RU2575073C2 - Compositions and methods comprising use of version microbial proteases - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention also relates to isolated nucleic acid encoding the subtilisin version, expression vector comprising the nucleic acid, a host cell capable to express the version of subtilisin, and also the cleaning composition comprising the subtilisin version and the method of cleaning.

EFFECT: invention enables to obtain a version of subtilisin with improved washing properties.

15 cl, 17 tbl, 17 ex

Description

The present invention provides methods for producing recombinant proteins and variant proteases. Specifically, the present invention provides methods for using site-determining libraries. The present invention also provides subtilisin variants suitable for various applications.

BACKGROUND OF THE INVENTION

Serine proteases constitute a subgroup of carbonyl hydrolases, which is a separate class of enzymes with a wide range of specificity and biological functions. Subtilisins are the subject of numerous studies, mainly due to their use in the cleaning and food industries. Additional efforts are aimed at harmful environmental conditions (such as exposure to oxidizing agents, chelating agents, extreme temperatures and / or pH), which can adversely affect the functioning of these enzymes in different applications. However, in this area there remains a need for enzymatic systems that are resistant to these adverse conditions while maintaining the same activity, or with increased activity compared to enzymes known in the art.

Various methods for producing recombinant proteins are known in the art. As a rule, proteins are modified to obtain the desired properties. In most methods, mutations are introduced into the nucleotide sequence of the cloned gene encoding the protein, after which the modified gene is expressed to produce mutants that are screened for activity of interest. The properties of the mutants are usually compared with the properties of the wild-type protein.

Historically, the process of constructing proteins has been used as an equivalent approach to solving the problem associated with the discovery in space of a whole protein of the sequence most suitable for the intended use. This problem is extremely complex and "NP-heavy." In complexity theory, problems that belong to class P are considered simple and amenable to effective solutions; polynomial time algorithms exist for their solution. NP-heavy problems are problems for which polynomial time algorithms are not currently available, and if one NP-heavy problem is solved, all NP-heavy problems can be solved (see, for example, Pierce and Winfree, Protein Engineer. 15: 779-782 [2002]). Current library creation and screening strategies typically include obtaining multiple protein sequences that contain random differences throughout the sequence, or differences that occur in a controlled random manner at specific positions of the protein. Such libraries usually contain a large number of members that are “negative” with respect to the original property of interest, therefore, in order to detect a relatively small number of members with positive mutations, a large number of objects must be screened. As a rule, negative mutations are ignored and sequence information is obtained only for positive members.

Saturated mutagenesis (Estell et al., In World Biotech Report 1984, vol. 2: USA, Online Publications, London [1984], pages 181-187; and Wells et al., Gene 34: 315-323 [1985]) represents is a method that can be used to search for mutations in the protein space that optimize some properties of the protein. Several groups have developed strategies for identifying sites to be altered by saturation mutagenesis (Reetz et al., Agnew. Chem. Int. Edn., 44: 4192-4196 [2005]; Kato et al., J. MoI. Biol., 351: 683-692 [2005]; and Sandberg et al., Proc. Natl. Acad. Sci., 90: 8367-8371 [1993]), but there is no general site identification system.

In addition, since most methods of producing recombinant proteins lead to the generation of many amino acid mutations, the screening of a large number of variants is required to obtain a protein with the desired property. Typically, to obtain a useful option, the screening is repeated again and again. Therefore, most methods are time consuming and time consuming. In this area, there continues to be a need for effective methods for producing recombinant proteins that give the desired results.

SUMMARY OF THE INVENTION

The present invention provides methods for producing recombinant proteins. Specifically, the present invention provides methods for using site-determining libraries. In particular, the present invention provides methods for applying the obtained information on a number of target properties, which allow rational and efficient design of libraries to optimize these properties. In some embodiments, the present invention provides methods for constructing libraries improved in at least two target properties.

The present invention provides methods for identifying amino acid sequence positions of a protein that are associated with improving target properties of the protein. In some particularly preferred embodiments, the present invention provides methods for determining mutations resulting in a protein with the specified target properties, as well as with improved properties. In some other particularly preferred embodiments, the present invention provides methods for identifying amino acid positions and mutations leading to a certain percentage of improvement in properties compared to a wild-type protein (for example, one of the properties is improved by 110% compared to a wild-type protein). In further preferred embodiments, the present invention provides methods for identifying mutations that result in a protein, at least one property of which is significantly improved, and at least one other property is not greatly degraded compared to a wild-type protein (for example, one property better by 110% compared to the wild-type protein, and another property is worse by no more than 90% compared to the wild-type protein). In further preferred embodiments, libraries are constructed based on this information. In some embodiments, libraries are constructed using all identified mutations, while in some other embodiments, libraries are constructed using a subgroup of identified mutations. Indeed, it is believed that libraries are not limited to any particular number and / or type of mutation.

The present invention provides methods for producing recombinant proteins, comprising the following steps: obtaining a library of protein variants; testing a library of protein variants with at least one property of interest using an analysis of interest; identifying a range of values for said at least one property of interest; identification of the minimum range of values associated with the favorable result of the analysis of interest; and obtaining a variety of protein variants containing at least one mutation that exceeds the specified minimum range of values characterizing at least one property of interest that comprise a library of protein variants containing at least one mutation, where the library is enriched components that give a favorable result in the analysis of interest. In some embodiments, a favorable result corresponds to a value in excess of about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the maximum value observed in the testing described in the above first step. In some alternative embodiments of the methods of the present invention, several assays of interest are used. In some preferred embodiments, the protein is an enzyme. In some particularly preferred embodiments, the enzyme is selected from the group consisting of proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases and lipases.

The present invention also provides methods for producing recombinant proteins, comprising the following steps: obtaining a library of protein variants; testing a library of protein variants with at least two properties of interest using an analysis of interest; identifying a range of values for at least two properties of interest; identification of the minimum range of values associated with the favorable result of the analysis of interest; and obtaining a plurality of protein variants that exceed the minimum range in at least two properties of interest that comprise a library of protein variants enriched with components characterized by favorable results in the analysis of interest. In some embodiments, a favorable result corresponds to a value in excess of about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the maximum value observed in the testing described in the above first step. In some preferred embodiments, the protein is an enzyme. In some particularly preferred embodiments, the enzyme is selected from the group consisting of proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases and lipases.

The present invention also provides methods for producing recombinant proteins, comprising the following steps: obtaining a wild-type protein and a library of wild-type protein variants; testing a library of variants of the protein and wild-type protein, at least one property of interest using analysis of interest; identifying a range of values for at least one property of interest; identification of the minimum range of values associated with the favorable result of the analysis of interest; identification of protein variants having a favorable result compared with the results obtained for wild-type protein, where a favorable result is associated with an improved property of interest; and obtaining a variety of protein variants that exceed the minimum range of at least one property of interest that comprise a library of improved protein variants enriched with components characterized by favorable results in the analysis of interest. In some preferred embodiments, the methods further include a step of determining a performance indicator, where the performance indicator is determined by dividing the value obtained for each improved variant of the protein by the value obtained for the wild-type protein. In some particularly preferred embodiments, the methods further include the step of identifying improved protein variants, where the improved protein variants have a functional index value greater than 1.1 based on the results of the analysis of interest. In some other embodiments, the protein is an enzyme. In some particularly preferred embodiments, the enzyme is selected from the group consisting of proteases, transferase, metalloprotease, esterase, amylase, cellulase, oxidase, cutinase and lipase. In some alternative embodiments, the protein is selected from antibodies and growth factors. In further preferred embodiments, the wild-type protein is a mature form of an enzyme selected from the group consisting of proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases and lipases. In some preferred embodiments, the property of interest is selected from the group consisting of charge, washing efficiency, cleaning efficiency of hard surfaces, solubility, thermal stability, storage stability, detergent resistance, substrate binding, enzyme inhibition, expression level, reaction rate and degradation substrate. In some embodiments, the wild-type protein and variant protein are components of at least one detergent composition. In some preferred embodiments, the washing efficiency is tested using a detergent composition that is in powder or liquid form, having a pH of from 5 to 12.0.

The present invention also provides methods for producing an improved variant of an initial protein with a specific folding, comprising the steps of: analyzing a plurality of variants of a test protein with a specific folding corresponding to a range of properties of interest according to an analysis of interest; identification of the minimum range of the property of interest associated with the favorable result of the analysis of interest; analysis of the source protein with a specific styling using the method of interest; and obtaining an improved variant of the starting protein by introducing an amino acid substitution into the starting protein, where the improved version is characterized by values that exceed the minimum range of the properties of interest. In some preferred embodiments, the starting protein is different from the test protein. In some embodiments, the methods further include a step of determining a functional indicator, wherein the functional indicator is determined by dividing the value obtained for the improved variant protein by the value obtained for the starting protein. In some embodiments, the test proteins and parent proteins are enzymes. In some particularly preferred embodiments, the enzymes are selected from the group consisting of proteases, transferase, metalloprotease, esterase, amylase, cellulase, oxidase, cutinase and lipase. In some alternative embodiments, the test and parent proteins are selected from antibodies and growth factors. In further preferred embodiments, the starting protein is a mature form of an enzyme selected from the group consisting of proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases and lipases. In some preferred embodiments, the property of interest is selected from the group consisting of charge, washing efficiency, cleaning efficiency of hard surfaces, thermal stability, solubility, storage stability, detergent resistance, substrate binding, enzyme inhibition, expression level, reaction rate and substrate degradation . In some embodiments, test and parent proteins are components of at least one detergent composition. In a separate alternative embodiment, an improved variant of the protein is a component of the detergent composition. In some preferred embodiments, the washing efficiency is tested using a detergent composition that is in powder or liquid form, having a pH of from 5 to 12.0.

