DK2390321T3 - Anvendelse og fremstilling af en opbevaringsstabil neutral metalloprotease - Google Patents

Anvendelse og fremstilling af en opbevaringsstabil neutral metalloprotease Download PDF

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DK2390321T3
DK2390321T3 DK11165353.1T DK11165353T DK2390321T3 DK 2390321 T3 DK2390321 T3 DK 2390321T3 DK 11165353 T DK11165353 T DK 11165353T DK 2390321 T3 DK2390321 T3 DK 2390321T3
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neutral metalloprotease
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ala
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Andrew Shaw
Louise Wallace
David A Estell
Sang-Kyu Lee
Eugene Steven Sadlowski
Hiroshi Oh
Ronald Hommes
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Procter & Gamble
Danisco Us Inc
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
    • C11D1/02—Anionic compounds
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38663—Stabilised liquid enzyme compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C14—SKINS; HIDES; PELTS; LEATHER
    • C14C—CHEMICAL TREATMENT OF HIDES, SKINS OR LEATHER, e.g. TANNING, IMPREGNATING, FINISHING; APPARATUS THEREFOR; COMPOSITIONS FOR TANNING
    • C14C11/00—Surface finishing of leather
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms

Claims (14)

  1. Anvendelse og fremstilling af en opbevaringsstabil neutral metalloprotease
    1. Isoleret neutral metalloproteasevariant med mindst 70% aminosyreidentitet med den neutrale metalloprotease omfattende SEQ ID NO: 3 eller SEQ ID NO: 18, og med en aminosyresekvens, der mindst omfatter én mutation svarende til en mutation i en neutral metalloprotease, der omfatter aminosyresekvensen ifølge SEQ ID NO: 18, hvor mutationerne er udvalgt fra T004C, T004E, T004H, T004I, T004K, TOML, T004M, T004N, T0041\ TtXMR, T004S, T004V, T004W, T004Y, G012D, G012B, G0121, GØ]2K. G0.12L, GG12M, G0P2Q, G012T, GQJ.2V, G012W, K013C,K013D, KOBE, KOBE, KOI30, Κ0Ϊ3Η, K0131, KOBL., KO.BM, K013N, K013Q, K013S, K013T, K013V, KO 13 Y, TO MF, T0l4G,TO14H, TO141.T0MK,TOHL,TØI4M,TO.14P, T014Q, T014R, T014S, T014V, TO14W,T014Y. S023A, S023D, S023F, S0231, S023K, S023L, S023M, S023N, S023P, S023Q, S023R, S023T, SQ23V, S023W, S023Y, G024A, G024D, G024F, G024G, G024H, G024I, G024K, G024L, G024M. G024N, G024P, G024R, G024S, G024T, 