さらにレプリカーゼ遺伝子(reppUB)、ネオマイシン/カナマイシ耐性遺伝子(neo) 、及びブレオマイシン耐性マーカ(bleo)を備える、pUB110 (McKenzie et al., Plasmid, 15:93-103, 1986)由来の別の複数のエレメントが含まれる。Pure YieldTM Plasmid Midiprep kit (Promega社)を用いて、形質転換されたバクテリアからプラスミドDNAを精製し、公知のUV分光分析法によりDNA濃度を求めた。得られたベクター構築体をGCI-P036 遺伝子のDNA 配列により検証したところ、予想される配列に対し100%の配列同一性が認められた。pHPLT-GCI-P036のGCI-P036 プロテアーゼ遺伝子のDNA配列を以下に示し、クローン化部位SacI及びHindIIIに下線を付して示す:
プロテアーゼ発現ベクターを備えるB. スブチリスのクローンを、鉄製の96-ウエルのレプリケータを用い、グリセロールのストック溶液から、200 μl の LB培地 + 25 μg/ml クロラムフェニコールを入れた96-ウエルの培養プレート(BD, 353075)へ移し、密閉湿潤条件下、37℃、220rpmにおいて一夜培養した。一夜培養液からサンプル200μlを採取し、5mlのプラスチック培養チューブ中の2000μl合成培地+ 25μg/mlクロラムフェニコールに植菌した。培養培地はMOPS緩衝液に基づき、主要な窒素源としての尿素、主要な酸素源としての炭素、及び補助成分として細胞を健全に成長させるための1%ソイトンを含む半合成富栄養化培地であった。培養チューブは、37℃、220rpmにおいて60時間インキュベートした。インキュベーション後、培養ブロスを8000 x RCF以上で遠心分離した。上清液を15mlのポリプロピレン製コニカルチューブに静かに移して貯蔵した。これ以上の精製又は濃縮は行わなかった。上清ストック溶液を長期間安定化させるために、最終濃度40%プロピレングリコールとなるように処方し、4℃で保管した。
また、をMOPSに基づく合成培地(MBD)を200μL入れた96ウエルマイクロタイタープレート中でB.スブチリス形質転換体を培養することによりプロテアーゼ変異体を調製した。MBD培地は、基本の培地からNH4Cl2, FeSO4, 及び CaCl2 を除去し、3 mM K2HPO4 を用い、基本の培地に60 mM 尿素, 75 g/L グルコース、及び1% ソイトンを添加した点を除き、公知の方法(Neidhardt et al., J Bacteriol, 119: 736-747, 1974参照)により調製した。また微量栄養素を、100Xストック溶液中の1リットル中に400 mg FeSO4 .7H2O, 100 mg MnSO4 .H2O, 100 mg ZnSO4.7H2O, 50 mg CuCl2.2H2O, 100 mg CoCl2.6H2O, 100 mg NaMoO4.2H2O, 100 mg Na2B4O7.10H2O, 10 ml の 1M CaCl2,及び 10 ml の 0.5 Mクエン酸ナトリウム含まれるように添加した。プレートを37℃で68時間インキュベートし、細胞を遠心分離により分離し、培養培地から対象とするタンパク質を得た。
<GCI-P036変異体の生成>
この実施例に、プロテアーゼ変異体を生成させる方法を開示する。
SELの構築は、独自の方法(WO 2004/059556A3)を用いるGENEARTにより行った。PCRによりタンパク質を発現させるためにヌクレオチド配列を最適化するための方法及び装置、及びDNA分子の製造には、GENEART (European Patent No. 0 200 362 及び 0 201 184; 及び US Patent No. 4,683,195, 4,683,202 及び6,472,184)により所有されているか、又はライセンスされている技術を用いた。この実施例に開示するGCI-P036 SELの構築は、GENEARTの遺伝子最適化、遺伝子合成、及びライブラリー構築及び分析技術及び方法を用いて行った。
予備PCR 1:
FW SacI プライマー、及び特異的 GCI-P036リバース突然変異誘発プライマー − いずれも1 μL (10 μM);及び
予備PCR 2:
RV HindIIIプライマー、及び特異的GCI-P036フォワード突然変異誘発プライマー − いずれも1 μL (10 μM)。
この実施例で用いたプライマー配列を下記に示す。
各合成GCI-P036多重突然変異ライブラリーには、2以上の選択されたコドンが特定のDNA配列で置き換えられた、GCI-P036遺伝子の混合物が含まれている。組み合わせGCI-P036遺伝子のクローン化は、Sloning BioTechnology GmbH社によりSlonomax Technologyを用いて実施された。次のTables 3-1 及び 3-2に、MMLのメンバー中に存在する置換のリストを示す。これらの表中の番号はそれぞれ、GCI-P036参照配列及びBPN’ 参照配列に対応して賦与されている。
<GCI-P036の性能>
種々の単一置換GCI-P036変異体の汚れ除去性能を評価するために、BMI分析(TIDE 2XTM 低温水; 32℃, pH8) 及び CS-38小見本片分析 (CALGONITTM ; WE ADW)により行った実験を説明する。また、LAS/EDTA安定性、AAPF活性の性能指数、及びタンパク質含有量も測定した。全ての分析は実施例1の方法を用いて行った。
<GCI-P036変異体のデータの比較評価>
この実施例では、GCI-P036及びGCI-PO36変異体のタンパク質発現、汚れ除去活性、LAS/EDTA安定性、及びAAPF活性 (対象とする性質の試験)について評価した実験結果を対比する。本願に開示するように、GCI-P036変異体の機能を、変異体及び参照プロテアーゼの性能の比である性能指数(PI)として定量した。本願でのPIの分類は:上昇突然変異(PI>1.0);中立突然変異(PI>0.5);非有害突然変異(PI>0.05);及び有害突然変異(PI≦0.05)である。
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X108S, X108T, X108V, X109A, X109C, X109E, X109F, X109G, X109H, X109I, X109K, X109L, X109M, X109N, X109P, X109Q, X109R, X109S, X109T, X109W, X109Y, X110A, X110G, X110S, X111C, X111E, X111F, X111I, X111L, X111M, X111V, X111Y, X112A,
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X224E, X224G, X224I, X224L, X224N, X224P, X224S, X224T, X225A, X225C, X225G, X225N, X225P, X225S, X225T, X225V, X226C, X226F, X226G, X226H, X226I, X226L, X226M, X226N, X226R, X226S, X226T, X226V, X226Y, X227A, X227C, X227F, X227G, X227I, X227L, X227M, X227Q, X227S, X227T, X227V, X227Y, X228A, X228C, X228G, X228I, X228L, X228M, X228S, X228V, X229A, X229G, X229P, X229S, X230A, X230D, X230E, X230F, X230G, X230H, X230I, X230L, X230N, X230P, X230Q, X230S, X230T, X230V, X230W, X230Y, X231A, X231C, X231F, X231G, X231H, X231I, X231L, X231S, X231T, X231W, X231Y, X232A, X232G, X232H, X232L, X232M, X232S, X232V, X233A, X233C, X233E, X233