The present invention also provides isolated Bacillus subtilisin variants, wherein the subtilisin variant is a mature form having proteolytic activity and containing a substitution at one or more positions selected from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 65, 66, 67, 71, 72, 73, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 14 1, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 203, 204, 206, 207, 209, 210, 212, 213, 214, 215, 216, 217, 218, 219, 220, 222, 223, 224, 225, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 269, 270, 271, 272, 273 and 274, where the positions are numbered according to the amino acid sequence of subtilisin B amyloliquefaciens BPN ', described as SEQ ID NO: 2. In some embodiments, one or more positions are selected from the following group: 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 33, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 65, 66, 67, 71, 72, 73, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 122, 123, 124, 125, 126, 127, 128, 129, 131, 133, 134, 135, 136, 137, 139, 141, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 203, 204, 206, 207, 212, 213, 214, 215, 216, 217, 218, 219, 220, 222, 223, 224, 225, 228, 22 9, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 269, 270, 271, 272, 273 and 274, and where the positions are numbered according to the amino acid sequence of subtilisin B. amyloliquefaciens BPN ', described as SEQ ID NO: 2. In some embodiments, one or more positions are selected from the following group: 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 21, 22, 23, 25, 26, 28, 29, 30, 31, 33, 35, 36, 37, 39, 40, 41, 42, 44, 46, 47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 62, 65, 66, 67, 71, 73, 77, 78, 79, 81, 82, 83, 84, 85, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 102, 105, 106, 110, 111, 112, 113, 114, 115, 116, 122, 123, 124, 125, 126, 128, 129, 131, 133, 134, 135, 136, 137, 139, 141, 143, 145, 146, 147, 148, 149, 150, 151, 152, 153, 156, 157, 158, 159, 160, 162, 165, 166, 167, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 187, 189, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 203, 204, 206, 207, 212, 214, 216, 217, 218, 219, 220, 222, 223, 224, 225, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 249, 250, 251, 253, 254, 25 5, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 269, 270, 271, 272, 273 and 274, and where the positions are numbered according to the amino acid sequence of B. amyloliquefaciens BPN subtilisin 'described as SEQ ID NO: 2. In some embodiments, the subtilisin variant also comprises a substitution at one or more positions selected from 18, 52, 72, 117, 119, 127, 144, 185, 209 and 213, and where the positions are numbered according to the amino acid sequence of B. amyloliquefaciens subtilisin BPN 'described as SEQ ID NO: 2. In some preferred embodiments, the replacement includes one or more selected from the following group: A001C, A001D, A001E, A001F, A001G, A001H, A001I, A001K, A001L, A001M, A001N, A001Q, A001R, A001S, A001V, A001V, A001V, A001V Q002A, Q002D, Q002E, Q002G, Q002S, S003A, S003D, S003F, S003G, S003I, S003K, S003L, S003M, S003N, S003P, S003Q, S003R, S003T, S003V, P003, S003V, P003, S003V, P003, S003V, P003, S003V, P003, S003V, P003, S003V, P003, S003V P005Q, P005T, Y006A, Y006C, Y006E, Y006F, Y006G, Y006K, Y006M, Y006N, Y006P, Y006Q, Y006R, Y006T, Y006V, Y006W, S009A, S009C, S009E, S009E, S009E, S009E, S009E, S009E, S009E, S009E, S009E, S0 S009P, S009Q, S009R, S009T, S009V, S009W, Q010A, Q010C, Q010E, Q010F, Q010G, Q010H, Q010I, Q010K, Q010L, Q010M, Q010R, Q010S, Q010T, Q01101, Q01001, Q01001, Q01101, 010T I011Y, K012A, K012C, K012D, K012E, K012F, K012G, K012H, K012I, K012N, K012Q, K012S, K012T, K012V, K012W, K012Y, A013C, A013F, A013G, A013G, A013G, A01301 13R, A013S, A013T, A013V, P014A, P014C, P014D, P014E, P014F, P014G, P014I, P014K, P014L, P014M, P014N, P014Q, P014R, P014S, P014T, P014, A01501, P01401, P01401, P01401, P01401, P01401, P01401 A015F A015G H017W, H017Y, S018A, S018E, S018F, S018G, S018H, S018I, S018K, S018L, S018M, S018N, S018P, S018Q, S018R, S018T, S018Q, Q01901, Q01901, Q01901, Q01901 Q019K, Q019M, Q019R, Q019T, Q019V, G020A, G020C, G020D, G020E, G020F, G020H, G020K, G020M, G020N, G020P, G020Q, G020R, G02021, Y0210, 021W, 021W, 021F Y021H; Y021K; Y021M; Y021R; Y021S; Y021T; Y021V; Y021W; T022A; T022C; T022E; T022F; T022G; T022H; T022I; T022K; T022L; T022M; S024F, S024G, S024H, S024I, S024K, S024L, S024M, S024N, S024Q, S024R, S024T, S024W, S024Y, N025C, N025E, N025F, N025G, N025H, N025I, N025K, N025L, N025M, N025P, N025R, N025S, N025T, N025V, N025W, K027A, K027D, K027E, K027F, K027G, K027H, K027L, K027M, K027P, K027R, K027S, K027W, K027Y, S033A, S033F, S033G, S033H, S033P, S033T, S033W, I035A, I035C, I035D, I035R, I035S, I035T, I035V, D036A, D036C, D036E, D036Q, D036S, D036W, S037A, S0370, S0370, S0370, S0370, S0370, S0370, S0370, S0370, S0370, S0370, S0370, S0370 S037L, S037M, S037P, S037Q, S037R, S037T, S037W, H039C, H039Q, H039T, H039V, P040A, P040C, P040E, P040F, P040G, P040G, P040H, P0400, P4040, P4040, P4040, P40, P40 P040S, P040T, P040V, P040W, D041E, L042I, L042M, L042V, K043A, K043C, K043D, K043E, K043F, K043G, K043I, K043L, K043M, K043N, K043R, K043R, K0430, K043R, K0430, K0430, K0430, K0430, K0430, K0430, K0430, K0430, K0430, K0430, K0430, K0430, K0430, K0430, V044c, v044i, v044l, v044m, v044p, v044s, v044t, a045c, a045e, a045f, a045h, a045i, a045k, a045l, a045m, a045n G046H, G046K, G046M, G046N, G046Q, G046T, G046W, G046Y, A048C, A048D, A048E, A048F, A048H, A048I, A048K, A048L, A048M, A048 Q, A048R, A048S, A048T, A048V, A048W, A048Y, M050A, M050C, M050F, M050H, M050I, M050K, M050L, M050Q, M050R, M050S, M050T, M050V, M05051, M050W, M050B, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0501, M0500, V051H, V051I, V051L, V051P, V051Q, P052C, P052D, P052E, P052F, P052H, P052I, P052K, P052L, P052Q, P052R, P052S, P052T, P052V, P052W0, S052, S052, S052, S052, S052, S052 S053K, S053L, S053M, S053N, S053Q, S053R, S053T, S053V, S053W, E054A, E054N, E054Q, E054S, T055A, T055C, T055D, T055F, T055G, T055H, T5555, 555, 555, 555, 555, 555, 551 T055S, T055V, T055W, T055Y, N056D, N056M, N056P, N056Q, N056S, N056T, N056V, P057N, P057Q, P057T, P057V, P057W, F058C, F058E, F058G058, 0558 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058 F058V, F058Y, Q059A, Q059C, Q059D, Q059E, Q059F, Q059G, Q059H, Q059K, Q059L, Q059M, Q059N, Q059P, Q059R, Q059S, Q059T, Q059V, Q059W, 060, 060, 060, 060, 060, 060 N062E, N062F, N062G, N062H, N062I, N062K, N062L, N062M, N062Q, N062R, N062S, N062T, N062V, N062W, N062Y, T066S, T066W, T066Y, H0 67A, H067C, H067F, H067I, H067L, H067M, H067N, H067P, H067R, H067S, H067T, T071I, T071Y, N077S, S078A, S078C, S078D, S078F, S078G, S078, S078, 7878, 7878, 78 S078Q, S078R, S078T, S078V, S078W, S078Y, I079A, I079C, I079E, I079F, I079G, I079H, I079K, I079L, I079N, I079Q, I079R, I079S, I079T, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V, I079V V081M, V081T, G083F, G083P, Q084A, Q084C, Q084H, Q084I, Q084N, Q084T, A085C, A085F, A085G, A085R, A085S, P086A, P086D, P086E, P086G0, P086, P086P0, P086, P086P0, P086, P086P0, P086 P086W, P086Y, S089C, S089D, S089E, S089F, S089G, S089H, S089K, S089L, S089V, S089W, S089Y, L090A, L090D, L090E, L090G, L090H, L090M, 090, 090M, 090, 090, 090, 090, 090 Y091E, Y091F, Y091H, Y091I, Y091L, Y091M, Y091Q, Y091S, Y091T, Y091V, L096F, L096H, L096I, L096K, L096M, L096T, L096V, G097A, G09797, G09797, G09797, G09797, G09797, G09797, G09797, G09797, G09797 G097P, G097Q, G097R, G097S, G097T, G097V, A098C, A098D, A098F, A098G, A098H, A098I, A098L, A098P, A098Q, A098R, A098S, A098T, A 098V, A098Y, D099C, D099E, D099I, D099K, D099L, D099N, D099P, D099Q, D099R, D099S, D099T, S101A, S101C, S101E, S101F, S101G, S101I, S101, S101, S01, 101 S101R, S101T, S101V, S101Y, G102A, G102C, G102E, G102F, G102I, G102N, G102S, G102V, G102Y, Q103A, Q103C, Q103E, Q103F, Q103G, Q103I, Q10, Q10, Q10, Q10, Q10, Q10, Q3, Q10, Q3, Q10, Q3, Q3, Q3 Q103T, Q103V, Q103W, S105A, S105C, S105D, S105E, S105F, S105G, S105I, S105K, S105L, S105M, S105N, S105P, S105R, S105V, S105W, W106A, W10, 610, 610, 610, 610 W106l, w106m, w106n, w106r, w106s, w106t, w106v, w106y, n109a, n109c, n109e, n109f, n109g, n109h, n109l, n109m, n109p I111F, I111L, I111M, I111N, I111P, I111T, I111V, I111W, E112A, E112C, E112D, E112F, E112G, E112I, E112L, E112M, E112N, E112Q, E112R, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, E1121, W113E, W113F, W113N, W113P, W113Q, W113S, W113V, W113Y, I115E, I115H, I115N, I115R, I115T, I115V, I115W, I115Y, A116C, A116D, A116E, A116F, A116G, A116H, A116I, A116K, A116L, A116M, A116N, A116P, A116Q, A116R, A116S, A116T, A116V, A116W, A116N, N118G, N118G, N118H, N118H, N118H, N118H, N118H, N118H N118K, N118L, N118M, N118Q, N118R, N118S, N118T, N118V, N118W, N118Y, I122A, I122C, I122H, I122K, I122L, I122M, I122N, I122P, N1233, N1233, N1233, N1233, N1233, N12312 N123M, N123Q, N123S, N123T, N123V, M124C, M124D, M124I, M124L, M124S, M124T, M124V, M124W, S125A, S125C, S125E, S125F, S125I, S125K, S125, S12512, S12512, S12512, S12512, S12512 ,12512, S12512 S125W, L126A, L126C, L126I, L126K, L126N, L126R, L126S, L126T, L126V, L126W, L126Y, G127A, G127C, G127E, G127F, G127I, G127K, G127M, G127G127T, G127G127G127G127G127G12 G127W, G127Y, G128A, G128D, G128F, G128H, G128L, G128N, G128P, G128S, G128T, G128V, G128W, G128Y, P129A, P129C, P129D, P129E, P129F, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912, P12912 P129Q, P129S, P129T, P129V, P129Y, G131A, G131C, G131E, G131F, G131K, G131L, G131M, G131N, G131P, G131Q, G131R, G131S, G131T, G131v, g131w, g131y, a133c, a133d, a133e, a133f, a133g, a133i, a133k, a133l, a133m, a133p, a133r, a133s, a133t A134L, A134M, A134P, A134Q, A134R, A134S, A134T, A134V, A134W, L135D, L135E, L135M, L135R, L135W, L135Y, K136E, K136H, K136L, K136M, K136 K136 K136 K1361313 K136V K136Y A137C A137E A137F A137H K141D, K141E, K141F, K141G, K141H, K141I, K141L, K141M, K141N, K141Q, K141R, K141S, K141V, K141W, K141Y, V143A, V143C, V143D, V143E, V143E, V143E, V143E, V14314 ,14314 ,14314 V143Q, V143R, V143S, V143T, V143W, A144C, A144D, A144E, A144G, A144I, A144K, A144L, A144M, A144R, A144S, A144T, A144V, A144W, S1451, S45, S45, S45, S45, S45, S45, S45, S45 S145I, S145L, S145M, S145Q, S145R, S145T, S145V, S145W, S145Y, G146A, G146C, G146D, G146E, G146M, G146Q, G146R, G146S, G146 T, G146Y, A153S, G154L, G154P, G154T, E156A, E156C, E156F, E156K, E156L, E156N, E156Q, E156R, E156S, E156T, E156V, E156W, E156Y, T158T, 1515T, 1515T, 1515T, 1515T, 1515T T158K, T158L, T158M, T158N, T158Q, T158S, T158V, T158Y, S159A, S159C, S159D, S159E, S159G, S159H, S159I, S159K, S159M, S159Q, S159R, G160, G160, G160, G160, G160 G160Q, G160S, G160T, S162A, S162C, S162E, S162H, S162K, S162L, S162M, S162N, S162Q, S162R, S162T, S162V, S163G, S163P, Y167A, Y167F16, Y167161616 P16 P16 P168s, p168y, g169a, g169e, g169f, g169h, g169i, g169m, g169n, g169r P172A, P172C, P172E, P172K, P172L, P172M, P172N, P172Q, P172R, P172S, P172T, P172V, P172Y, S173A, S173C, S173E, S173I, S173T, S173V4, V17, V17, V17, V17, V17, V17, V17, V17, V17, V17, V17, V17 I175L, I175M, I175R, I175T, I175V, A176C, A176E, A176F, A176K, A176M, A176S, A176Y, A179G, V180A, V180C, V180L, V180N, V180S, V1 80T, D181C, D181E, D181G, D181H, D181M, D181N, D181S, D181T, D181W, S182A, S182C, S182D, S182F, S182G, S182H, S182I, S182K, S182M, S182, S182, S182, 188, S182, 188, S182, 188, S182 S183C, S183E, S183F, S183G, S183H, S183I, S183K, S183L, S183M, S183N, S183Q, S183R, S183T, S183V, S183W, S183Y, N184C, N184D, N184E, N18, N18, N18, N18, N18, N18, N18, N18, N18 N184S, N184T, N184V, N184W, Q185A, Q185C, Q185E, Q185F, Q185G, Q185H, Q185I, Q185K, Q185L, Q185M, Q185N, Q185S, Q185T, Q185V, Q185W, R18, 188, R18, 1818, 187, 187 A187F, A187G, A187P, A187S, A187W, A187Y, S188A, S188C, S188D, S188E, S188F, S188G, S188H, S188I, S188K, S188L, S188M, S188P, S188Q, S18818, S18818, S18818, S188, S188, S188, S188, S188, S188, S188, S188, S18818 S190I, S190K, S191A, S191G, S191N, S191P, V192A, V192S, V192T, V192Y, G193F, G193H, G193I, G193N, G193P, G193R, G193T, P194C, P194E, P19419, P19419, P19419, P19419 P19 P194T, P194V, P194W, L196A, M199P, M199S, A200C, A200G, A200K, A200Y, G202D, G202E, G202F, G202L, G202P, G202V, G202Y, Q206A, Q206C, Q206D, Q206E, Q206F, Q206G, Q206H, Q206I, Q206L, Q206M, Q206N, Q206P, Q206R, Q206S, Q206T, Q206V, Q206W, Q206Y, S207D, S207E7, S20720, 720P, 720P, 720P P210S, P210T, N212A, N212C, N212E, N212F, N212G, N212H, N212K, N212L, N212M, N212P, N212Q, N212R, N212S, N212V, K213A, K213C, K213D, K213, K213, K213, K213, K213, K213, K213, K213, K213, K213, K213, K213, K213, K213, K213, K213, K213 K213N, K213Q, K213R, K213S, K213T, K213V, K213Y, Y214W, G215A, G215C, G215D, G215E, G215I, G215M, G215N, G215Q, G215S, G215T, G215V21616 A21616 A216 A2161616 A216M, A216N, A216P, A216Q, A216R, A216S, A216V, A216W, Y217A, Y217C, Y217D, Y217E, Y217F, Y217G, Y217H, Y217I, Y217K, Y217L, Y217M, Y217N, Y217, Y217T, Y217T Y217W, N218A, N218C, N218E, N218F, N218G, N218H, N218K, N218M, N218R, N218S, N218T, N218W, N218Y, G219A, G219C, G219D, G219H, G21921, G21921, G21921, G21921, G21921, G21921 G219V, G219W, T220D, T220E, T220F, T220G, T220K, T220M, T220S, T220Y, A223E, A223F, A223L, A223M, A223R, A223S, A223V, A223W , A223Y, S224A, S224C, S224F, S224G, S224H, S224M, S224N, S224Q, S224R, S224T, P225A, P225C, P225F, P225G, P225H, P225I, P225K, P225L, P225M, P225R, P225S, P225T, P225V , A228P, A228R, A228S, A228T, A228W, G229A, G229H, G229I, G229S, A230C, A230E, A230G, A230Q, A230R, A230S, A230T, A230V, A231C, A231I, A231P, A231R, I234, 2323A, 2323A, 2323A I234N, I234P, I234Q, I234S, I234T, I234V, L235A, L235C, L235G, L235I, L235K, L235M, L235N, L235Q, L235R, L235S, L235T, L235V, L235236, S23623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623623 , S236H, S236N, S236Q, S236T, S236V, S236Y, K237A, K237E, K237F, K237G, K237H, K237I, K237L, K237M, K237N, K237Q, K237R, K237S, K237238, K237238, K237238, K237238, K237238, K237238, K23723 H238F, H238M, H238R, H238S, P239C, P239D, P239E, P239F, P239H, P239L, P239M, P239N, P239Q, P239R, P239S, P239T, P239V, P239W, P23A, N240, N40 , N240L, N240Q, N240R, N240S, N240T, N240V, N240W, N240Y, W241A, W241F, W241G, W241H, W241I, W241K, W241M, W241Q, W241T, W 241v, t242a, t242c, t242e, t242f, t242g, t242k, t242m, t242n, t242p, t242r, t242s, t242w, t242y, n243c, n243e, n243f, n243g T244A, T244D, T244F, T244G, T244H, T244K, T244L, T244M, T244N, T244P, T244q, t244r, t244s, t244v, t244w, t244y Q245R, Q245T, Q245V, Q245Y, R247W, S248A, S248E, S248F, S248G, S248H, S248I, S248L, S248M, S248N, S248Q, S248R, S248T, S248V, S248W, S248W, S249W, S248W, S248W, S248W, S248W, S248W, S248W, S248W, S248W, S248W, S248W, S248W, S248W S249L, S249M, S249N, S249Q, S249R, S249T, S249V, S249W, S249Y, L250D, L250F, L250H, L250I, L250M, L250T, L250V, E251A, E251T, E251W, N25, N25, N25, N25, N25, N25, N25, N25, N25, N25, N25, N2 N252L, N252M, N252Q, N252R, N252S, N252T, N252V, N252Y, T253A, T253C, T253E, T253G, T253H, T253K, T253M, T253S, T253V, T254A, T254C, T25L, 2525, T25L, T25L, T25L T255C, T255D, T255E, T255F, T255G, T255H, T255I, T255K, T255L, T255M, T255R, T255S, T255V, K256A, K256C, K256D, K256E, K256F, K256H, K256I, K256L, K256M, K256N, K256P, K256Q, K256R, K256S, K256T, K256V, K256W, K256Y, L257A, L257C, L257F, L257G, L257H, L257I25, L25725, L2572525 L257252525252525252525 L257V, L257W, L257Y, G258Q, D259A, D259E, D259F, D259G, D259L, D259N, D259P, D259Q, D259R, D259S, D259T, D259V, D259W, D259Y, S260A, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S26060, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260, S260 S260L, S260M, S260N, S260P, S260R, S260V, S260W, S260Y, F261H, F261W, Y262A, Y262C, Y262E, Y262F, Y262H, Y262L, Y262M, Y262N, Y263F, Y263M, Y263, 642, G26, G64, G64 K265C, K265E, K265G, K265H, K265L, K265M, K265N, K265Q, K265R, K265S, K265W, K265Y, G266C, G266F, G266L, G266M, G266P, G266R, G266V726, 26626, 266, 266, 266 L267H; L267I; L267M; L267N; L267Q; L267S; L267T; L267V; I268A; I268C; I268K; I268L; I268M; I268P; I268R; Q271D, Q271E, Q271F, Q271G, Q271H, Q271I, Q271K, Q271L, Q271M, Q271N, Q271P, Q271R, Q271S, Q271T, Q271V, Q271W, Q271Y, A272E, A272f A274V.