0024V, G024W, G024Y, K033H, Q045C, Q045D, Q045E, Q045F, Q045H, Q045I, Q04SK, Q045L, Q045M, Q04SN, Q045P, Q045R, Q045T, Q045W, N04GA, N046C, N046E, N046F, N046G, N0461I, N046I, N046K, N046L, N046M, N046P, ND46Q, N046R, N0465, N046T, N046V, N046W, N046Y, R047E, RG47K, R047L, R047M, R47Q, R047S, R047T, Y049A, Y049C, Y049D, Y049E, Y049P, Y049H, Y049I, Y049K, Y049L, Y049N. Y049R, Y049T, Y049V, Y049W, NGSOD, N050F, N050G. NOSOH, Ν050Ϊ. N950K, N050L, N050M, N050P, N050Q, N050W, N050Y, T054C, T054D, T054E. T054P, T054G, T054H, T0541 TQ54K, T054L, T054M, T0S4N, T054P, T054Q, T054R, T054S, T0S4V, T054W, T054Y, S05SD, S058H, S058I, S058L, S058N, S058P, S058Q. T059A, T059C, T059E, TO59G, TO59H, T059I, ΊΉ59Κ, ΊΌ59Ε Ί059Μ, T059N, T059P, TO59Q, T059R, TQ59S, TO59V,T059W, TO60D, T060F, 10(50)., ΪΟόΟΚ, Τ06ί)Ε, T060N, TO60Q, T060R, T060V, T060W, T060Y, T06SC, T065E,T065F, T065H, ΊΌ651, T065K, T065L, T065M, T065P, TQ65Q, T065R, T065V, T065Y, S066C, S066D, S066E, S066F, S066H, S066I, S066K, S066l„S066N, S066P, S066Q, S066R, S06ffr, S066V, S066W, S066Y, Q087A, Q087D, Q087B, QCS7H, Q0871, Q087K, Q087U Q087M, Q087N, Q087R, Q087T, Q087V. Q087W, N090C, N090D, N090E, N090F, N090G, N090H, NG90L, N090R, N090T, N096G, N096H. N096K, N096R, K097H, K097Q, K097W, KIOOA. KiOOD, KiOOE, K100F, K100H, K100N, K100P, K100Q. K100R, KIOOS, KIOOV, K100Y, RI 10A, RI 10C, RI IDE, RI 10H, RI iOK, R! SOL. RI lOM, RI ION, RliOS. RL10Y, Dl I9E, Dl 191 i. Dl 191, D l I9L, Di 19Q, D1I9R, D119S, Dl 19T, Dl 19V, Dl 19W, G128C, G128F, G12811, G128K.G128L, G128M, G128N, GI28Q, G128R, GI28W, GI28Y, SI29A, S129C.S129D. S129F, SJ29G, S129H, S1291, S129.K, S129L, S129M, S129Q, S L29R, S129T, S129V, S129W, S129Y, Γ1301, F130K, F130L, E130M, F13(X}, EI30R, F130T, F130V, F130Y, S135P, GI.361, G13«L, GI36P, G136V,G136W, G136Y, S137A, Ml381, M138K, M138Q, M138V.D139A, D139C, D139E, D139G, DI39FI, D139L DI39K, D139L, D139M,D139P,DI39R,D139S, D139V, D139W, Dl39Y, V140C, QI5II, E152A, E152C,E.B2D, E152F, E152G, B152H, E152E, E152M,Bi52N, E152R, E152S,E152W, N155D, N15SK,N155Q, N155R, D178A, D178C, D178G, D178H, D178K, DI78I.,, D178M, D178N, DI78P, D178Q, D178R, D178S, D178T, DI78V, D178W, D178Y, T1.79A, T179F, T179H,T179I, T179K, T179L, T179M, TI79N, TI79P, T179Q, T179R, T179S, TJ79V, T179W, T.179Y, Ε186Λ, EI86C, E186D, EJ86G, E186H, E186K, E186L, E186M, EI86N,EI86P, EI86Q.E186R, E186S, E186T, EI86V, E1S6W, E186Y, V190H, Vi901, V190K, V190U V.190Q, V190R, S191F, S191G, S191H, S191I, S191K, S191L, S191N. S19IQ, S191R, S19IW, LI9SN4, L198V, S199C S199D, S199P, S199F, S199I, S199K, S199L, S199N, S199Q, SI99R, S199V, Y204H, Y204T, G205F, G205H, G2051,, G205M, G205R, G205S, G205Y, K21 !C, K21 ID, K21S G, K211M, K21 IN, K211Q, K211R, K21 IS, K21 