F, X233G, X233I, X233L, X233M, X233N, X233P, X233Q, X233R, X233S, X233T, X233V, X233Y, X234D, X234F, X234G, X234H, X234L, X234M, X234N, X234P, X234Q, X234S, X234T, X234V, X234Y, X235C, X235D, X235E, X235F, X235G, X235H, X235I, X235K, X235L, X235M, X235N, X235Q, X235R, X235S, X235V, X235W, X235Y, X236A, X236C, X236E, X236F, X236G, X236H, X236K, X236L, X236N, X236P, X236Q, X236R, X236S, X236T, X236V, X236W, X236Y, X237A, X237C, X237F, X237G, X237H, X237I, X237K, X237L, X237M, X237P, X237Q, X237R, X237S, X237T, X237V, X237W, X237Y, X238C, X238D, X238E, X238F, X238G, X238H, X238I, X238K, X238L,
X238M, X238N, X238Q, X238R, X238S, X238T, X238V, X238Y, X239C, X239D, X239F, X239G, X239H, X239I, X239K, X239L, X239M, X239N, X239P, X239Q, X239R, X239S, X239T, X239V, X239W, X239Y, X240A, X240C, X240E, X240F, X240I, X240K, X240L, X240M, X240N, X240Q, X240R, X240S, X240T, X240W, X240Y, X241A, X241C, X241D, X241E, X241F, X241G, X241H, X241I, X241K, X241L, X241M, X241N, X241P, X241Q, X241R, X241S, X241T, X241V, X241W, X241Y, X242A, X242C, X242D, X242F, X242G, X242H, X242I, X242L, X242M, X242P, X242Q, X242R, X242S, X242T, X242V, X242W, X243C, X243D, X243E, X243F, X243G, X243H, X243I, X243K, X243L, X243M, X243N, X243P, X243Q, X243R, X243S, X243T, X243V, X243W, X244A, X244D, X244E, X244F, X244H, X244I, X244L, X244M, X244N, X244P, X244Q, X244R, X244S, X244T, X244V, X244W, X244Y, X245A, X245C, X245E, X245G, X245H, X245K, X245L, X245P, X245Q, X245R, X245S, X245V, X245W, X245Y, X246A, X246C, X246E, X246F, X246G, X246H, X246I, X246L, X246M, X246N, X246P, X246Q, X246R, X246S, X246T, X246V, X246W, X246Y, X247A, X247C, X247D, X247E, X247F, X247G, X247H, X247I, X247K, X247L, X247M, X247N, X247P, X247Q, X247R, X247S, X247T, X247V, X247W, X247Y, X248C, X248D, X248E, X248G, X248H, X248I, X248K, X248L, X248M, X248N, X248P, X248R, X248S, X248T, X248V, X248W, X248Y, X249A, X249D, X249E, X249F, X249G, X249H, X249I, X249K, X249L, X249M, X249N, X249Q, X249R, X249S, X249T, X249V, X249W, X249Y, X250A, X250C, X250E, X250F, X250G, X250H, X250I, X250L, X250M, X250N, X250Q, X250S, X250V, X250Y, X251A, X251D, X251E, X251F, X251G, X251K, X251L, X251M, X251P, X251Q, X251R, X251S, X251T, X251V, X251Y, X252A, X252C, X252D, X252F, X252G, X252H, X252I, X252K, X252L, X252M, X252N, X252P, X252R, X252S, X252V, X252W, X252Y, X253A, X253D, X253E, X253F, X253G, X253H, X253I, X253K, X253M, X253R, X253S, X253T, X253V, X253W, X254A, X254C, X254D, X254G, X254N, X254P, X254S, X254T, X254V, X255A, X255C, X255D, X255E, X255F, X255H, X255I, X255L, X255N, X255P, X255Q, X255R, X255S, X255T, X255V, X255W, X255Y, X256A, X256C, X256D, X256E, X256G, X256H, X256I, X256K, X256L, X256M, X256N, X256P, X256R, X256S, X256T, X256V, X256W, X256Y, X257A, X257C, X257E, X257F, X257G, X257H, X257I, X257K, X257L, X257M, X257P, X257S, X257T, X257V, X257W, X257Y, X258A, X258C, X258D, X258E, X258F, X258G, X258H, X258I, X258L, X258M, X258P, X258Q, X258R, X258S, X258T, X258V, X258W, X258Y, X259A, X259C, X259E, X259G, X259I, X259L, X259M, X259P, X259Q, X259R, X259S, X259T, X259V, X260A, X260D, X260E, X260F, X260H, X260I, X260L, X260M, X260N, X260P, X260R, X260S, X260T, X260V, X260Y, X261A, X261C, X261E, X261F, X261G, X261I, X261K, X261L, X261N, X261P, X261Q, X261R, X261S, X261T, X261V, X261W, X261Y, X262A, X262C, X262D, X262F, X262G, X262H, X262I, X262K, X262L, X262M, X262P, X262Q, X262R, X262S, X262T, X262V, X262W, X262Y, X263A, X263C, X263D, X263F, X263G, X263H, X263I, X263L, X263M, X263N, X263P, X263Q, X263R, X263W, X263Y, X264A, X264C, X264E, X264F, X264G, X264H, X264I, X264P, X264Q, X264S, X264V, X264Y, X265A, X265C, X265D, X265F, X265G, X265H, X265I, X265K, X265L, X265M, X