[发明专利]储存稳定的中性金属蛋白酶的用途和制备

摘要

本发明提供含有至少一种中性金属蛋白酶的方法和组合物，所述中性金属蛋白酶具有改善的储存稳定性。在一些实施方式中，中性金属蛋白酶在清洗和其它应用中应用。在一些特别优选的实施方式中，本发明提供含有从芽孢杆菌属物种得到的中性金属蛋白酶(一种或多种)的方法和组合物。在一些更特别优选的实施方式中，中性金属蛋白酶是从解淀粉芽孢杆菌中得到的。在又进一步优选的实施方式中，中性金属蛋白酶是解淀粉芽孢杆菌中性金属蛋白酶的变体。又在其它实施方式中，中性金属蛋白酶是解淀粉芽孢杆菌中性金属蛋白酶的同系物。本发明在包括但不限于清洗、漂白和消毒的应用中具体应用。

主权项

1.分离的中性金属蛋白酶变体，其中，在与SEQ ID NO:18所示的中性金属蛋白酶中的位置等同的位置上，所述蛋白酶与SEQ ID NO:3或SEQ ID NO:18的不同在于：(a)选自下述的多个突变：S023W/G024M,T004V/S023W/G024W,S023W/G024Y/A288V,T004L/S023W/G024Y,N046Q/N050F/T054L,N050Y/T059R/S129Q,S023W/G024W,A273H/S285P/E292G,S023Y/G024Y,S023Y/G024W,T004S/S023Y/G024W,N046Q/T054K,S023W/G024Y,T004V/S023W,T059K/S066N,N046Q/N050W/T054H/T153A,T004V/S023W/G024Y,L282M/Q286P/A289R,N046Q/R047K/N050Y/T054K,L044Q/T263W/S285R,T004L/S023W/G024W,R047K/N050F/T054K,A273H/S285R,N050Y/T059K/S066Q,T054K/Q192K,N046Q/N050W,L282M/Q286K,T059K/S066Q,T004S/S023W,L282M/Q286R/A289R/K011N,L282M/A289R,N046Q/N050W/T054H,T059K/S129Q,T004S/S023N/G024Y/F210L,T004V/S023W/G024M/A289V,L282M/Q286K/A289R/S132T,N050W/T054H,L282M/Q286R,L282F/Q286K/A289R,T059R/S066Q,R047K/N050W/T054H,S265P/L282M/Q286K/A289R,L282M/Q286R/T229S,L282F/Q286K,T263W/S285R,S265P/L282M/Q286K,T263H/A273H/S285R,T059R/S129V,S032T/T263H/A273H/S285R,T059R/S066Q/S129Q,T004S/G024W,T004V/S023W/G024M,T059K/S066Q/S129Q,L282M/Q286K/A289R/I253V,T004V/S023Y/G024W,T059R/S066N/S129Q,N050F/T054L,T004S/S023N/G024W,T059R/S066N,T059R/S066N/S129V,Q286R/A289R,N046Q/R047K/N050F/T054K,S265P/L282M/Q286P/A289R,S265P/L282M/Q286R/A289RQ062K/S066Q/S129I,S023N/G024W,N046Q/R047K/N050W/T054H,R047K/T054K,T004L/G024W,T014M/T059R/S129V,T059R/S066Q/N092S/S129I,R047K/N050W/T054K,T004V/G024W,N047K/N050F/T054K,S265P/L282F/Q286K/N061Y,L282F/Q286K/E159V,T004V/S023Y/G024M,S265P/L282F/A289R/T065S,T059K/F063L/S066N/S129V,T004L/S023W,N050F/T054H,T059R/S066Q/S129V,V190I/D220E/S265W/L282F,T004S/S023Y/G024M,T004L/S023N/G024Y,T059K/S066N/S129I,T059R/S066N/S129I,L282M/Q286R/A289R/P162S,N046Q/N050F/T179N,T059K/Y082C/S129V,T059K/S129I,N050Y/T054K,T059K/S066Q/V102A/S129Q,T059R/S066Q/S129I,T059W/S066N/S129V/S290R,T059R/S129I,T059K/S066Q/S129I,T059K/S066Q/S129V,S265P/L282M/Q286R/A289R/T202S/K203N,T004V/S023N/G024W,S265P/Q286