In some preferred embodiments, the replacement includes a combination selected from the following group: Y021H-A045V-Y217E, Y021W-S101E-G128R-Y217Q, Y021H-Y217E, Y021H-A045V-S101N-Y217Q, Y021H-A045I-Y217E -S101E-Y217Q, Y021W-A045I-S101E-Y217E, D036N-S101E-Y217L, Y021H-A045V-Y217Q, Y021H-A045I-S101E-Y217L, Y021H-A045I-Y217E, Y021H-Y217Q, Y021W-S101E-Y217L, Y021W -A045V-S101E-Y217L, Y021H-A045V-S101E-Y217E, S101E-Y217L, Y021H-A045V-S101E-Y217Q, Y021H-Y217L, Y021W-A045I-S101N-Y2121N1-Y111101101101101101101101 , Y021H-S101E-Y217E, M119F-K213K-Y217Q, Y021W-A045V-Y217E, Y021W-A045I-Y217E, M119F-K213I-Y217Q, Y021W-Y217E-N212S, M119Q-Y21I21I21-Y1I21-Y2I21-Y2I21-Y2I-Y2EI-N212S Y021W-A045V-S101E-Y217Q; Y021H-A045V-S101E-Y217L; S101S-M119F-K213I-Y217Q; K213N-Y217Q; M119F-Y217Q; -Y217Q, S101N-M119F-K213K-Y217L, S101N-M119F-K213I-Y217L, Y 021H-A045V-S101E, S101N-K213L-Y217E, A045V-Y217L, S101N-K213I-Y217L, S101S-M119M-K213K-Y217L, Y021H-Y217L, S101E-K213L-Y11711111111111111111111111111111111111 Y021W-A045V-Y217L, A092G-A114G, S024H-A092G-Q103E, Y021W-S101E, V26Q-K213I, Y021H-S101N, S101E-K213N-Y217E. In some preferred embodiments, the replacement includes a combination selected from the following group: S024S-V028V-M050M-A092A-Q103E-A114G-V246V, S024S-V028V-M050V-A092A-Q103Q-A114A-V246V, S024S-V0280-A245V -Q103Q-A114G-V246V, S024H-V028V-M050V-A092A-Q103Q-A114A-V246T, S024H-V028V-M050V-A092A-Q103E-A114A-V246V, S024H-V028V0A4A024V0A6A024V0A6A024V0A0A6A024V0A0A6V0A6A024V0 V028V-M050M-A092A-Q103E-A114A-V246V S024H-V028V-M050M-A092A-Q103E-A114A-V246T S024S-V028V-M050M -A114G-V246T, S101E-M119N-K213N, S101N-K213I, S101N-M119H, M119H-K213I-Y217L, S101N-M119H-K213N-Y217L, S101N-K213L-Y21711-M1113111211 M1113 K113 S101P-K213N; S101N-M119H-K213N; S101N-M119F-K213I-Y217L; K213L; M119N-K213N; K213N; K213I-Y217E; S101N-M119H-Y217Q; , M119H-K213N, S101N-K213L-Y217Q, S101P-K213N, S101E-K213N-Y217E, S1 01E-M119H-K213I-Y217Q; S101E-M119H-K213N; K213I-Y217Q; S101N-K213I-Y217Q; M119H-Y217Q; Y217L; M119F-K213L; M119H-K213N-Y217E; S101N-M119N-K213N-Y217Q; S101N-M119H-K213N-Y217Q; Y217Q; S101N-M119F-K213I-Y217Q; S101N-K213I-Y217L; S101N-M119H-K213N-Y217L; S101E-K213L-Y217L; K213N-Y217L; M119F-K213I-Y217Q; S101N-K213I-Y217Q; S101E-M119H-K213I-Y217L; S101N-M119H-K213I-Y217Q; M119H-K213N-Y217E; A048H-K213L, V026Q-A048Y-K213L, K213N, V026N-K213L, V026N-K213L, V026Y-K213N, A048D-K213N, V026Q-A048E-K213L, A048H-K213N, KL1313, V026H-K213N, V026H-K213N, V026 K213N, V147D-K213L, K213I, V026Q-A048E-K213N, V026N-A048E-K213N, A048E-K213L, V026Y-A048E-K213I, A048D-K213N, K213I, A048H-012N1 K113N2132N1 K1131, K1131 Y217L, Y021H-Y217L, Y021H-A045V-S101E-Y217Q, Y 021H-A045I-S101E-Y217L, Y021H-Y217Q, Y021H-A045V-Y217E, A045V-Y217L, Y021H-A045V-S101N-Y217Q, Y021W-S101P-Y217L, Y021W-A0E-Y021W-A0E1010-Y021W-A0E0 Y021H-A045I-S101E-Y217L; Y021H-A045I-S101E-Y217Q; Y021H-A045V-Y217E; Y021W-S101E-Y217L; S101E-Y217L; A045I-Y217Q; Y021H-A045V-Y217E; Y021W-A045V-S101E-Y217Q; Y021H-S101E-Y217E; Y021W-S101N-Y217L; Y021w-a045v-s101e-y217l K213I-Y217Q, S101S-M119M-K213K-Y217L, Y021H-A045I-S101E-Y217Q, Y021H-A045V-S101E-Y217E, Y021H-A045V-S101E, Y021H-Y21721-Y1I0-Y021I0-Y021I0-Y021I0-Y021I0-Y021I0-Y021I1-Y0I0 Y217E; S101N-M119F-K213K-Y217L; S101E-M119H-K213N-Y217E; Y021H-A045I-Y217E; S101S-M119H-K213L-Y217L; Y021H-Y217E; Y217L. In some preferred embodiments, the replacement includes a combination selected from the following group: S024S-V028T-M050V-A092G-Q103E-A114G-V246T, S101N-M119H, M119N-K213N-Y217L, Y217L, M119N-K213N, K213N S101E-K213N-Y217E S101E-M119N-Y217L Y217Q S101N-M119N-K2113E -K213K-Y217L, Y021H-A045I-S101E-Y217L.