265P, X265Q, X265R, X265S, X265T, X265V, X265W, X265Y, X266G, X266W, X267A, X267C, X267D, X267E, X267F, X267G, X267H, X267I, X267K, X267L, X267M, X267N, X267S, X267T, X267V, X267Y, X268A, X268D, X268E, X268G, X268H, X268K, X268L, X268M, X268N, X268P, X268Q, X268R, X268S, X268V, X268W, X268Y, X269C, X269D, X269F, X269G, X269H, X269I, X269L, X269M, X269N, X269Q, X269R, X269S, X269T, X269V, X270A, X270C, X270D, X270E, X270F, X270G, X270H, X270I, X270L, X270M, X270N, X270P, X270Q, X270S, X270T, X270V, X271A, X271C, X271E, X271F, X271G, X271H, X271I, X271K, X271L, X271M, X271N, X271P, X271T, X271V, X271Y, X272A, X272C, X272D, X272E, X272F, X272G, X272H, X272K, X272L, X272M, X272N, X272P, X272R, X272S, X272T, X272W, X272Y, X273A, X273C, X273D, X273E, X273F, X273G, X273H, X273I, X273K, X273L, X273R, X273S, X273T, X273V, X273W, X273Y, X274A, X274C, X274D, X274E, X274G, X274H, X274K, X274L, X274M, X274N, X274P, X274Q, X274R, X274S, X274T, X274W, X275A, X275C, X275D, X275E, X275F, X275G, X275H, X275K, X275L, X275M, X275P, X275Q, X275R, X275V, X275W, X275W。
一方、位置64, 65 及び 70の最初のスクリーニングでは、少なくとも1の性質について性能指数が1.0より大きくなる(PI>1.0)突然変異は認められなかった。
X139M, X139T, X139V, X140A, X140C, X140D, X140E, X140F, X140G, X140I, X140L, X140M, X140N, X140Q, X140R, X140S, X140T, X140V, X140Y, X141D, X141E, X141G, X141H, X141I, X141K, X141L, X141N, X141Q, X141R, X141S, X141V, X141Y, X142A, X142C, X142L, X142T, X142V, X142Y, X143C, X143D, X143F, X143G, X143H, X143I, X143K, X143L, X143M, X143N, X143S, X143T, X143V, X143W, X143Y, X144A, X144C, X144D, X144G, X144H, X144I, X144L, X144M, X144N, X144Q, X144R, X144S, X144T, X144V, X144W, X144Y, X145A, X145C, X145D, X145E, X145F, X145G, X145K, X145L, X145M, X145N, X145Q, X145R, X145S, X145T, X145W, X145Y, X146A, X146C, X146D, X146E, X146G, X146K, X146Q, X147I, X147L, X147M, X147T, X147V, X148A, X148C, X148E, X148F, X148H, X148I, X148L, X148M, X148N, X148S, X148T, X148V, X148Y, X149A, X149C, X149I, X149L, X149M, X149P, X149S, X149T, X149V, X150A, X150F, X150L, X150T, X150V, X151A, X151G, X151S, X152A, X152S, X153A, X153G, X153S, X153V, X156A, X156C, X156D, X156E, X156F, X156L, X156M, X156N, X156S, X156T, X158A, X158C, X158E, X158F, X158H, X158K, X158L, X158M, X158Q, X158R, X158S, X158T, X158V, X158W, X159A, X159C, X159E, X159G, X159H, X159M, X159P, X159Q, X159R, X159S, X159W, X160A, X160C, X160D, X160F, X160G, X160I, X160L, X160M, X160N, X160Q, X160R, X160S, X160T, X160V, X160Y, X165I, X165L, X165T, X165V, X166A, X166C, X166D, X166E, X166H, X166M, X166N, X166S, X167C, X167D, X167E, X167F, X167P, X167V, X167W, X167Y, X169A, X169G, X169S, X170A, X170D, X170E, X170G, X170H, X170K, X170L, X170P, X170Q, X170R, X170S, X170V, X170W, X170Y, X171C, X171F, X171L, X171N, X171Y, X172A, X172C, X172D, X172F, X172G, X172I, X172K, X172L, X172M, X172P, X172Q, X172R, X172S, X172T, X172V, X172Y, X173A, X173C, X173D, X173E, X173F, X173G, X173H, X173K, X173L, X173M, X173N, X173Q, X173R, X173T, X173V, X173W, X174A, X174G, X174S, X174T, X174V, X175A, X175C, X175I, X175L, X175M, X175Q, X175T, X175V, X175Y, X176A, X176G, X176S, X177A, X177C, X177I, X177T, X177V, X178A, X178G, X179A, X179G, X180C, X180I, X180L, X180S, X180T, X180V, X181D, X181N, X182A, X182D, X182E, X182F, X182G, X182H, X182I, X182K, X182L, X182M, X182N, X182P, X182Q, X182R, X182S, X182T, X182V, X182W, X182Y, X183A, X183D, X183F, X183G, X183H, X183I, X183K, X183L, X183M, X183N, X183Q, X183R, X183S, X183T, X183V, X183W, X183Y, X184D, X184N, X185A, X185C, X185E, X185F, X185G, X185H, X185I, X185K, X185L, X185M, X185N, X185Q, X185R, X185S, X185T, X185V, X185Y, X186H, X186I, X186K, X186L, X186R, X187A, X187P, X187W, X188A, X188D, X188E, X188F, X188G, X