K,S265P/L282F/A289R,D220P/S265W,L055F/T059W/S129V,T059R/S129Q/S191R,N050W/T054K,T004S/S023W/G024M,R047K/N050F/T054H,T059K/S066N/K088E,T059K/S066Q/S129I/V291L,L282M/Q286R/A289R,T059R/S066N/F085S/S129I,L282F/Q286P/A289R,L282F/Q286R/A289R,G099D/S265P/L282F/Q286K/A289R,N046Q/N050F,N050Y/T059W/S066N/S129V,T009I/D220P/S265N,V190F/D220P/S265W,N157Y/T263W/A273H/S285R,T263W/A273H/S285R,T263W/S285W,T004V/S023Y,N046Q/R047K/N050W,N050W/T054L,N200Y/S265P/L282F/Q286P/A289R,T059R/S066Q/P264Q,T004V/G024Y,T004L/G024Y,N050Y/S191I,N050Y/T054L,T004L/S023W/G024Y/N155K,F169I/L282F/Q286R/A289R,L282M/Q286K/A289R,F130L/M138L/E152W/D183N,N046Q/R047K/N050Y/T054H,T004V/G024M,N050Y/T059W/S066Q/S129V,S023N/G024Y,T054H/P162Q,T004S/S023W/G024Y,N050Y/T054H,L282F/Q286R/A289R/F169I,R047K/N050W,V190F/D220P,L282M/F173Y,T004L/S023Y,N050W/A288D,V190I/D220P/S265Q,S265P/L282F/Q286P/A289R,S265P/L282F/Q286R/A289R,N046Q/N050Y/T054K,T059W/S066Q,T263W/A273H/S285WT263W/A273H/S285P,S023Y/G024M,T004L/S023N/G024W,T004V/S023N/G024Y,T059W/S066N/S129Q,T004S/S023Y,T004S/S023N/G024M,T059W/S066N/A070T,T059W/S066Q/S129Q,T263W/A273H,A273H/285P,N046Q/R047K/N050Y/T054L,N046Q/R047K/N050Y,R047K/N050Y,T263H/S285W,R047K/N050F,N046Q/R047K/N050F/T054H,S023N/G024M,T004S/G024Y,R047K/N050Y/T054H,T059W/S066N/S129I,R047K/T054L,T004S/S023W/G024W,M138L/E152F/T146S,D220P/S265N,T004S/G024M,T004V/S023N,N046Q/N050F/T054K,N046Q/N050Y/T054H,Q062H/S066Q/S129Q,T059W/S129Q,T059W/S129V,N050F/T054K,R047K/N050F/T054L,V190I/D220P/S265W,N112I/T263H/A273H/S285R,T059W/S066N/S129V,T059W/S066Q/S129I,T059W/S129I,T263W/S285P,V190I/D220P,A289V/T263H/A273H,T263H/A273H/S285P,N90S/A273H/S285P,R047K/N050Y/T054L,T004S/S023N,T059R/S129Q,N046Q/R047K/T054H,T059W/S066Q/S129V,E152W/T179P,N050Y/S066Q/S129V,T202S/T263W/A273H,T263W/A273H/S285P,M138L/E152W/T179P,N046Q/R047K,N046Q/T054H/F176L,T004L/G024M,T004S/L282M,T263H/A273H,T263H/A273H/S285W,T004L/S023Y/G024M,L282F/Q286P,T004V/S023Y/G024Y,V190F/S265W,M138L/E152F,V190F/D220E/S265W,N046Q/N050F/T054H,N157Y/S285W,T004F/S023Y/G024M,T004V/S023N/G024M,L198I/D220E/S265Q,N046Q/N050Y/T054K/A154T,S016L/D220E/S265W,D220E/S265W,D220E/A237S/S265W,S066Q/S129Q,V190F/D220E/S265Q/T267I,L282M/F173Y/T219S,E152F/T179P,V190I/S265W,M138L/S066Q,M138L/E152W,T059W/S066Q/A070T/S129I,V190F/D220E/S265N,V190F/S265N,N046Q/N050Y,和M138L/E152F/T179P，或(b)(a)中所述的多个突变和选自如下的至少一个突变，其中所述变体与包含SEQ ID NO:3或SEQ ID NO:18的中性金属蛋白酶具有至少95％或至少98％氨基酸同一性：T004C,T004E,T004H,T004I,T004K,T004L,T004M,T004N,T004P,T004R,T004S,T004V,T004W,T004Y,G012D,G012E,G012I,G012K,G012L,G012M,G012Q,G012R,G012T,G012V,G012W,K013A,K013C,K013D,K013E,K013F,K013G,K013H,K013I,K013L,K013M,K013N,K013Q,K013S,K013T,K013V,K013Y,T014F,T014G,T014H,T014I,T014K,T014L,T014M,T014P,T014Q,T014R,T014S,T014V,T014W,T014Y,S023A,S023D,S023F,S023G,S023I,S023K,S023L,S023M,S023N,S023P,S023Q,S023R,S023S,S023T,S023V,S023W,S023Y,G024A,G024D,G024F,G024G,G024H,G024I,G024K,G024L,G024M,G024N,G024P,G024R,G024S,G024T,G024V,G024W,G024Y,K033H,Q045C,Q045D,Q045E,Q045F,Q045H,Q045I,Q045K,Q045L,Q045M,Q045N,Q045P,Q045R,Q045T,Q045W,N046A,N046C,N046E,N046F,N046G,N046H,N046I,N046K,N046L,N046M,N046P,N046Q,N046R,N046S,N046T,N046V,N046W,N046Y,R047E,R047K,R047L,R047M,R47Q,R047S,R047T,Y049A,Y049C,Y049D,Y049E,Y049F,Y049H,Y049I,Y049K,Y049L,Y049N,Y049R,Y049S,Y049T,Y049V,Y049W,N050D,N050F,N050G,N050H,N050I,N050K,N050L,N050M,N050P,N050Q,N050R,N050W,N050Y,T054C,T054D,T054E,T054F,T054G,T054H,T054IT054K,T054L,T054M,T054N,T054P,T054Q,T054R,T054S,T054V,T054W,T054Y,S058D,S058H,S058I,S058L,S058N,S058P,S058Q,T059A,T059C,T059E,T059G,T059H,T059I,T059K,T059L T059M,T059N,T059P,T059Q,T059R,T059S,T059V,T059W,T060D,T060F,T060I,T060K,T060L,T060N,T060Q,T060R,T060V,T060W,T060Y,T065C,T065E,T065F,T065H,T065I,T065K,T065L,T065M,T065P,T065Q,T065R,T065V,T065Y,S066C,S066D,S066E,S066F,S066H,S066I,S066K,S066L,S066N,S066P,S066Q,S066R,S066T,S066V,S066W,S066Y,Q087A,Q087D,Q087E,Q087H,Q087I,Q087K,Q087L,Q087M,Q087N,Q087R,Q087S,Q087T,Q087V,Q087W,N090C,N090D,N090E,N090F,N090G,N090H,N090K,N090L,N090R,N090T,N096G,N096H,N096K,N096R,K097H,K097Q,K097W,K100A,K100D,K100E,K100F,K100H,K100N,K100P,K100Q,K100R,K100S,K100V,K100Y,R110A,R110C,R110E,R110H,R110K,R110L,R110M,R110N,R110Q,R110S,R110Y,D119E,D119H,D119I,D119L,D119Q,D119R,D119S,D119T,D119V,D119W,G128C,G128F,G128H,G128K,G128L,G128M,G128N,G128Q,G128R,G128W,G128Y,S129A,S129C,S129D,S129F,S129G,S129