In addition, the present invention provides the options described in the tables in the Examples section, as well as compositions containing these options.

The present invention also provides isolated nucleic acids encoding a subtilisin variant, expression vectors containing said nucleic acids, and host cells containing said vectors. In addition, the present invention provides cleaning compositions comprising a subtilisin variant. In some embodiments, the cleaning composition is a laundry detergent. In some embodiments, the laundry detergent is a cold water detergent, a low pH detergent, or a compact detergent. In addition, the present invention provides methods for producing a Bacillus subtilisin variant, comprising: transforming the host cell with an expression vector containing a nucleic acid encoding a subtilisin variant; and culturing the transformed host cell under conditions suitable for obtaining a variant of subtilisin. In some embodiments, the methods further include the step of isolating the resulting subtilisin variant. In some embodiments, the host cell is a Bacillus species, and in a subset of these embodiments, the Bacillus species is B. subtilis. The present invention also provides a cleaning method comprising the step of bringing into contact a surface and / or fabric containing article with a cleaning composition comprising an isolated subtilisin variant.

In addition, the present invention provides methods for producing a recombinant protease, comprising the following steps: a) obtaining a set of site-determining libraries (SEL), each of which contains many variants of the protease containing different substitutions at the same amino acid positions; b) testing protease variants from SEL and a standard protease for a property of interest; c) determination of a performance indicator (PI) for all protease variants tested; d) identifying two or more amino acid positions as non-limiting, wherein at least one of the plurality of protease variants of each of the two SELs has a PI above 0.5; and f) obtaining a library of multiple mutations containing a plurality of protease variants with multiple substitutions, each variant containing substitutions in two or more non-limiting positions. In some embodiments, the test includes two or more different assays selected from the group consisting of stain removal analysis (from microsamples), LAS resistance analysis, detergent resistance analysis, and specific activity analysis. In some embodiments, the protease is selected from the group consisting of bacterial serine protease, bacterial subtilisin, and bacterial neutral metalloprotease.

In other embodiments, the present invention provides methods for producing a Bacillus multi-substitution subtilisin variant, comprising: testing a plurality of one-substitution subtilisin variants using a first analysis of the first property and a second analysis of the second property, where the property of the original subtilisin is assigned a value of 1.0 in each test , a favorable first or second property has a value greater than 1.0, and an excessively unfavorable first or second property has a value of less than about 0.80, or, in some sim ilar embodiments, less than about 0.60; identification of a substitution in at least one embodiment of subtilisin with one substitution that is associated with a favorable first property and which is not associated with an unnecessarily unfavorable second property; identification of a substitution in at least one variant of subtilisin with one substitution that is associated with a favorable second property and which is not associated with an unnecessarily unfavorable first property; and introducing a substitution identified in the previous steps into subtilisin to form a multiple substitution subtilisin variant. In some embodiments, the methods further include testing multiple-substitution variants of subtilisin using a first analysis and a second analysis, where an improved version of subtilisin reaches values greater than 1.0 based on the results of both the first and second analyzes, values greater than 1.0 in the first analysis and values from 0.80 to 1.0 in the second analysis. In some embodiments, the methods further include providing improved subtilisin variant (s). In some embodiments, the first and second properties have a negative correlation. In some embodiments, the favorable first or second property has a value of greater than about 1.2. In some embodiments, an excessively unfavorable first or second property has a value of less than about 0.40. In some embodiments, the first property is stability, and the second property is cleaning efficiency. In the subset of these properties, stability includes detergent resistance, and cleaning efficiency includes the removal of blood, milk, ink (BMI) in the detergent. In some embodiments, the original bacterial subtilisin is a mature form of B. amyloliquefaciens BPN 'subtilisin having the amino acid sequence described as SEQ ID NO: 2. In other embodiments, the original bacterial subtilisin is a mature form of wild-type B. lentus GG36 subtilisin having the amino acid sequence described as SEQ ID NO: 562 or BPN "containing the substitution Y217L (SEQ ID NO: 565), alone or in combination with In some embodiments of the present invention, the washing efficiency is tested in a powder or liquid detergent composition having a pH of from 5 to 12.0. It is assumed that the order of the steps is not limited to the exact ones listed above, since in the present invention You can use any suitable order. However, in some preferred embodiments greater than about 50%, or greater than about 65% of substitutions are at positions constituting Original subtilisin solvent accessible surface (SAS).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a pHPLT-VAAcl map, and FIG. 1B shows a pHPLT-BPN 'map.