188H, X188I, X188K, X188L, X188P, X188Q, X188R, X188S, X188T, X188V, X188W, X188Y, X191D, X191Q, X192W, X192Y, X194A, X194C, X194D, X194E, X194F, X194G, X194H, X194I, X194L, X194M, X194P, X194Q, X194R, X194S, X194T, X194V, X194W, X194Y, X195A, X195C, X195D, X195E, X195G, X195Q, X195Y, X196L, X196M, X197A, X197C, X197D, X197E, X197G, X197N, X197Q, X197S, X197T, X198A, X198F, X198G, X198H, X198I, X198L, X198M, X198N, X198R, X198S, X198T, X198Y, X199A, X199C, X199I, X199M, X199S, X199T, X199V, X200A, X200C, X200G, X200S, X203A, X203C, X203E, X203I, X203L, X203S, X203T, X203V, X204A, X204C, X204E, X204F, X204G, X204I, X204K, X204L, X204N, X204R, X204S, X204T, X204W, X204Y, X205I, X205T, X205V, X206A, X206C, X206D, X206E, X206G, X206H, X206I, X206K, X206L, X206N, X206P, X206Q, X206R, X206S, X206T, X206V, X206W, X206Y, X207A, X207S, X208C, X208L, X208S, X208T, X208V, X209A, X209C, X209F, X209G, X209H, X209I, X209K, X209L, X209M, X209N, X209R, X209S, X209V, X209W, X209Y, X210A, X210C, X210E, X210G, X210H, X210I, X210L, X210M, X210N, X210P, X210R, X210S, X210V, X210Y, X211A, X211C, X211E, X211F, X211G, X211H, X211I, X211L, X211M, X211P, X211Q, X211R, X211T, X211V, X211W, X211Y, X212C, X212F, X212G, X212H, X212I, X212M, X212N, X212P, X212R, X212S, X212T, X212V, X212Y, X213A, X213C, X213D, X213E, X213F, X213G, X213I, X213K, X213L, X213M, X213N, X213Q, X213R, X213S, X213T, X213V, X213W, X213Y, X214F, X214L, X214W, X214Y, X215A, X215C, X215D, X215E, X215F, X215G, X215H, X215I, X215K, X215M, X215N, X215P, X215R, X215S, X215T, X215V, X215W, X215Y, X216A, X216C, X216D, X216E, X216F, X216G, X216H, X216I, X216K, X216L, X216M, X216N, X216P, X216Q, X216R, X216S, X216V, X216W, X216Y, X217A, X217C, X217E, X217F, X217G, X217I, X217K, X217L, X217M, X217Q, X218C, X218D, X218E, X218F, X218G, X218H, X218I, X218M, X218N, X218Q, X218R, X218S, X218T, X218V, X218Y, X220S, X220T, X222A, X222M, X222Q, X223A, X223G, X223S, X224A, X224G, X224L, X224N, X224S, X224T, X225C, X225P, X226C, X226F, X226H, X226M, X226V, X227A, X227C, X227G, X227I, X227L, X227M, X227S, X227T, X227V, X228A, X228C, X228G, X228I, X228S, X228V, X229A, X229G, X229P, X229S, X230A, X230D, X230E, X230G, X230H, X230I, X230L, X230N, X230Q, X230S, X230T, X230V, X231A, X231C, X231F, X231G, X231H, X231I, X231L, X231S, X231T, X231Y, X232A, X232G, X232H, X232L, X232M, X232S, X232V, X233A, X233C, X233E, X233F, X233G, X233I, X233L, X233M, X233N, X233P, X233Q, X233S, X233T, X233V, X233Y, X234D, X234F, X234G, X234H, X234L, X234M, X234N, X234Q, X234S, X234T, X234V, X234Y, X235C, X235D, X235E, X235F, X235G, X235H, X235I, X235K, X235L, X235M, X235N, X235Q, X235R, X235S, X235V, X235W, X235Y, X236A, X236C, X236E, X236F, X236H, X236K, X236N, X236P, X236Q, X236R, X236S, X236T, X236V, X236W, X236Y, X237A, X237C, X237F, X237G, X237H, X237I, X237K, X237L, X237M, X237P, X237Q, X237R, X237S, X237T, X237V, X237W, X237Y, X238C, X238D, X238E, X238F, X238G, X238H, X238I, X238K, X238L, X238M, X238N, X238Q, X238R, X238S, X238T, X238V, X238Y, X239C, X239D, X239F, X239G, X239H, X239I, X239K, X239L, X239M, X239N, X239P, X239Q, X239R, X239S, X239T, X239V, X239W, X239Y, X240A, X240C, X240E, X240F, X240I, X240K, X240L, X240M, X240N, X240Q, X240R, X240S, X240T, X240W, X240Y, X241A, X241C, X241D, X241E, X241F, X241G, X241H, X241I, X241K, X241L, X241M, X241N, X241P, X241Q, X241R, X241S, X241T, X241V, X241W, X241Y, X242A, X242C, X242D, X242F, X242G, X242H, X242I, X242L, X242M, X242P, X242Q, X242R, X242S, X242T, X242V, X242W, X243C, X243D, X243E, X243F, X243G, X243H, X243I, X243K, X243L, X243M, X243N, X243P, X243Q, X243R, X243S, X243T, X243V, X243W, X244A, X244D, X244E, X244F, X244H, X244I, X244L