IT, K21IV, K2I4A, K214C, K214E, K214I, K214I , K214M, K214N, K214Q, K214S, K214V, L216A, 1.,2160, L216F, L216H, L216Q, L216R, L216S, L216Y, N2I8K, N2I8P, T219D, D220AJD220E, D220H, D220K, D220N, D22QP, A221D, A221F, A221I, A221K, A221L, A221M, A221N, A221S, A221V, A221Y, G222C G222H. G222N, G222R, Y224F, Y224H, Y224N, Y224R, T243C, T243G, T243H, T243L T243K, T243L. T243Q, T243R, T243W, T243Y, K244A, K244C, K244D, K244E, K244F. K244G, K244E, K244M, K244N, K244Q, K244S, K244T, K244V, K244W, K244Y, V260A, V260D, V260E, V260G, V260H, V2G0I, V260K, V200L.V260M, V260R, V260Q, V260R V260S, V260T, V260W, V260Y, Y26iC, Y26JF. Y261I, Y26SL, T263E, T263P, T263H, Τ263Ϊ, T263L, T263M, T263Q, T263V, T263W, T263Y, S265A, S265C S265D, S265E, S265K. S265N, S265P, S265Q, $265R,S265T, S265V, S265W, K269E, K269F, K269G, K269H, K269I. K269L, K269M, K269N, K269P, K269Q, K269S, K269T, K269V, K269W, K269Y, A273C, A273IX A273H, A273I. A273K, A273L, A273N. A273Q, A273R, A273Y, R280A, R280C, R280D. R280E, R280F, R280G, R280H, R280K, R280L, R280M, R280S, R28OT, R280V, R280W, R280Y, L282F, L282G, L282H, L282I, L282K, L282M, L2S2N, L282Q, L282R, L282V, L282Y, S285A, S2S5C, S285D. S285E, S28SK, S285P, S28SQ. S285R, S285W, Q286A, Q286D, Q2S6E, Q286K, Q286P, Q286R, A289C, A289D, A289E, A289K, A289L, A289R, A293C, A293R, N296C, N296D, N296B, N296K, N296R, N296V, A297C, A297K, A297N, A297Q, A297R, og G299N; hvor aminosyresekvensidentiteten med SEQ ID NO: 3 eller SEQ ID NO: 18 er bestemt ved anvendelse af BLAST med standardparametre; hvor den neutrale metalloproteasevariant har forbedret termostabilitet sammenlignet med vildtype B. amyloliquefaciens neutral metalloprotease med sekvensen SEQ ID NO: 18; forbedret performance under lavere eller højere pH-forhold sammenlignet med vildtype B. amyloliquefaciens neutral metalloprotease med sekvensen SEQ ID NO: 18; eller forbedret autolytisk stabilitet sammenlignet med vildtype B. amyloliquefaciens neutral metalloprotease med sekvensen SEQ ID NO: 18.
  2. 2. Isoleret neutral metalloproteasevariant ifølge krav 1, hvor proteasen omfatter flere mutationer udvalgt fra S023W/G024M, T004V/SG23W/G024W, S023W/G024Y/A288V, TQ041/8023 W/G024Y, N046Q/N050I7TO54L, NO5OY/T059R/SI29Q, S023W/GQ24W, A273H/S285P/E292G, S023Y/G024Y, S023Y/G024W, T004S/SG23Y/G024W, N046Q/T054K, S023W/G024Y, T004 V/S023W, T059K/S06SN, N046Q/N050W/T054H/T153A. T004V/S023 W/G024Y, L282M/Q286P/A2S9R,N046Q/R047K/N050Y/T054K, L044Q/T263W/S285R, 10041,./8023W/GS24W, RS4?K/N050F/I054Kj A273B/S285R, N050Y/T059K/S066Qi T0S4K/QI92K, N046Q/N050W, L2S2M/Q286K, T059K/S066Q, T004S/S023W, E282M/Q286R/A289R/K0 i 1N, L282M/A289R, N046Q/N0SØW/T054K,T059K/S129Q, T004S/S023N/G024Y/F210L, TQ04V/S023W/G024M/A289V, L282M/Q286K/A289R/;S 132T, N05GW/T054H, L282M/Q2S6R, L282SF/Q286K/A2S9R, T059R/S056Q, R047K/N050W/T054H, S265P/L282M/Q286K/A289R, L282M/Q286R/T229S, L2S2F/Q286K, 'S'263W/S285R, S26SP/L2821ttiiQ2«6K,T263H7A273H/S28S'R>.T059R/Sl29Vi 80321717631-1/ A273H/S2S5R, TOS 9R/ S06 6Q/S i 29 Q, T004.§/G024'W, T004V/S023W/G024M, T0S9!C/S066Q/S129Q; L282M/Q286K/A289R/1253V, T0O4V/SO23Y/GO24W, T0S9R/S006N/S129Q; N050F/T054Li TOD4S/SG23N/G024W, T059R/S066N, T059R/SG66N/S129V, Q286S/A289R, N046Q/R047K/N050F/T054K, S26SP/L282M/Q286P/A289R, $265P/L282lvi/Q286R/A289K Q062K/S066Q/S! 291. S023N/G024W, N046Q/R047K/N050W/T054H, RQ47K/T054K, T004L/G024 W, 10 i 4M/T059R/S129V, T059R/S066Q/N092 S/S129f, R047K/N050W/T054K, TQ04V/G024W, N047K7NQ5QF/TØS4K, S265P7L282F /Q286R/N06 l Y, L282F/Q286K/B i 59'V, T004V7S023 Y/G024M, S265P/L282F/A289R/T06S5, T059K7F063L/S066N/S129 V, T004L/S023· W, N05 0F/T054H, T059R/S066Q/S129V, V190I/D220E/S265W/L2S2F, T004S/S023 Y/GG24M, T004L/S023N/G024Y, T059K/S066N/S1291, T059R/S066N/5129J, L282M/Q286R/A289R/Pi62S, N046Q/N050F/T179N, T059K/Y082DS129V, T059K/S1291, N050Y/T054K, Tø59K/SØ<$6Q/V i 02Å/5129Q, TC>59R/S066Q/S129i, T059W/S066N/S i 29V/S290R, T059R/S129J, T059K/S066Q/S129I, TØS9K/S066Q/S129V, S26:5P/L282M/Q286R/A2&amp;9R/T202S/K203'N, T004V/S023N/G024W, S265P/Q286K, S265P/L2&amp;2F/A289R, D220P/S265W, LD55F/T059W/S i29V, T059R/S129Q/S19IR, N050W/T054K, T0Q4S/S023 W/G024M, R047K/NQ50F/TC54H, T059K/S066N/R088E. T059K/S066Q/S 1.29J/V291L, L282M/Q286R/A289R, .TQ59R/S066N/F085S/S1291, L282F/Q286P/A289R, L282F/Q286RM289R, G099D/S205P/L282F/Q286K/A289R;, N046Q/N050F. N050Y/T059W/S066N/S ! 29V, T009i/D220P/S2$5N, V190F/D220P/S265 W, N15 7Y/T263 W/A273 H/S285R, 1263 W/A273 H/S285R, T263W/S285W, T004V/S023Y, N046Q/R047K/N050W, N050W/T0S4L, N200Y/S265P/L2S2E/Q286P/A289R)'r0S9R/SO66Q/P264QI T004V/G024Y, T004L/G024Y, N050Y/SI91 i, N050Y/T054L, Τ0Ο4Ε/'5Ο23\ν/αθ24Υ/Ν155Κ, F1695ZL282P/Q286R/A289R, L2S2M/Q286K/A2S9R, F130L/M138IÆ152W/0183N, NO46Q/'RO47K/N05OY/TO54H! T004V/G024M, N050Y/'rø59W/S066G/S 129V, 5023 N/G024Y,T0S4H/P; 62Q, T004S/S023W/G024Y, N0S0Y/T054H, L282F/Q286R/A289R''F!69!, R047K/N050W, V190F/D220P, L282M/F173Y, T004L/S023 Y, N050W/A2S8D, V190RD220P/S26SQ, S265P/L282F/Q286P/A289R> S265P/L2&amp;2F/Q286R/A289R, N04«OW50Y/T»S4K, T059W/S066Q,· T263 W/A273H/S285W T263W/Å273H/S285P, S023Y/G024M, T004L/S023N/G024W,. T004V/S023 