On figa depicts the dependence of the cleaning ability of BPN'-Y217L CCL in the North American washing detergent against BMI from changes in charge. Similarly, FIG. 2B depicts the dependence of the cleaning ability of GG36 CCL in a North American washing detergent on BMI on a change in charge.

FIG. 3A depicts the dependence of the cleaning ability of BPN'-Y217L CCL in West European liquid washing detergent on BMI versus charge change. Similarly, FIG. 3B depicts the dependence of the cleaning ability of GG36 CCL in a West European liquid washing detergent on BMI versus charge change.

FIG. 4A shows the dependence of the cleaning ability of BPN'-Y217L CCL in Japanese BMI detergent powder detergent on charge change. Similarly, FIG. 4B shows the dependence of the cleaning ability of GG36 CCL in Japanese BMI detergent powder detergent on charge change.

Fig. 5A shows the dependence of the cleaning ability of the BPN'-Y217L CCL in dishwasher detergent on sintered egg yolk against a change in charge. Similarly, FIG. 5B depicts the dependence of the cleaning ability of GG36 CCL in dishwasher detergent on sintered egg yolk on a change in charge.

Figure 6 shows the dependence of resistance to LAS / EDTA on the change in the total charge compared to the original BPN'-Y217L, for a library containing 80 options.

DESCRIPTION OF THE INVENTION

The present invention provides methods for producing recombinant proteins. Specifically, the present invention provides methods for using site-determining libraries.

In practice, there is usually no need to identify the best sequence in protein space in order to create a protein that is optimal for a particular application. In most applications, the problem to be solved is the identification of at least one protein sequence, which is characterized by a value equal to or greater than the minimum value required for a number of properties. To solve this problem, information about mutations that have a beneficial effect on a particular property is needed, as well as information about mutations that have an adverse effect on one of the target properties. The present invention provides methods for achieving the goal by identifying positions in the protein, the change of which can lead to an improvement in the basic properties and the preservation of other properties within the desired range.

The present invention provides methods for determining all protein positions involved in the formation of all properties of interest by creating "site-determining libraries" for each site. In preferred embodiments, such libraries contain 9-19 mutations at each position and are used to analyze each position for applicability in methods for producing recombinant proteins and constructing libraries. Each property is measured in comparison with the starting enzyme and the apparent difference in free energy of each mutant and wild-type enzyme is calculated. The indicated apparent values of delta delta G ("ie, ΔΔG") are used to determine additivity.

The ideal way to analyze options is to determine the difference in free energy between the option and the source protein in the process of interest. Gibbs free energy for a process is defined as the maximum amount of work that the system can perform. The change in free energy compared to the starting enzyme (ΔΔG) is calculated using the following formula:

ΔΔG = -RT ln (k _variant / k _parent )

where k _variant is the reaction rate constant of the variant enzyme, and k _parent is the reaction rate constant of the starting enzyme, R is the gas constant, and T is the absolute temperature. Most analyzes are not designed to determine the true free energy, so the authors use the following formula for calculations:

ΔΔG = -RT ln (P _variant / P _parent )

where P _variant is the performance of the variant, and P _parent is the performance of the original enzyme, measured under the same conditions. It can be expected that from the point of view of data distribution and additivity, the ΔΔG _app values behave the same as the ΔΔG values. However, since ΔΔG represents the maximum amount of work that the option can perform compared to the original enzyme, the value of ΔΔG _app , as a rule, may be lower than the value of ΔΔG, indicating that the results can be synergistic, since the properties of the two additive positions can be higher than the value obtained by adding the values of ΔΔG _app .

The methods of the present invention can be used to obtain effective libraries that are used to develop several properties simultaneously. Although some enzymes are described herein, these methods can be applied to any protein of interest from the point of view of genetic engineering. Site-determining libraries (SELs) are obtained by the methods described herein by introducing from 12 to 19 substitutions for each position. The resulting mutations are analyzed using analyzes of activity and stability. For each position, the wild-type amino acid is given as a comparison point. For each property of the starting enzyme, the value and standard deviation ΔΔG _{app are} determined. The value obtained for the starting enzyme (μ _parent ) is normalized to 0 and the standard deviation (σ _parent ) for ΔΔG _{ap is} determined. The indicated values are used for comparison when measuring each property for each position of the molecule. Site determination results are analyzed for evidence of correlation of properties. Graphs are built of the dependence of the ΔΔG _app values of one property on the ΔΔG _app values of another property and the correlation coefficients are calculated. A correlation of the two activities with respect to the substrates of the two proteins is observed.

To analyze the positions of the amino acid sequence, two types of sites are determined. "Unproductive" sites do not contain mutations responsible for the improvement of properties compared with the original enzyme, while "productive" sites contain at least one substitution leading to improved properties compared to the original enzyme. The probability that a site is productive is determined by dividing the number of productive sites by the total number of sites. Although the likelihood that a mutation leads to an improvement over the original enzyme is low (i.e., 6% -28%), the likelihood that a given site contains at least one upward mutation is very high.

It is important to determine the relationship between the distribution of productive and non-productive sites and structural features (such as hidden amino acids, interacting amino acids, positions near the active center, etc.) of the protease, as well as conservative or variable parts of the sequence during evolution. This determination is carried out by analyzing the structure and comparing the sequence with the main homologues.

As indicated in the examples, mutations that have a harmful effect on one property correlate with mutations that have a harmful effect on another property, regardless of the ratio of properties. Only in a small number of positions (5-10%) can mutations be present that have a harmful effect on all properties. Such provisions define “styling” and are conservative in terms of evolution. The significance of this phenomenon lies in the fact that, in contrast to the identification of improving mutations, which requires the use of, in essence, predictive screening for this property, the identification of mutations that are supposed to have a harmful effect on any property can be performed using any screening. A simplified recombinant protein strategy involves constructing SEL and screening using simple activity and / or stability assays. The harmful mutations are identified and then the positions in which several harmful mutations may be present are used to obtain libraries and combinatorial mutations with the improvement of several properties. In addition, the choice of sites that are on the surface of the protein interact with a few other elements and are variable according to the results of aligning sequences, which allows to achieve a high ratio of productive sites. Areas that are located in the inner part of the molecule come into contact with many other elements and are highly conservative from the point of view of evolution, are characterized by a high probability of harmful mutations, so they should be avoided. It is implied that in the present invention, you can use any suitable method for analyzing the sequence and / or information about the structure, including, without limitation, computer and / or electronic methods and / or programs.

The methods allow for pairwise comparison of a number of options, the activity of which is more than 5% of the mass. and less than 5% for each of the two properties, and determine the correlation coefficients for the two properties.

Getting Libraries

In some particularly preferred embodiments, the results of using site-determining libraries are used to produce combinatorial libraries. The traditional directed evolution includes obtaining statistical libraries and screening a large number of libraries for individual properties, combining these processes and repeating them. Several researchers have found (see, for example, Bloom et al, Curr. Opin. Struct. Biol., 15: 447-452 [2005]; Bloom et al., Proc. Natl. Acad. Sci. USA 103: 5869-5874 [2006]; and Guo et al., Proc. Natl. Acad. Sci. USA 101: 9205-9210 [2004]) that the accumulation of positive mutations in one property usually leads to deterioration of other properties. The likelihood that a mutation will lead to an improvement in a property is low, the likelihood that the mutation will be deteriorating is high (> 85%), and the likelihood that more than three (3) mutations increase will increase activity, will lead to a deterioration of several other properties, is very high. This problem can be avoided by using site-specific data to build libraries with several advanced features. Non-productive sites are not used to create combinatorial libraries, and productive sites are additionally classified by the percentage of improving mutations.

Cleaning compositions

The cleaning compositions of the present invention are preferably used, for example, for washing, cleaning hard surfaces, as means for dishwashers, and also as cosmetics, for example, for dentures, teeth, hair and skin. However, due to the unique advantages of increased efficiency in solutions at a reduced temperature, the enzymes of the present invention are ideally suited for washing. In addition, the enzymes of the present invention can be used in the composition of both granular and liquid compositions.

Variant proteases of the present invention can also be used as auxiliary products for detergents. In some embodiments, cleaning compositions that are effective in solution at low temperatures are used. The auxiliary product may include, in its simplest form, one or more proteases. In some embodiments, the adjuvant is incorporated into a medicament to be added to a cleaning process. Any suitable unit dosage form may also be used in the present invention, including, without limitation, pills, tablets, capsules, or other forms, such as dosage powders or liquids. In some embodiments, to increase the volume of such a composition, a filler (s) or a carrier (s) are added to the composition. Suitable fillers or carriers include, but are not limited to, various salts such as sulfates, carbonates and silicates, as well as talc, clay, and the like. Suitable excipients or carriers for liquid compositions include, without limitation, water or low molecular weight primary and secondary alcohols, including polyols and diols. Examples of such alcohols include, without limitation, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions comprise from about 5% to 90% of such substances. Acidic fillers are used to reduce pH. Alternatively, cleaning additives include additional ingredients, which are described in more detail below.

The cleaning compositions and cleaning additives of the present invention comprise an effective amount of at least one of the protease variants described herein, used alone or in combination with other proteases and / or other enzymes. The desired level of enzyme is achieved by adding one or more protease variants of the present invention. Typically, the cleaning compositions of the present invention contain at least about 0.0001 weight percent, about 0.0001 to 1, about 0.001 to 0.5, or even about 0.01 to 0.1 weight percent, at least one of the variant proteases of the present invention.