, X244M, X244N, X244P, X244Q, X244R, X244S, X244T, X244V, X244W, X244Y, X245A, X245C, X245E, X245G, X245H, X245K, X245L, X245P, X245Q, X245R, X245S, X245V, X245W, X245Y, X246A, X246C, X246E, X246F, X246G, X246H, X246I, X246L, X246M, X246N, X246Q, X246S, X246T, X246V, X246W, X246Y, X247A, X247C, X247D, X247E, X247F, X247G, X247H, X247I, X247K, X247L, X247M, X247N, X247P, X247Q, X247R, X247S, X247T, X247V, X247W, X247Y, X248C, X248D, X248E, X248G, X248H, X248I, X248K, X248L, X248N, X248P, X248R, X248S, X248T, X248V, X248W, X248Y, X249A, X249D, X249E, X249F, X249G, X249H, X249I, X249K, X249L, X249M, X249N, X249Q, X249R, X249S, X249T, X249V, X249W, X249Y, X250A, X250C, X250F, X250I, X250L, X250M, X250Q, X250S, X250V, X251A, X251D, X251E, X251F, X251G, X251K, X251L, X251M, X251Q, X251R, X251T, X251V, X251Y, X252A, X252C, X252D, X252F, X252G, X252H, X252I, X252K, X252L, X252M, X252N, X252R, X252S, X252V, X252W, X252Y, X253A, X253D, X253E, X253F, X253G, X253H, X253I, X253K, X253M, X253R, X253S, X253T, X253V, X253W, X254A, X254C, X254G, X254S, X254T, X255A, X255C, X255D, X255E, X255F, X255H, X255I, X255L, X255N, X255P, X255Q, X255R, X255S, X255T, X255V, X255W, X255Y, X256A, X256C, X256D, X256E, X256G, X256H, X256I, X256K, X256L, X256M, X256N, X256P, X256R, X256S, X256T, X256V, X256W, X256Y, X257C, X257F, X257I, X257K, X257L, X257M, X257V, X258A, X258C, X258D, X258E, X258F, X258G, X258H, X258I, X258L, X258M, X258P, X258Q, X258R, X258S, X258T, X258V, X258W, X258Y, X259A, X259C, X259E, X259G, X259I, X259L, X259M, X259P, X259Q, X259R, X259S, X259T, X259V, X260A, X260D, X260E, X260F, X260H, X260I, X260L, X260M, X260N, X260P, X260R, X260S, X260T, X260V, X260Y, X261A, X261C, X261E, X261F, X261G, X261I, X261K, X261L, X261N, X261P, X261Q, X261R, X261S, X261T, X261V, X261W, X261Y, X262A, X262C, X262D, X262F, X262G, X262H, X262I, X262K, X262L, X262M, X262Q, X262R, X262S, X262T, X262V, X262W, X262Y, X263C, X263F, X263L, X263Y, X264A, X264G, X265A, X265C, X265D, X265F, X265G, X265H, X265K, X265M, X265Q, X265R, X265S, X265T, X265W, X265Y, X267G, X267I, X267L, X267M, X267N, X267V, X268A, X268G, X268H, X268L, X268M, X268N, X268P, X268Q, X268S, X268V, X269C, X269D, X269F, X269G, X269H, X269I, X269L, X269M, X269N, X269Q, X269R, X269S, X269T, X269V, X270A, X270C, X270D, X270G, X270I, X270L, X270M, X270N, X270S, X270T, X270V, X271A, X271C, X271E, X271F, X271G, X271H, X271I, X271K, X271L, X271M, X271N, X271P, X271T, X271V, X271Y, X272A, X272C, X272D, X272E, X272F, X272G, X272H, X272K, X272L, X272M, X272N, X272P, X272R, X272S, X272T, X272W, X272Y, X273A, X273C, X273D, X273E, X273F, X273G, X273H, X273I, X273L, X273S, X273T, X273V, X274A, X274C, X274D, X274E, X274G, X274H, X274L, X274M, X274N, X274P, X274Q, X274R, X274S, X274T, X274W, X275A, X275C, X275D, X275E, X275F, X275G, X275H, X275K, X275L, X275M, X275P, X275Q, X275R, X275V, 及び X275W。
M, X241N, X241Q, X241R, X241S, X241T, X241V, X241W, X241Y, X242A, X242C, X242D, X242G, X242H, X242I, X242L, X242M, X242P, X242Q, X242S, X242T, X242V, X243C, X243D, X243E, X243F, X243G, X243H, X243I, X243L, X243M, X243N, X243P, X243Q, X243S, X243T, X243V, X243W, X244D, X244E, X244F, X244H, X244I, X244L, X244M, X244N, X244Q, X244S, X244T, X244V, X244W, X245A, X245C, X245E, X245G, X245H, X245K, X245L, X245P, X245Q, X245S, X245V, X245W, X245Y, X246A, X246C, X246I, X246L, X246M, X246T, X246V, X247A, X247C, X247F, X247G, X247H, X247K, X247L, X247M, X247N, X247R, X247S, X247T, X247V, X247W, X247Y, X248C, X248D, X248E, X248G, X248H, X248I, X248L, X248N, X248P, X248R, X248S, X248T, X248V, X248Y, X249A, X249D, X249E, X249F, X249G, X249H, X249I, X249K, X249L, X249M, X249N, X249Q, X249S, X249T, X249