N/G024Y, T059W/S056N/S129Q, T004S/S023Y, T004S/SO23N/GO24M, T059W/S066N/A070T, T0S9W/S.066Q/S129Q, T263W7A273H, A273H/285P, NO46Q/RO47K/N0SOY/TO54L, N046Q/R047K/N05GY, R047K/N050Y, T263H/S2 8 SW, R047K/N0SQFj. N046Q/RQ47K/NQ50F/T054H, S023N/G024M, T0045/G024Y, R047K/N050Y/T054H, T059W/'S066N/S!29i! R047K/T0S4L, T004S/S023W/G024W, 'M138L/B1S2F/T146S, D220P/S265N, TØQ45/G024M, T004V/S023N, N046Q/N050F/T054K, N046Q/N050Y/TO54R Q0-52H/SOS6Q/S129Q, T0S9W/SI29Q, T0S9W/5129V, N050F/T054K, R047K/Nr0S0F/TG54L, V190I/D220P/S265W, NU2I/T263H/A273H/S2S5R, T059W/S066N/S129V, T059W/S0<56Q/S!29I, T0S9W/S1291, T263W/S285P, V190I/D220P, A2S9V/T263H/A273H, T263H/A273E/S285P, N90S/A273H/S285F, R047K/N050Y/T054L* T004S/S623N, T059RZS129Q, N046Q/&amp;047K/T654H> T059W/'S06SQ/S129¥! E152W/T179P, N050Y/$066Q/S129V, T202S/T263W/A273H, T2S3W/A273H/S285P, Mi 3 8L/E152 W/TI79P, N046Q/R047K, N046Q/T054H/F176L, T004L/G024M, T004S/L282M, T263H/A273H, T263H/A273H/S285W, TQ04L/S023Y/G024M, L282F/Q286P, T004V/S023Y/GG24Y, VI90F/S265W, M138L/Ei52F, V190F/D220E/S265W, N046Q/N050F/T054H, N157Y/S285W, T004F/S023 ΥΛ3024Μ, T004V/S023N/G024M, Lt98I/D220E/S2<S5 Q, N046Q/NO50Y/T054K/A154T, SO 16L/D220E/S265W, D220E/S265'% 0220E/A237S/S265 W, S066Q/SI29Q, V190F/D220E/S265Q/T2671, L282M/FI73Y/T2!9% E152F/T179P, V190I/S265W, M138L/S066Q, M138IÆ152W, T059W/SO66Q.'Ad7OT/Si29i, V190F/D220E/S265N, V190F/S26SN, N046Q/N050Y, og M138L/B1S2F/T179P.
  3. 3. Isoleret neutral metalloproteasevariant ifølge krav 1, hvor proteasen omfatter flere mutationer udvalgt fra V1901/D220P, VI901/D220P/S265Q, V190L/D220.E, V190X/D220E/S265Q, V1903/D220E/S265W/L282F, V190L/D220B/S265Q, V1 M?D22QE/S265W, V190UD220E/S265N, T059R/S066Q/S1291, V190J7D220E/S265N, Vi9033/D22øEfS265W(:Vl90PD220gj T059W/S066N/Si29V, T059K/S066Q/Si29V, T059K/Y082C/S129V, T059R/S066N/S129I, S066Q/S129V, T0S9R2S06AQ/S129Y, T059R/S1291, N050Y/T059W/S066N/S129V, D22QP/S265R S066Q/S129S, T0S9 W/5066Q7S129V, T059K/S066Q/S1291, T059R/S129V, N050Y/S066Q/5129V, T059W/S066Q/SI29], NO50Y/T059W/S066Q/SI29V, T0S9K/S129I, D220P/5265W, F13 OL/Μ i 3 S L/T17 9P, S066N/S1291, T059R/SQ66N/S129V, F13W/M138L/T179P, T059R/S066Q/N092S/S129S, S066N/S129V, D220E/S265Q, F130L/M138L/B152W/T179P, T059W/Si29Y, S26SP/L282M/Q286R/A289R, S26SP/L282F7Q2S6R/A289R, T059W/SQ66N/S1291, V i 90I/D220P/S26S W, Fi3 OLÆ i 52 W/Π 79P, Fi30L/M138L/:Ei 52F/T379P, Q062K./S060Q/S129L T059K/S066N/5 i 29Ϊ, Ei52K/TI79Pf S2«5P/L282Mf'Q286K/A289R, Fi30LÆvil3SL/E152H/Tl 79P, T263WM273H/S2SSR, D22GE/S265R Ρ130ί/Μ138Ε/Β152Η/Π79Ρ, F130V/M138'L/E152W/T] 