The cleaning compositions described herein are usually prepared in view of the fact that during water cleaning operations, the washing water has a pH from about 5.0 to 11.5, or even from about 7.5 to 10.5. The pH of the undiluted liquid compositions is usually from about 3.0 to 9.0, or even from about 3 to 5. Granular laundry detergents usually have a pH from about 9 to 11. Methods for maintaining the recommended pH are well known to those skilled in the art and include the use of buffers, alkalis, acids, etc.

Suitable cleaners with low pH in undiluted form typically have a pH of about 3 to 5, and typically do not contain surfactants that hydrolyze in the indicated pH range. Such surfactants include sodium alkyl sulfate-based compounds that contain at least one ethylene oxide moiety, or even from about 1 to 16 moles of ethylene oxide. Said cleaning compositions typically contain an amount of a pH modifier, such as sodium hydroxide, monoethanolamine or hydrochloric acid, sufficient to provide a pH of the undiluted cleaning composition of about 3 to 5. Such compositions usually contain at least one acid resistant enzyme. In some embodiments, the compositions are liquid, and in other embodiments they are in solid form. The pH of the liquid composition is usually measured undiluted. The pH of the solid composition is measured as a 10% solution of the specified composition in distilled water. In these embodiments, all pH measurements are carried out at 20 ° C.

In some embodiments, if the variant protease (variant proteases) is used as part of a granular or liquid composition, it is desirable that the variant protease be encapsulated to protect it from other components of the granular composition during storage. In addition, encapsulation allows you to control the availability of the variant protease during the cleaning process. In some embodiments, encapsulation enhances the efficacy of variant protease (variant proteases) and / or other enzymes. In this regard, the variant proteases of the present invention are encapsulated using any suitable encapsulating agent known in the art. In some embodiments, at least a portion of the variant protease catalyst (variant proteases) of the present invention is typically encapsulated. Typically, an encapsulating substance is capable of dissolving and / or dispersing in water. In some embodiments, the glass transition temperature (Tg) of the encapsulating substance is 0 ° C or higher. The glass transition temperature is described in more detail in WO 97/11151. The encapsulating substance is selected from the group consisting of carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes, and combinations thereof. If the encapsulating substance is a carbohydrate, it is usually selected from monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. Typically, the encapsulating substance is starch. Suitable starches are described in EP 0922499; US 4,977,252; US 5354559 and US 5935826. In some embodiments, the encapsulating substance is a microsphere obtained from plastic, such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof; suitable commercially available microspheres include the supplied EXPANCEL® (Stockviksverken, Sweden) and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL® and SPHERICEL® (PQ Corp., Valley Forge, PA )

As described herein, variant proteases of the present invention can be used, in particular, in the cleaning industry, including, without limitation, in the composition of detergents and detergents for washing dishes. In this application, the enzymes are exposed to different environmental conditions. Variant proteases of the present invention have advantages over many currently used enzymes, which are stable under different conditions.

In fact, there are different washing conditions that the proteases involved in washing are exposed to; these conditions include the composition of the detergent composition, the volume of washing water, the temperature of the washing water, and the duration of the wash. In addition, detergent compositions used in different geographical regions provide different concentrations of the respective components in the washing water. For example, a European detergent typically provides approximately 4,500-5,000 ppm. detergent components in washing water, while Japanese detergent typically provides approximately 667 ppm. detergent components in washing water. In North America, especially in the United States, detergents typically provide approximately 975 ppm. detergent components in washing water.

A low detergent concentration system includes detergents that provide less than about 800 ppm. detergent components in washing water. Japanese detergents are typically low detergent systems because they provide approximately 667 ppm. detergent components in washing water.

A system with an average concentration of detergent includes detergents that provide approximately 800 ppm. up to 2000 ppm detergent components in washing water. North American detergents are generally considered systems with an average concentration of detergent, as they provide approximately 975 ppm. detergent components in washing water. Brazilian detergents typically provide approximately 1,500 ppm. detergent components in washing water.

A system with a high concentration of detergent includes detergents that provide more than about 2000 ppm. detergent components in washing water. European detergents are generally considered systems with a high concentration of detergent, as they provide approximately 4,500-5,000 ppm. detergent components in washing water.

Latin American detergents, which usually contain phosphate components and are characterized by high foaming, can be classified both as systems with an average and as a system with a high concentration of detergent, since they provide from 1500 to 6000 ppm. detergent components in washing water. As indicated above, Brazilian detergent typically provides approximately 1,500 ppm. detergent components in washing water. However, in other geographical regions, not limited to Latin America, detergents containing phosphate components and characterized by high foaming can be produced, which are systems with a high concentration of detergent and provide up to 6000 ppm. detergent components in washing water.

In light of the above, it is obvious that the concentrations of detergent components in typical washing solutions of different countries vary from less than 800 ppm. ("geographic varieties with a low concentration of detergent"), for example, about 667 ppm in Japan, up to about 800-2000 ppm ("geographical varieties with an average concentration of detergent"), for example, about 975 ppm. in the USA and approximately 1,500 ppm in Brazil, and up to more than about 2000 ppm ("geographic varieties with a high concentration of detergent"), for example, from about 4,500 to 5,000 ppm. in Europe and approximately 6000 ppm in geographical varieties that contain phosphate components and are characterized by high foaming.

Concentrations in typical washing solutions are determined empirically. For example, in the United States, a typical washing machine holds approximately 64.4 liters of washing solution. Accordingly, in order to obtain a detergent concentration in the washing solution of approximately 975 ppm, approximately 62.79 g of the detergent composition must be added to 64.4 L of the washing solution. This amount is usually measured by the consumer in the washing solution using a measuring cup supplied with the detergent.

On the other hand, different washing regions use different washing temperatures. Japan usually uses a lower temperature of washing water than in Europe. For example, in North America and Japan, the temperature of the washing water is usually about 10 to 30 ° C (for example, about 20 ° C), while in Europe the temperature of the washing water is usually about 30 to 60 ° C (for example, about 40 ° C)

In another example, in different geographical regions, water has different hardness. Water hardness is usually described in terms of the amount of mixed granules Ca ²⁺ / Mg ²⁺ per gallon. Water hardness is a measure of the amount of calcium (Ca ²⁺ ) and magnesium (Mg ²⁺ ) in water. Most of the water in the United States is hard, but the degree of hardness varies. Water with a hardness from medium (60-120 ppm) to high (121-181 ppm) contains from 60 to 181 parts per million (parts per million are converted into grains per US gallon by dividing the ppm # 17.1) stiffening minerals.

Water Grains per gallon Ppm Soft less than 1.0 less than 17 Low rigidity from 1.0 to 3.5 from 17 to 60 Medium hard from 3.5 to 7.0 from 60 to 120 Tough from 7.0 to 10.5 from 120 to 180 Very tough more than 10.5 more than 180

The hardness of European water is usually more than 10.5 (for example 10.5-20.0) mixed Ca ²⁺ / Mg ²⁺ grains per gallon (for example, about 15 mixed Ca ²⁺ / Mg ²⁺ grains per gallon). Water hardness in North America is usually greater than water hardness in Japan, but less than water hardness in Europe. For example, water hardness in North America can range from 3 to 10 grains, 3-8 grains, or about 6 grains. Water hardness in Japan is generally lower than water hardness in North America, usually less than 4, for example 3 mixed grains of Ca ²⁺ / Mg ²⁺ per gallon.

Accordingly, in some embodiments, the present invention provides variant proteases that exhibit unexpected performance when washing in at least one set of conditions (including water temperature, water hardness and / or detergent concentration). In some embodiments, the variant proteases of the present invention are comparable in wash characteristics to other subtilisin proteases. In some embodiments, the variant proteases of the present invention have improved washing characteristics compared to currently available subtilisin proteases. Thus, in certain preferred embodiments of the present invention, variant proteases are provided that have enhanced oxidation resistance, increased thermal stability and / or increased resistance to chelating agents. In addition, the variant proteases of the present invention can be used to obtain cleaning compositions that do not contain detergents, again, alone or in combination with detergent components and stabilizers.

In some embodiments of the present invention, the cleaning compositions comprise at least one variant protease of the present invention in an amount of from about 0.00001% to about 10% based on the weight of the composition, and the rest (e.g., from about 99.999% to approximately 90.0% based on the weight of the composition), including auxiliary cleaning agents. In other aspects of the present invention, the cleaning compositions of the present invention comprise at least one variant protease in an amount of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2% from about 0.005% to about 0.5% based on the weight of the composition, and the rest of the cleaning composition (for example, from about 99.9999% to about 90.0%, from about 99.999% to about 98%, from about 99.995% to about 99.5% by weight) including auxiliary cleaning agents.

In some embodiments, preferred cleaning compositions, in addition to one or more of the variant proteases described herein, comprise one or more other enzymes or derivative enzymes that provide improved cleaning and / or tissue care performance. Such enzymes include, without limitation, other proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g. laccases) and / or mannases.

Any other suitable protease may be used in the compositions of the present invention. Suitable proteases include animal, plant or microbial proteases. In some particularly preferred embodiments, microbial proteases are used. In some embodiments, chemically or genetically modified mutants are used. In some embodiments, the protease is a serine protease, preferably an alkaline microbial protease or trypsin-like protease. Examples of alkaline proteases include subtilisins, especially those derived from Bacillus (e.g., subtilisin, lentus , amyloliquefaciens , subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168). Other examples include mutant proteases described in US Patent Nos. RE 34606, 5955340, 5700676, 6312936 and 6482628, which are incorporated herein by reference. Other examples of proteases include, but are not limited to, trypsin (e.g., porcine or bovine) and the Fusarium protease described in WO 89/06270. Preferred commercially available protease enzymes include MAXATASE®, MAXACAL ^™ , MAXAPEM ^™ , OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT® and PURAFECT® OXP (Genencor); ^{ALCALASE®, SAVINASE®, PRIMASE®, DURAZYM TM} , RELASE® and ESPERASE® (Novozymes); and ^{BLAP TM (Henkel Kommanditgesellschaft auf Aktien,} Duesseldorf, Germany). Various proteases are described in WO95 / 23221, WO 92/21760 and US patents Nos. 5801039, 5340735, 5500364, 5855625, US RE 34606, 5955340, 5700676, 6312936 and 6482628, as well as other patents.