W, X249Y, X250C, X250I, X250L, X250M, X250V, X251A, X251D, X251E, X251F, X251G, X251K, X251L, X251M, X251Q, X251R, X251T, X251V, X251Y, X252A, X252C, X252D, X252F, X252G, X252H, X252I, X252K, X252L, X252M, X252N, X252R, X252S, X252V, X252W, X253A, X253E, X253H, X253M, X253S, X253T, X253W, X255A, X255C, X255D, X255E, X255I, X255L, X255N, X255Q, X255T, X255V, X255Y, X256A, X256C, X256D, X256E, X256G, X256H, X256I, X256K, X256L, X256M, X256N, X256P, X256S, X256V, X256W, X256Y, X258D, X258G, X259A, X259C, X259E, X259P, X259Q, X259S, X260A, X260D, X260E, X260H, X260I, X260N, X260P, X260S, X260T, X260V, X261A, X261C, X261E, X261F, X261I, X261K, X261L, X261N, X261P, X261Q, X261S, X261T, X261V, X261W, X261Y, X262A, X262C, X262D, X262F, X262H, X262I, X262L, X262M, X262Q, X262T, X262V, X262Y, X265A, X265D, X265S, X267I, X267L, X268A, X268L, X268V, X269D, X269H, X269N, X269Q, X269S, X270A, X270C, X270G, X270I, X270L, X270N, X270S, X270T, X270V, X271C, X271E, X272A, X272C, X272D, X272E, X272F, X272G, X272H, X272L, X272M, X272N, X272P, X272S, X272T, X272W, X272Y, X273A, X273C, X273G, X273S, X274A, X274C, X274G, X274L, X274M, X274N, X274S, X274T, X275D, X275E, X275F, X275H, X275K, X275L, X275M, X275P, X275Q, X275R。
<プロテアーゼ変異体のデータの対比評価>
この実施例では、BPN’、GCI-P036及び変異体プロテアーゼの洗浄性能、LAS/EDTA安定性、 AAPF活性及びタンパク質含有量 (対象とする性質の試験) について評価した実験結果を対比する。使用した洗剤及び条件は実施例1に開示されている。下記の表では、「EGG」の結果は、Table 1-1に記載したCALGONITTM (WE ADW) におけるCS-38小見本片分析結果を意味する。本願に開示するように、GCI-P036変異体の機能を変異体及び参照プロテアーゼの性能の比である性能指数 (PI) として定量する。本願でのPIの分類は:上昇突然変異 (PI>1.0);中立突然変異(PI>0.5);非有害突然変異(PI>0.05);及び有害突然変異(PI≦0.05)である。BPN’変異体は、本願に参照として組み込むPCT/09/046066及びPCT/US09/046156に開示された方法で調製し、評価した。
<GCI P036組み合わせライブラリー変異体の汚れ除去効果の評価>
この実施例には、GCI-P036の変異体の汚れ除去効果を、食器洗浄機及び液体衣類洗剤について評価した結果を示す。組み合わせライブラリーのクローン化は、Sloning BioTechnologyによりSlonomax Technologyを用いて実施された。プロテアーゼ変異体のサンプルは、上記のようにして調製した。簡単にいうと、組み合わせライブラリー変異体を、洗濯及び食器洗い機(ADW)用途の双方について、様々な地域市場を代表する洗剤(例えばpH、温度及び/又は水硬度の違い)に関して、血、ミルク、インク(BMI)小見本片及びCS-38小見本片分析を行った。本願に開示するように、プロテアーゼ変異体の機能を、変異体及び参照プロテアーゼの性能の比である性能指数(PI)として定量した。置換をBPN’の位置番号を用いてGCI-P036参照プロテアーゼに対し示し、PIはGCI-P036参照プロテアーゼとの関係において求めた。データをTables 7-1 から7-5に示す。
GCI-P036の変異体の汚れ除去効果を、洗浄用途について評価した。組み合わせライブラリーのクローン化は、Sloning BioTechnologyによりSlonomax Technologyを用いて実施された。プロテアーゼ変異体のサンプルは、上記のようにして調製した。簡単にいうと、MML変異体について、実施例1に示した方法により血、ミルク、インク(BMI)小見本片及びCS-38小見本片分析を行った。本願に開示するように、プロテアーゼ変異体の機能を、変異体及び参照プロテアーゼの性能の比である性能指数(PI)として定量した。置換をBPN’の位置番号を用いてGCI-P036参照プロテアーゼに対し示し、PIはGCI-P036参照プロテアーゼとの関係において求めた。データをTables 7-6と7-7に示す。
LAS 及び EDTAを含有する組成物中で GCI-P036変異体をインキュベートした後、GCI-P036変異体の安定性を評価した。組み合わせライブラリーのクローン化は、Sloning BioTechnologyによりSlonomax Technologyを用いて実施された。プロテアーゼ変異体のサンプルは、上記のようにして調製した。簡単にいうと、MML変異体について、実施例1に示した方法によりLAS/EDTA安定性分析を行った。本願に開示するように、プロテアーゼ変異体の機能を、変異体及び参照プロテアーゼの性能の比である性能指数(PI)として定量した。置換をBPN’の位置番号を用いてGCI-P036参照プロテアーゼに対し示し、PIはGCI-P036参照プロテアーゼとの関係において求めた。結果をTables 7-8に示す。
GCI-P036の変異体の組換え発現を、実施例1のTCA分析における培養物のタンパク質含有量を測定することにより評価した。組み合わせライブラリーのクローン化は、Sloning BioTechnologyによりSlonomax Technologyを用いて実施された。プロテアーゼ変異体のサンプルは、上記のようにして調製した。簡単にいうと、MML変異体について、実施例1に示した方法によりTCA分析を行った。本願に開示するように、プロテアーゼ変異体の機能を、変異体及び参照プロテアーゼの性能の比である性能指数(PI)として定量した。置換をBPN’の位置番号を用いてGCI-P036参照プロテアーゼに対し示し、PIはGCI-P036参照プロテアーゼとの関係において求めた。結果をTables 7-9に示す。
<GCI-P036の多重突然変異体ライブラリー(MML)による汚れ除去効果の評価>
Table 8-1に、GCI-P036の変異体について行った、汚れ除去活性(CS-38見本片を用い、食器洗浄機用洗剤の洗浄性能を評価する小見本片分析)、LAS/EDTA安定性、熱安定性、及びTCA分析によるタンパク質の同定 (対象とする性質の試験)の評価結果を示す。これらの結果は、実施例1に記載した方法を汚れ除去性能分析に関して次のように修正した方法により求めたものである。試験に用いた洗剤を熱不活性化(CALGONITTM 5 in 1及び CASCADETM Complete)し、MTPを粘着性膜でシールし、IEMSインキュベーターに取り付けて所定の温度で30分間振蕩した。
<多重突然変異体ライブラリー(MML)による洗濯用途における汚れ除去効果の評価>