79P, F130I/M138L/E152W/T1 79P, T059W/S1291, D220E/S265W, Fi30V/Mt3:SL/Tl?9F, F130L/Ei52VΓΤ179?, T059R/SI29Q, T263W/S285P, F1301/M138L/B152F/T179P,. BIS2W/T179P, V190L/S265Q, FI30IÆ152F/T179P, L282M/Q286R7A289R/Pi62S, D22GP/S265Q, Μί3SL/E152F/Tϊ79Ρ, F130I/E152Η/Τ! 79F, Mi3SL/Bi52W/T179Ρ, F13 OL/ΤΙ 79P, F13QL/M13«L/E152W7T179P/'Q286H; F130UM13SL/Bi52:H, T263W/A273H/S2SSW, S265P/Q2S6K, T059W7S066Q/$i29Q, T263W/5285R, T059W/S066N/S129Q, T263 W/S28S W, TQ59R/S06SN/S129Q, $265 P/L282M/Q286R/A2 89R/T202S/K203N, 1059 W/S i 29Q, QQ62H/S0e6Q/S 129Q, L282M/Q286RM.289R, VS9øt/D220PYS265N/V291I, V190L/S26SN, F130l2M138IÆ1.52W,N050Y/T059R/S129Q/F130FTi79P/r059K/S066Q/Sl29Qv T059K/Si29Q, S265P/L282M/Q2S6P/A2S9R, S265P/L2S2F/Q286P/A289R, T263W/A273H/S285P, $245?/L282M/Q286K, S016L/D220E/S265W, S0«6Q/SI29Q, S265P/L2S2M/Q286P, L282P/Q286R/A289R, F130V/E152W/T179P, LD44Q/T263W/S285R.L05SF/T0S9W/S129V, Vi90L/S265W, -Q28«.^'A289R> G99.D/S265P/L282F/Q2S6K/A289R, f 130L/Mi38IÆi 52F, T059R/S066Q/S129Q, FI3QI/E152H, S06SN/S129Q, T004S/SG23KG024M/K269N, S26SP/L2S2M, El52F/TI79P, TOS9W/S066N/S129V/S299R, L282P/Q286K/A289R, FISOL/MISSL, F130RML18L/E152W, S265PÆ.282F, P130f/MI38L/Ei52H, F130V/M !38L/Ei 52H, V19GJ/S265Q, M138L/Ei52M, S265P/L282F/Q286?, MI38L/E1S2H, T059K/‘S06FN/K:OS8B, VI90I/S265W, F130L/G152W, L282M/Q286K./A2 89R, L2S2M/Q286K/Å289S/I253V, T263W/A273H, V190I/S265N, M13.3L/B152W, A273H/S2S5R, FBOI/MMSL, FT.1GL/EI S2F, F!3QV/Vf:i38t/E152W, T059K/S06SQ/V102 A/S i 29Q, F130V/E152H/T179F,. FU0I/M13 SUE i 52F, FI30V/M13SL/E152P, M138L/E152F, L2S2WQ286R, F130I/E1S2H, S265P/L282F/A2 89R/T06SS, T263H/A273H/S285R! F13W/M138L, T0I4M/TO59R/S129V, L282M/Q286R/A289IVK] IR A273K/S285P, L282M/Q2 S6K/A289R/S Ϊ32T, T263H/A273H/S285W, f 130V/E5 52W, S265P/L282P/Q286K/N06 S Y, F13{h7£l52W, L198ED220E/S205G, V190I/S26SL T263H/S28SW, S265P/L282P/A289R, Μ13«.25066ζ)> Fl30i/E152F, N90S/A273H/S285P, S032'i7T263Fi/A273H/S285R, L282F/Q286P/A289R, N1S7Y/T263W/A273H/S285R, V105A/S129V. T263H/A273H/S285P, S129Q/L282H, T0S9W/SO66Q, F130V/E152H, 5023397(3024Y, T0C4WSQ23N, T059R/S066Q, NO50W/TOS4L, L282M/Q286P/A289R, A H5 VAG 90L/S265W, L2S2M/Q286K, T0S9R/S066N, L2S2F/Q286P, T004V/S023W/G024M, S265P/L282P/Q286R/L78H, L282P/Q286R, TO04V/S023W/G024Y, S023W/G024M, T059R/R256S, F330V/E152F, T004V/G024W, N05QW/T054K, S023Y/G024M, 1004V/S023Y, T004WSQ33Y/G024M, M050Y/T054R S.023 W/G024W, T004V/S023 Y/G024Y, T004V/S023N/G024W, FI30L/M138L/E152F/T · 79P/V291L NO50Y/TO59K/SO66Q, T004V/S023Y/G024W, T059K/S066H TQ04V/S023N/G024Yv S023Y/G024W, N050F/T054L, R047K/T054K, S023N/G024W, L282M/A289R, S023Y/G024Y, T004V/G024M,, RO47K/N0S0P/T054K, N050F/T054K, T059K/S066Q, S023N/G024M, S023WG024Y, TQ04L/S023H, R047K/N050W/T054H, T004IJS023W/G024Y, T004S/S023W, N046Q/N050W/T054H/A142T, TQ94L/S023Y, T004V/S023W. N05QW/TDS4M, T004S/S023I4, T004S/L282M, T004L/S023W, N050F/T054H, N050Y/T054L·, og R047K^0503Y/T054K,