In addition, any suitable lipase may be used in the present invention. Suitable lipases include, but are not limited to, bacterial or fungal enzymes. Chemically or genetically modified mutant forms are within the scope of the present invention. Examples of suitable lipases include lipase Humicola lanuginosa (see., E.g., EP 258068 and EP 305216), lipase Rhizomucor miehei (see., E.g., EP 238023), lipase Candida lipase, such as a lipase C. antarctica (e.g. lipase C antarctica A or B; see, for example, EP 214761), Pseudomonas lipase such as P. alcaligenes and P. pseudoalcaligenes lipase (see, for example, EP 218272), P. cepacia lipase (see, for example, EP 331376 ), P. stutzeri lipase (see, for example, GB 1372034), P. fluorescens lipase, Bacillus lipase (eg B. subtilis lipase [Dartois et al., Biochem. Biophys. Acta 1131: 253-260 [1993]) ; B. stearothermophilus lipase [see, for example, JP 64/744992]; and B. pumilus lipase [see, for example, WO 91/16422]).

In addition, in some embodiments of the present invention, a number of cloned lipases may be used, including, but not limited to, Penicillium camembertii lipase (see Yamaguchi et al., Gene 103: 61-67 [1991]), Geotricum candidum lipase (see , Schimada et al, J. Biochem., 106: 383-388 [1989]), and various Rhizopus lipases, such as R. delemar lipase (see, Hass et al, Gene 109: 117-113 [1991]), lipase SL R. niveus (Kugimiya et al., Biosci. Biotech. Biochem. 56: 716-719 [1992]) and lipase R. oryzae .

Other types of lipolytic enzymes, such as cutinases, which include, without limitation, cutinase derived from Pseudomonas mendocina (see WO 88/09367) and cutinase derived from Fusarium solani pisi, can also be used in some embodiments of the present invention. (see WO 90/09446).

Other suitable lipases include commercially available enzymes such as MI LIPASE ^™ , LUMA FAST ^™ and LIPOMAX ^™ (Genencor); LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE P ^TM "Amano" (Amano Pharmaceutical Co. Ltd., Japan).

In some embodiments of the present invention, the cleaning compositions further comprise lipases in an amount of from about 0.00001% to about 10% of the additional lipase relative to the weight of the composition, where the rest of the weight of the composition is supplemented by auxiliary cleaning agents. In other aspects of the present invention, the cleaning compositions further comprise lipases in an amount of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to about 0 5% based on the weight of the composition.

Any amylases (alpha and / or beta) suitable for use in alkaline solutions may also be used in some embodiments of the present invention. Suitable amylases include, but are not limited to, enzymes of bacterial or fungal origin. Some embodiments encompass the use of chemically or genetically modified mutant forms. Amylases suitable for use in the present invention include, but are not limited to, α-amylases derived from B. licheniformis (see, for example, GB 1296839). Commercially available amylases suitable for use in the present invention include, without limitation, DURAMYL®, TERMAMYL®, FUNGAMYL® and BAN ^™ (Novozymes), as well as RAPIDASE® and MAXAMYL® P (Genencor).

In some embodiments of the present invention, the cleaning compositions further comprise amylases in an amount of from about 0.00001% to about 10% additional amylase based on the weight of the composition, where adjuvants are supplemented with the rest of the weight of the composition. In other aspects of the present invention, the cleaning compositions of the present invention further comprise amylases in an amount of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to about 0.5% by weight of the composition.

In some other embodiments, the cleaning compositions of the present invention may contain any suitable cellulase. Suitable cellulases include, without limitation, enzymes of bacterial or fungal origin. Some embodiments encompass the use of chemically or genetically modified mutant forms. Suitable cellulases include, but are not limited to, Humicola insolens cellulases (see, for example, US Pat. No. 4,435,307). Particularly preferred are cellulases that provide better color retention (see, for example, EP 0495257). Commercially available cellulases suitable for use in the present invention include, without limitation, CELLUZYME® (Novozymes) and KAC-500 (B) ^TM (Kao Corporation). In some embodiments, cellulases are used as parts or fragments of mature wild-type or variant cellulases from which a part of the N-terminus has been removed (see, for example, US Pat. No. 5,874,276). In some embodiments, the cleaning compositions of the present invention further comprise cellulases in an amount of about 0.00001% to about 10% additional cellulase based on the weight of the composition, where adjuvants are supplemented with the rest of the weight of the composition. In other aspects of the present invention, the cleaning compositions of the present invention further comprise cellulases in an amount of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to about 0.5% by weight of the composition.

Any suitable mannanases may also be used in the detergent compositions of the present invention. Suitable mannanases include, but are not limited to, enzymes of bacterial or fungal origin. Some embodiments encompass the use of chemically or genetically modified mutant forms. There are various mannanases that can be used in the present invention (see, for example, US patent No. 6566114, US patent No. 6602842 and US patent No. 6440991, which are incorporated into this description by reference). In some embodiments, the cleaning compositions of the present invention further comprise mannanases in an amount of about 0.00001% to about 10% additional mannanase relative to the weight of the composition, where adjuvants are supplemented with the rest of the weight of the composition. In other aspects of the present invention, the cleaning compositions of the present invention further comprise mannanases in an amount of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to about 0.5% by weight of the composition.

In some embodiments, the compositions of the present invention comprise peroxidases that can be used in combination with hydrogen peroxide or a source thereof (such as percarbonate, perborate or persulfate). In some alternative embodiments, oxidases are used in combination with oxygen. Both types of enzymes are used to “bleach the solution” (i.e., to prevent the transfer of textile dye from the dyed fabric to another fabric if these fabrics are washed together in a washing solution), preferably in combination with an agent that increases effectiveness (see, for example WO 94/12621 and WO 95/01426). Suitable peroxidases / oxidases include, but are not limited to, enzymes of plant, bacterial or fungal origin. Some embodiments encompass the use of chemically or genetically modified mutant forms. In some embodiments, the cleaning compositions of the present invention further comprise peroxidase and / or oxidase in an amount of from about 0.00001% to about 10% additional peroxidase and / or oxidase based on the weight of the composition, where adjuvant cleaning agents complete the rest of the weight of the composition. In other aspects of the present invention, the cleaning compositions of the present invention further comprise peroxidase and / or oxidase in an amount of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to about 0.5% based on the weight of the composition.

Other enzymes can be used in some embodiments, including but not limited to perhydrolases (see, for example, WO 05/056782). In addition, some particularly preferred embodiments cover the use of mixtures of the above enzymes, for example, one or more other proteases, amylases, lipases, mannanases and / or at least one cellulase. Indeed, it is contemplated that various mixtures of these enzymes can be used in the present invention. It is also contemplated that levels of variant protease (variant proteases) and one or more other enzymes can independently vary up to 10%, where auxiliary cleaning substances supplement the mass of the cleaning composition. Specific auxiliary cleaning agents can be easily selected taking into account the surfaces, products or fabrics to be cleaned, as well as the form of the composition desired for the cleaning conditions present during use (such as the use of a detergent).

Examples of suitable auxiliary cleaning agents include, but are not limited to, surfactants, detergents, whitening agents, bleaching activators, whitening catalysts, other enzymes, stabilizing enzyme systems, chelating agents, optical brighteners, dirt-removing polymers, transfer inhibiting agents colorants, dispersants, foaming suppressants, colorants, perfumes, pigments used as salt fillers, hydrotropic substances, photosensitives, fluorescent substances, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrink agents, smoothing agents, germicides, fungicides, stain removers, silver-containing fabric care products, anti-tarnish and / or anticorrosion agents, sources of alkalinity, solubilizing agents, carriers, processing aids, pigments and pH adjusting agents (see, for example, US Pat. Nos. 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5 695679, 5686014 and 5646101, which are incorporated herein by reference). Embodiments of specific cleaning compositions are described in detail below. If the auxiliary cleaning agents are not compatible with the variant proteases of the present invention in the cleaning compositions, methods are used in which the auxiliary cleaning substances and the protease (s) are kept separate (i.e., they do not contact each other) until the two components are combined, if necessary . Such separate use methods include any suitable methods known in the art (for example, capsule use, encapsulation, tablet use, physical separation, etc.).

In some preferred embodiments, an effective amount of one or more of the variant proteases described herein is incorporated into compositions used to clean different surfaces that need to be removed from protein stains. Such cleaning compositions include compositions for cleaning hard surfaces, fabrics and utensils. Indeed, in some embodiments, the present invention provides compositions for cleaning fabrics, while in other embodiments the present invention provides compositions for cleaning products other than fabrics. It should be noted that the present invention provides cleaning compositions for personal hygiene, including cleaning compositions for the care of the oral cavity (including dentifrices, toothpastes, mouthwashes, etc., as well as compositions for cleaning dentures ), skin and hair. It is intended that the present invention cover detergent compositions in any form (for example, in the form of liquids, granules, bars, semi-solids, gels, emulsions, tablets, capsules, etc.).

As an example, below are described in more detail some cleaning compositions, which may include variant proteases of the present invention. The cleaning compositions of the present invention for use in washing machines preferably contain at least one surfactant and at least one detergent component, as well as one or more cleaning aids, preferably selected from organic polymeric compounds, bleaching agents, other enzymes, foaming suppressants, dispersing agents, dispersing agents based on lime soap, anti-suspension agents and To Re-deposition of dirt, and corrosion inhibitors. In some embodiments, washing compositions also contain emollients (as other auxiliary cleaning agents). The compositions of the present invention can also be used as detergents in solid or liquid form. Such additives are intended to supplement and / or enhance the action of traditional detergent compositions, they can be added at any stage of the cleaning process. In some embodiments, the density of the detergent compositions of the present invention varies from about 400 to about 1200 g / liter, while in other embodiments, it varies from about 500 to about 950 g / liter at 20 ° C.

In separate embodiments, the compositions of the present invention intended for washing dishes by hand, contain at least one surfactant and, preferably, at least one other auxiliary cleaning agent selected from organic polymeric compounds, foaming agents, ions Group II metals, solvents, hydrotropes and other enzymes.