Table 9-1に、GCI-P036変異体 (実施例8に示したクローン8, 47)の汚れ除去活性 (洗濯用途における洗浄効果を評価するための小見本片分析)を評価するために行った実験の結果を示す。実施例1の方法により、これらの結果を得た。試験に用いた洗剤は、熱不活性化したP&G TIDETM 2X (NA HDL) 及び P&G TIDETM (NA HDG)であった。BMI汚染小見本片による洗浄性能の評価は、0.2ppmの変異体を用いて、200uLの容積を25℃、1400rpmで30分間振蕩して行った。本願に開示するように、GCI-P036変異体の機能を、変異体と親GCI-P036タンパク質の性能の比である性能指数(PI)として定量した。スブチリシンFNA (BPN’-Y217L) 及び GCI-P036-S87N-G118V-S128L-P129Q-S130Aタンパク質も評価した。
<増加した産出力価を有するプロテアーゼ>
GCI-P036の成熟領域内の突然変異を、タンパク質力価の向上、又はマイクロタイタープレートで行ったスーパースクリーン(superscreen)において観察された小見本片洗浄性能の向上に基づいて選択した。前述の実施例で評価したプロテアーゼのサンプルを、複製プラスミド発現系を用いて調製した。この実施例に示す45種の変異体の発現には、GCI-P036ciでラベルされ、野生型GCI-P036タンパク質をコードする遺伝子を用いた。この「ci」合成遺伝子は、FNAコドンの使用(非常に高レベルで発現されるタンパク質)に基づく改良コドンからなる。これらの変異体をコードする核酸を、下記に説明するGCI-P036ci遺伝子テンプレート中に形質転換してp2JH骨格 (p2JH-GCI-P036ci, 図 3)に一連のプラスミド、大スケールでのタンパク質産出に従う組み込みベクターシステムを生成させる。この組み込みバシラス発現システムを用いて、振蕩フラスコ培養物からの培養ブロスについて、Table 9-1に示す変異体の評価を行った。タンパク質発現及び小見本片性能を再び評価した。いくつかの変異体のsuc-AAPF-pNA基質に対するプロテアーゼ活性が野生型と比べて低い値であったたため、エグリンc滴定分析によりプロテアーゼ濃度を求めた。エグリンc分析及びBMI分析の結果を、それぞれ図4と図5に示す。
組み込みプラスミドp2JH-GCI-P036ciを、AprEシグナル配列の8盤面のコドンに融合され、AprE プロモータ及びFNBaseターミネータの制御下にあるGCI-P036 コドン改良合成遺伝子(GCI-P036ci)を用いて組み立てた(Wells et al., Nucleic Acids Res, 11:7911-25, 1983)。以下に示すp2JH-GCI-P036ciのGCI-P036ci配列では、太字イタリック体はAprEプロモータを表し、標準字体はシグナル配列領域を表し、下線部はGCI-P036 プロ配列領域を表し、太字はGCI-P036成熟プロテアーゼ領域をコードするDNAを表し、下線を付したイタリック体はFNBaseターミネータを表す。
Multi-site QuickChange Kit (Stratagene社)による部位特異的突然変異誘発を、テンプレート及び部位特異的プライマーセットとしてp2JH-GCI-P036ciを用い、Table 9-2に示す配列のフォワード(F)及びリバース(R) を用いて行った。5マイクロリットルの突然変異生成物を用いて化学的コンピテントTop10 E.coli 細胞 (Invitrogen社)を形質転換し、LA + 50 ppm カルベニシリンに塗布して選択条件下で培養した。プレートを37℃で一夜培養した。
振蕩フラスコによるタンパク質発現の分析を、濃度が既知の精製GCI-P036の検量線に基づく力価により、SDS-PAGEを用いて行った。SDS-PAGEでは、45μLの培養液上清に10μLの2M HClを添加し、氷上で10分間インキュベートした。次に、各サンプルに2X グリシンサンプル緩衝液50μLを添加し、各サンプル混合液10μLを10 Bis-Trisゲル上に塗布し、MES緩衝液を用いて200Vで30分間展開した。ゲルを脱イオン水で3回洗浄し、SimplyBlue Safe染料(Invitrogen社)で1時間染色し、脱イオン水で一夜脱色した。
<GCI-P036の電荷ラダー変異体の産出>
この実勢例では、GCI-P036の電荷ラダー及び組み合わせ電荷ライブラリーの生成について説明する。本願のプロテアーゼ変異体の試験は、プロテアーゼ変異体の性能指数(PI)を求めるために、参照プロテアーゼと比較しながら行った。
対幅広い範囲の象とする物性を有する多数のタンパク質変異体を、既存のライブラリーから選択するか、又は公知の部位特異的突然変異誘発(例えばUS Appln. Ser/ No., 10/576,331, 11/581,102, 及び 11/583,334参照) により調製した。このように定義されたタンパク質プローブの集団を、対象とする試験により分析した。プロテアーゼの電荷ラダーの例を以下の表に示すとともに、本願の方法により分析した。下記の表では、電荷の変化は参照プロテアーゼに対するものであり、この実施例では参照プロテアーゼは野生型プロテアーゼである。
コドン改良遺伝子を備えるpAC-GG36ciプラスミドを用いて、DNA 2.0によりCCLを生成させた。形質転換には、バシラス・スブチリス株 (遺伝子型: ΔaprE, ΔnprE, ΔspoIIE, amyE::xylRPxylAcomK-phleo) を用いた。さらに、Table 10-2に示す、GCI-P036プロテアーゼ中の4つの部位のそれぞれについての位置ライブラリ(positional library)を生成させた。変異体は、96-ウエルプレート中のグリセロールストック溶液として供給された。
<GCI-P036電荷変異体の汚れ除去性能>
この実施例では、様々な販売地域を代表する洗剤(例えば異なるpH、温度、及び/又は水硬度)であって、洗濯用洗剤及び食器洗浄機(ADW) 洗剤の双方について、GCI-P036変異体の血、ミルク、インク(BMI) 小見本片分析、及び加熱卵黄 小見本片分析を行った結果を説明する。この実施例の表に記載した販売地域には、Table 1-1に記載したNA(北米)、WE(西欧)、及びWE(ADW)が含まれる。Table 12-1では、食器洗浄(すなわち「PI食器洗浄」)の結果は、実施例1に示した40℃、12gpg、pH10でのCALGONITTM (Reckitt-Benckiser社)による加熱卵黄小見本片分析を行った結果である。
<液体洗濯洗剤組成物>
この実施例では、様々な液体洗濯洗剤組成物の処方を説明する。以下の本願の液体洗濯洗剤組成物は、下記のようにして調製される。これらの処方のいずれにおいても、少なくとも1の本願のプロテアーゼ変異体が、約0.0001から約 10重量パーセントの濃度範囲で含有されている。又いくつかの別の実施形態では、処方業者がニーズに基づいて決めた、異なる濃度で使用される。
<食器手洗い用液体洗剤組成物>
この実施例では、様々な食器手洗い用液体洗剤の処方を説明する。以下の本願の食器手洗い用液体洗剤組成物は、下記のようにして調製される。これらの処方のいずれにおいても、少なくとも1の本願のプロテアーゼ変異体が、約0.0001から約 10重量パーセントの濃度範囲で含有されている。いくつかの別の実施形態では、処方業者がニーズに基づいて決めた、異なる濃度で使用される。
<食器洗浄機用液体洗剤組成物>
この実施例では、様々な食器洗浄機用液体洗剤の処方を説明する。以下の本願の食器洗浄機用液体洗剤組成物は、下記のようにして調製される。これらの処方のいずれにおいても、少なくとも1の本願のプロテアーゼ変異体が、約0.0001から約 10重量パーセントの濃度範囲で含有されている。いくつかの別の実施形態では、処方業者がニーズに基づいて決めた、異なる濃度で使用される。
<顆粒状及び/又は錠剤状洗剤組成物>
この実施例では、様々な顆粒状及び/又は錠剤状洗剤の処方を説明する。以下の本願の顆粒状及び/又は錠剤状の洗剤組成物は、下記のようにして調製される。これらの処方のいずれにおいても、少なくとも1の本願のプロテアーゼ変異体が、約0.0001から約 10重量パーセントの濃度範囲で含有されている。いくつかの別の実施形態では、処方業者がニーズに基づいて決めた、異なる濃度で使用される。
<液体洗濯洗剤>