H,S129I,S129K,S129L,S129M,S129Q,S129R,S129T,S129V,S129W,S129Y,S135P,G136I,G136L,G136P,G136V,G136W,G136Y,S137A,D139A,D139C,D139E,D139G,D139H,D139I,D139K,D139L,D139M,D139P,D139R,D139S,D139V,D139W,D139Y,V140C,Q151I,E152A,E152C,E152D,E152F,E152G,E152H,E152L,E152M,E152N,E152R,E152S,E152W,N155D,N155K,N155Q,N155R,D178A,D178C,D178G,D178H,D178K,D178L,D178M,D178N,D178P,D178Q,D178R,D178S,D178T,D178V,D178W,D178Y,T179A,T179F,T179H,T179I,T179K,T179L,T179M,T179N,T179P,T179Q,T179R,T179S,T179V,T179W,T179Y,E186A,E186C,E186D,E186G,E186H,E186K,E186L,E186M,E186N,E186P,E186Q,E186R,E186S,E186T,E186V,E186W,E186Y,V190H,V190I,V190K,V190L,V190Q,V190R,S191F,S191G,S191H,S191I,S191K,S191L,S191N,S191Q,S191R,S191W,L198M,L198V,S199C,S199D,S199E,S199F,S199I,S199K,S199L,S199N,S199Q,S199R,S199V,Y204H,Y204T,G205F,G205H,G205L,G205M,G205N,G205R,G205S,G205Y,K211A,K211C,K211D,K211G,K211M,K211N,K211Q,K211R,K211S,K211T,K211V,K214A,K214C,K214E,K214I,K214L,K214M,K214N,K214Q,K214R,K214S,K214V,L216A,L216C,L216F,L216H,L216Q,L216R,L216S,L216Y,N218K,N218P,T219D,D220A,D220E,D220H,D220K,D220N,D220P,A221D,A221E,A221F,A221I,A221K,A221L,A221M,A221N,A221S,A221V,A221Y,G222C,G222H,G222N,G222R,Y224F,Y224H,Y224N,Y224R,T243C,T243G,T243H,T243I,T243K,T243L,T243Q,T243R,T243W,T243Y,K244A,K244C,K244D,K244E,K244F,K244G,K244L,K244M,K244N,K244Q,K244S,K244T,K244V,K244W,K244Y,V260A,V260D,V260E,V260G,V260H,V260I,V260K,V260L,V260M,V260P,V260Q,V260R V260S,V260T,V260W,V260Y,Y261C,Y261F,Y261I,Y261L,T263E,T263F,T263H,T263I,T263L,T263M,T263Q,T263V,T263W,T263Y,S265A,S265C,S265D,S265E,S265K,S265N,S265P,S265Q,S265R,S265T,S265V,S265W,K269E,K269F,K269G,K269H,K269I,K269L,K269M,K269N,K269P,K269Q,K269S,K269T,K269V,K269W,K269Y,A273C,A273D,A273H,A273I,A273K,A273L,A273N,A273Q,A273R,A273Y,R280A,R280C,R280D,R280E,R280F,R280G,R280H,R280K,R280L,R280M,R280S,R280T,R280V,R280W,R280Y,L282F,L282G,L282H,L282I,L282K,L282M,L282N,L282Q,L282R,L282V,L282Y,S285A,S285C,S285D,S285E,S285K,S285P,S285Q,S285R,S285W,Q286A,Q286D,Q286E,Q286K,Q286P,Q286R,A289C,A289D,A289E,A289K,A289L,A289R,A293C,A293R,N296C,N296D,N296E,N296K,N296R,N296V,A297C,A297K,A297N,A297Q,A297R,和G299N，其中所述中性金属蛋白酶变体与野生型解淀粉芽孢杆菌中性金属蛋白酶相比在较低或较高pH条件下具有提高的性能。