  4. 4. Isoleret neutral metalloproteasevariant ifølge krav 1, hvor proteasen omfatter flere mutationer udvalgt fra S066Q/S129V, S066Q/S1291, N050Y/S066Q/S129V, S066N/S129]t T059K/S066Q/S129V, S066N/S129V, F13OL/E i52W/Fi79P, S265P/L282M/Q286R/A289R, Fi30LÆ152V7Ti 79P, T059KyS066Q/S 1291, T263W/S285P. T059K7S066N/S129I, T2S3WM273H/S285P, S265P/L282F/Q286R/A289R, F130V/Ei52W/Tl 79P, T263WM.273H/S2S5R, V190J/D220P/S265W, F130L/E152H. S066N/S129Q, S265P/L282M/Q286K/A289R,. V19GJ/D220E, T059R/S066N/S129I, V190I/D220E/S265W, T059K/S i 29i, X059R/S066Q/SI291, Fl3Oi/M138L/E152ΡΪ/ΤΠ9Ρ, F! 30i/Ti79P, T263W/A273H/S285W, SO!617D220E/3205 V, S066Q/S!29Q, V19O1/D220E/S265Q, T059R/S066Q/S129V, D220E/S265N, VI90L/D220E, D220E/S265W, V i 90VD220P,, V i 90-L/D220E/S265N, L044Q/T263W/S285R, S265P/L282M/Q286E/A289R, FlieL/MiSSL/ElSSH/Tn?!3, T263W/S28SR, L282M/Q2S6R/A2S9R, T263W/S285W, F130VE152H/T179P, V i 9Q1/D220E/S265N. V i 90L/D22QE/S265W, VIM/D22QP/S2SSQ, T059R/SQ6SN/S 1-29V, V190L/B220B/S265Q, ΕΪ52Η/Τ179Ρ, Fl30L/Mi38L/BJ52P/T]79P, QQS2H/S060Q/$129Q.. T059K/S129V, Vi9<WD220E/S265W/L282F, VI90I/S265Q, FS301Æi 52P7Ti?9P, D220E/S265Q, BI 52W/T179P, T059K/S066Q/S129Q, PI 3OL/M13SL/TI79P, F130I/Mi3åiÆi52F/T179P, F130L/M138LÆ152W/T179P, NQ50Y/T059W/S066Q/S129V, S265P/L282M/Q286K, T059S/S129I, F130V/E152H/T179P, D220P/S265N, S2S5P/L282M/Q286P, F130I/B1S2H,. TG59R/S060Q/N092S/S \ 291, F i 30L/T ϊ 79P, 0990/δ2<$5Ρ&amp;282Ρ/<2286Κ/Α289Κ, T263W/A273H, V19Q1/S265N'.. D220P/S265 W, FijøLÆmW, Fi30L/M138L/EI52H, S265P/L282M, V190J7S265Q, F130L/ESS2F, T059K/S12-9Q, Q2 86R/A2S9R, Μ i 3 SL/E152W/Ti 79P, P i 3 ØFM i 3 8L/E i 52HS D220P/S265Q, V190L/S26SN, FI30I/M138L/E152W, S265P/Q2S6K, V190L/S2&amp;Q, V190I/S26SW, F130L/M138L/E1S2F, F130V/E152H, E152PTfl79P5 N050Y/T059W/S066N/S129V, T059R/S066N/S529Q, F130f/£!52W, Pi3øV/Ei52W, T059R/'S066Q/SI29Q, T263H/A273H/S2S5P, N90S/A273H/S285P, V190L/.D220E/S265N/V2911, T059K/S S 29Q, A273H/S2SSP, F130i/Mi3SL/EiS2W/Tn9P, F!30V7M138L/£tl52F, NOSOY/TOSPRy'Sl^SQ, T059W/S066Q/S129I, F13 OWM1381/1179P, F130Y/ML38L/EI52W/T179P, V1901/S265W, F130V/M13SL/S152 W, T059W/S066Q/S129V', VI90J/S265Q, F130V/MI38L/EI52H, FJ30J/E152F, NI