この実施例では、様々な液体洗濯洗剤の処方を説明する。以下の本願の液体洗濯洗剤は、下記のようにして調製される。これらの処方のいずれにおいても、少なくとも1の本願のプロテアーゼ変異体が、約0.0001から約 10重量パーセントの濃度範囲で含有されている。いくつかの別の実施形態では、処方業者がニーズに基づいて決めた、異なる濃度で使用される。
<高密度食器洗浄洗剤>
この実施例では、様々な高密度食器洗浄洗剤の処方を説明する。以下の本願の高密度食器洗浄洗剤は、下記のようにして調製される。これらの処方のいずれにおいても、少なくとも1の本願のプロテアーゼ変異体が、約0.0001から約 10重量パーセントの濃度範囲で含有されている。いくつかの別の実施形態では、処方業者がニーズに基づいて決めた、異なる濃度で使用される。
<錠剤状洗剤組成物>で圧縮すること
この実施例では、様々な錠剤状洗剤の処方を説明する。以下の本願の錠剤状洗剤組成物は、顆粒状食器洗浄洗剤を、標準的なヘッドが12個のロータリープレスを用いて圧力13KN/cm2で圧縮して調製される。これらの処方のいずれにおいても、少なくとも1の本願のプロテアーゼ変異体が、約0.0001から約 10重量パーセントの濃度範囲で含有されている。いくつかの別の実施形態では、処方業者がニーズに基づいて決めた、異なる濃度で使用される。
Claims (7)
- SEQ ID NO:2に示すバシラスレンタススブチリシン(Bacillus lentus subtilisin)のスブチリシン変異体を含む洗浄組成物であって、前記スブチリシン変異体のアミノ酸の置換が下記から選択される置換の組み合わせのみからなり:
S87N/G118V/S128L/P129Q/S130A/S24W/S101L/Q109K, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101L/Q109R/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/Q109R/S164I, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109R/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101Y/Q109L/N185R/A215R, S87R/G118R/S128L/P129Q/S130A/S101K/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/S24W/S101L/Q109L, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101Y/Q109R/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101L/Q109R/S164I, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109R/S188D/T213E/N248R, S87N/G118V/S128L/P129Q/S130A/N76D, S87N/G118V/S128L/P129Q/S130A/S24W/S101Y/Q109K, S87N/G118V/S128L/P129Q/S130A/N43S/P52V/S101R/Q109R, S87N/G118V/S128L/P129Q/S130A/I72V/S101Y/Q109R/S164I, S87N/G118V/S128L/P129Q/S130A/S101L/Q109L/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N76D/S101L, S87N/G118V/S128L/P129Q/S130A/S24W/S101Y/Q109L, S87N/G118V/S128L/P129Q/S130A/N76D/S164I, S87R/G118R/S128L/P129Q/S130A/N76D/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109L/S188D/N248R, S87R/G118R/S128L/P129Q/S130A/V118R/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N76D/S101M, S87N/G118V/S128L/P129Q/S130A/N18K/N76D/G97P/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101L/S164I, S87R/G118R/S128L/P129Q/S130A/S101L/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109L/S188D/T213E/N248R, S87R/G118R/S128L/P129Q/S130A/Q109L/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101L/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101Y/S164I, S87N/G118V/S128L/P129Q/S130A/S101Y/T213E/N248R, S87N/G118V/S128L/P129Q/S130A/I72V/S101L/Q109L/S164I, S87R/G118R/S128L/P129Q/S130A/S101V/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101Y/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101V/S164I, S87N/G118V/S128L/P129Q/S130A/S101L/T213E/N248R, S87N/G118V/S128L/P129Q/S130A/I72V/S101Y/Q109L/S164I, S87R/G118R/S128L/P129Q/S130A/S101H/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/Q109R/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101H/S164I, S87N/G118V/S128L/P129Q/S130A/S101L/Q109R/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101L/Q109L/N185R/A215R, S87R/G118R/S128L/P129Q/S130A/S101M/S188D/N248R, N76D/S87R/G118R/S128L/P129Q/S130A/S188D/N248K, S87N/G118V/G127S/S128L/P129Q/S130A, S87N/S101H/G118V/S128L/P129Q/S130A, S87N/S101K/G118V/S128L/P129Q/S130A, S87N/S101V/G118V/S128L/P129Q/S130A, S87N/S101Y/G118V/S128L/P129Q/S130A, S87N/S101L/G118V/S128L/P129Q/S130A, I72V/S87N/G118V/S128L/P129Q/S130A//S164I、ここで、これらの位置番号は、SEQ ID NO:1に示すB. アミロリケファシエンス・スブチリシンBPN’のアミノ酸配列に対応して賦与される、洗浄剤組成物。 - 前記洗浄組成物が、液体、ゲル、錠剤、粉末、及び/又は顆粒状洗剤からなる、請求項1の洗浄組成物。
- 前記洗浄組成物が衣類洗剤及び食器洗剤から選択される、請求項1または2の洗浄組成物。
- 前記洗浄組成物が衣類洗剤である、請求項3の洗浄組成物。
- 前記洗浄組成物が食器洗剤であり、前記食器洗剤が手洗い食器洗浄洗剤及び自動食器洗浄機用洗剤から選択される食器洗剤である、請求項3の洗浄組成物。
- さらに少なくとも1つの漂白剤を含有する、請求項1から5のいずれか1項の洗浄組成物。
- 前記洗浄組成物が低温水洗剤である、請求項1から6のいずれか1項の洗浄組成物。
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