S7Y/T263W/A273H/S285R, T263H/S285W, M138L/E152F/T! 79P, AS I5V/V190L/S265W, M138L/E152M, T263.H/Å273.H/S28SW» F]3QL/MmLÆ152W» T059K/S066N/K088E, F130I/M138L/E152F, F13GI/M138L/F179P, T004V/S023N, T059K/SG66Q/Vi02A/S S29Q, f 130L/MI38L, N047K/N050F/TO54K, T263H/A273H&amp;285R, F130L/M138L/EI52W/T179P/Q2S6H, MI3SL/E152 H, Mi 38L/S066Q, L282M/Q28&amp;R/A289R/P162S, 12S2F/Q286R/A289R, Q0«K/5066Q/Si29i, A273H/S285K, S26SP/L282P7Q286P, S265P/L282F/Q286F/A289R, S265P/L282M/Q286R/A289R/T202S/K203N, T059W/SG66N/S129.S, VI90I/S265L, T059W/S066N/S229V, FHOI/SVinSL, L282M/Q286K/A289R/J253V, R047K/N050F/T054K, MI38L/ESS2F, N050W/T054K, L19WD220E/S26SQ, U82F/Q286K/A289R, N050F/T054K, L282M/Q286R, M138L/ES52W, S265P/L282F, F130V/£i52F, T059W/S066N/SS29Q, FS3QV/M138L, T263H/A273H, L282M/Q286fC/A289R,N046Q/N050W/T054H/A142T,T059W/S066Q/S129Qi S265P/L282F/A289R/T065S, N050F/T054H, SS29Q/L282H, L282M/Q286K/A289R/S132T, L282M/Q286R/A289R/K1 IN, TQS9K/S066N, R047K/N050W/T054K» T059K/S066Q, T004V/S023Y, T059W/S066N/S129V/S290R, NOSOY/TOS9K/S066Q, og R047K/N050Y.
  5. 5. Polynukleotid, der koder for den neutrale metalloprotease ifølge et hvilket som helst af kravene 1 til 4.
  6. 6. Ekspressionsvektor omfattende nukleotidsekvensen ifølge krav 5.
  7. 7. Værtscelle omfattende ekspressionsvektoren ifølge krav 6.
  8. 8. Fremgangsmåde til fremstilling af et enzym med neutral metalloproteaseaktivitet, omfattende dyrkning af værtscellen ifølge krav 7 på betingelser, der er egnede til fremstillingen af den neutrale metalloproteasevariant.
  9. 9. Fremgangsmåde ifølge krav 8, der endvidere omfatter trinnet med høstning af den fremstillede neutrale metalloproteasevariant.
  10. 10. Fremgangsmåde ifølge krav 7 eller krav 8, hvor værtscellen er en Bacillus-art.
  11. 11. Rengøringssammensætning omfattende den neutrale metalloproteasevariant ifølge et hvilket som helst af kravene 1 til 4.
  12. 12. Fremgangsmåde til rengøring, omfattende trinnet med at bringe en overflade og/eller en artikel omfattende et stof i kontakt med rengøringssammensætningen ifølge krav 11.
  13. 13. Fremgangsmåde ifølge krav 12, hvor overfladen og/eller en artikel omfattende et stof omfatter en græsplet og trinnet med etablering af kontakt omfatter at bringe græspletten i kontakt med rengøringssammensætningen.
  14. 14. Dyrefodersammensætning, tekstilbehandlingssammensætning eller læderbehandlingssammensætning, der omfatter den isolerede neutrale metalloproteasevariant ifølge et hvilket som helst af kravene 1 til 4.
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