In some embodiments, different cleaning compositions can be used in combination with the variant proteases of the present invention, for example, those described in US Pat. No. 6,605,458. Thus, in some embodiments, a composition comprising at least one variant protease of the present invention is a granular cleaning composition fabrics, and in other embodiments, the composition is a granular fabric cleaning composition used to wash dyed fabrics, in the following embodiments, the composition is a granular composition for cleaning fabrics which provides softening to the entire volume of the wash in other embodiments, the composition is a liquid composition for cleaning fabrics in hard conditions. In some embodiments, compositions containing at least one variant protease of the present invention are tissue cleaning compositions, for example, those described in US Pat. Nos. 6,610,642 and 6,376,450. In addition, variant proteases of the present invention can be used to prepare granular laundry compositions agents especially suitable for use in washing conditions present in Europe or Japan (see, for example, US Pat. No. 6,610,642).

In some alternative embodiments, the present invention provides compositions for cleaning hard surfaces containing at least one variant protease described herein. Thus, in some embodiments, the composition comprising at least one variant protease of the present invention is a composition for cleaning hard surfaces, for example, one of those described in US patent No. 6610642, 6376450 and 6376450.

In further embodiments, the present invention provides dishwashing compositions comprising at least one variant protease as described herein. Thus, in some embodiments, the composition comprising at least one variant protease of the present invention is a composition for cleaning hard surfaces, for example, one of those described in US patent No. 6610642 and 6376450. In other embodiments, the present invention provides a composition for washing cookware containing at least one variant protease described herein. In some embodiments, compositions comprising at least one variant protease of the present invention include oral care compositions such as those described in US Pat. Nos. 6,376,450 and 6,376,450. The compositions, compounds, and auxiliary cleaning agents described in the above US patent No. 6376450, 6605458, 6605458 and 6610642, can be used in combination with the variant proteases described herein.

The cleaning compositions of the present invention are obtained in any suitable form using any methods selected by one of ordinary skill in the art, non-limiting examples of which are described in US Pat. as a reference. A low pH cleaning composition is prepared by adjusting the pH with a substance such as monoethanolamine or an acidic substance such as HCl.

The following is a non-limiting list of adjuvants suitable for preparing the cleaning compositions described herein, although their use is not mandatory for the purposes of the present invention. In some embodiments, these adjuvants are formulated to provide or enhance cleaning performance, to treat the base to be cleaned, or to modify the aesthetic characteristics of the cleaning composition, as in the case of flavoring agents, colorants, pigments, and the like. Obviously, such adjuvants are added to the variant proteases of the present invention. The specific types of these auxiliary components and their levels depend on the physical form of the composition and the type of cleaning operation for which they are used. Suitable adjuvants include, but are not limited to, surfactants, detergents, chelating agents, dye transfer inhibiting agents, precipitating agents, dispersing agents, other enzymes and enzyme stabilizers, catalytic agents, bleaching activators, bleaching enhancers, hydrogen peroxide , sources of hydrogen peroxide, pre-obtained peracids, polymer dispersants, agents for removing clay mud / preventing repetition CB sedimentation, brighteners, agents that suppress foaming, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids and / or pigments. In addition to the description below, suitable examples and levels of these other auxiliary agents can be found in US patent No. 5576282, 6306812 and 6326348, which are incorporated into this description by reference. The aforementioned adjuvants may constitute the remainder of the cleaning compositions of the present invention.

In some embodiments, the cleaning compositions of the present invention comprise a surfactant or surfactant system, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof.

In some other embodiments, the cleaning compositions of the present invention comprise one or more detergent components or detergent washing systems. Detergent components include, but are not limited to, polyphosphate salts of alkali metals, ammonium and alkanolammonium, alkali metal silicates, alkaline earth and alkali metal carbonates, polycarboxylate compounds of aluminosilicate components, hydroxypolycarboxylate ethers, copolymers of maleic anhydride and ethylene, or vinyl methyl ether. , 5-trihydroxybenzene-2,4,6-trisulfonic acid and carboxymethyloxy succinic acid, different salts of alkali metals, ammonium and substituted ammonium and polyacetic acid slot such as ethylenediaminetetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof.

In some other embodiments, the cleaning compositions of the present invention comprise a chelating agent. Suitable chelating agents include copper-containing, iron-containing and / or manganese-containing chelating agents and mixtures thereof.

In some further embodiments, the cleaning compositions described herein comprise a precipitating agent. Suitable precipitating agents include polyethylene glycol, polypropylene glycol, polycarboxylate, dirt-removing polymers such as polytephthalic acid, clays such as kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite and mixtures thereof.

In some other embodiments, the cleaning compositions of the present invention also comprise one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidonone polymers, polyamide N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyl oxazolidones and polyvinylimidazoles, or mixtures thereof.

In some further embodiments, the cleaning compositions of the present invention also comprise dispersants. Suitable water-soluble organic dispersants include homo- or copolymer acids or their salts, where the polycarboxylic acid contains at least two carboxyl radicals separated from each other by no more than two carbon atoms.

In some particularly preferred embodiments, the cleaning compositions comprise one or more detergent enzymes that provide cleaning effectiveness and / or improve tissue care. Examples of suitable enzymes include, but are not limited to, hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, cutinase, pectinase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, pullulanase, tannane, tannase, tannane, β-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases and amylases, or mixtures thereof. Typically, mixtures contain conventional enzymes, for example at least one protease, at least one lipase, at least one cutinase and / or at least one cellulase in combination with at least one amylase.

In some other embodiments, the enzymes used to prepare the cleaning compositions are stabilized by any suitable method. In some embodiments, the enzymes used in this invention are stabilized by adding water-soluble sources of calcium and / or magnesium ions to the final compositions that provide such ions with access to the enzymes.

In some further embodiments, the cleaning compositions of the present invention comprise catalytic metal complexes. One of the metal-containing bleaching catalysts is a catalytic system containing a transition metal cation with a specific catalytic activity with respect to bleaching, such as a cation of copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese, an auxiliary metal cation having low catalytic activity with respect to bleaching, or not having such activity, such as zinc or aluminum cation, and a component that provides certain stability constants for catalytic and an auxiliary metal cation, in particular ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid) and their water-soluble salts. Such catalysts are disclosed in US patent No. 4430243.

In some embodiments of the compositions described herein, a manganese compound is used as a catalyst. Such compounds and the levels at which they can be used are well known in the art and include, for example, manganese-based catalysts disclosed in US Pat. No. 5,576,282. In addition, cobalt bleaching catalysts suitable for use in the present invention are known. which are described, for example, in US Pat. Nos. 5,597,936 and 5,595,967. Such cobalt catalysts can be easily prepared using known methods, such as those described, for example, in US Pat. Nos. 5,597,936 and 5,595,967. In some embodiments, The compositions may also contain a transition metal complex with a macropolycyclic rigid ligand (ie, “MRL”). As a practical, but not limiting example, the composition of the compositions and the cleaning processes are selected so that they ensure the presence of MRL active elements in the water washing medium at a level of at least one part per hundred million, preferably from about 0.005 m .d. up to about 25 ppm, more preferably from about 0.05 ppm up to about 10 ppm, and most preferably from about 0.1 ppm up to about 5 ppm MRL in washing solution. Preferred transition metals used to produce soluble whitening catalysts include manganese, iron and chromium. Preferred MRLs for the present invention include a particular type of transverse bridge superrigid ligands, for example 5,12-diethyl-1,5,8,12-tetraazobicyclo [6.6.2] hexadecane. Suitable transition metal complexes with MRL can be easily obtained using known methods, such as described, for example, in WO 00/332601 and US patent No. 6225464.

As indicated above, the cleaning compositions of the present invention can be obtained in any suitable form using any methods selected by a person skilled in the art, non-limiting examples of which are described in US patent No. 5879584, 5691297, 5574005, 5569645, 5565422, 5516448, 5489392 and 5486303 included in this description by reference.

The cleaning compositions disclosed herein can be used to clean products (e.g., surfaces, dishes, or fabrics). Typically, at least a portion of the product is brought into contact with the embodiment of the cleaning composition of the present invention, in pure form or diluted with washing liquid, after which the product is optionally washed and / or rinsed. For the purposes of the present invention, the term “washing” includes, without limitation, friction and mechanical stirring. In some embodiments, cleaning compositions are typically used in concentrations ranging from about 500 ppm. up to about 15000 ppm in solution. If the washing solvent is water, the temperature of the water usually ranges from about 5 ° C to 90 ° C, and if the product contains tissue, the mass ratio of water to fabric is typically from about 1: 1 to about 30: 1.

In other embodiments, the present invention also provides compositions and methods comprising the use of various surfactants and mixtures of surfactants, which allow to determine and improve the effectiveness of the proteases included in detergent compositions. Using the experimental design software, a mixed experiment design with a simplex lattice is developed to determine the protease activity in the presence of surfactants most commonly used in liquid laundry detergents; such as linear alkylbenzenesulfonate (LAS), alkyl ethoxysulfate (AES) and alcohol ethoxylate (AE). LAS and AES are anionic surfactants, and AE is a nonionic surfactant.

Surprisingly, it has been found that the choice of a surfactant composition that provides optimal protease efficacy does not boil down to simply choosing a surfactant in which the protease is the most stable. Rather, the optimal composition of surfactants is based on a combination of different surfactants in a ratio that provides the best overall cleaning results, where the mixture includes surfactants that, when used in the absence of other surfactants, destabilize the protease.

For example, it has been found that in some embodiments, the protease may function better in the presence of a first surfactant if a second surfactant is present, but worse if a third surfactant is present. In other embodiments, the protease may be resistant to much higher levels of the first surfactant depending on the ratio of the second and third surfactants. The fact that enzymes can be used in the presence of different surfactants, if their ratio is carefully selected, contradicts the traditional belief that enzymes should be used in combination only with those surfactants that do not adversely affect their activity, which limits the use of enzymes with certain detergent compositions.

Partly on the basis of the observations described here, in the compositions and methods of the present invention, detergent compositions containing at least one protease and a mixture of surfactants are used in a pre-selected ratio that provides protease activity, where the choice of surfactants is not limited to measurement stability in every surfactant. In some cases, the protease is inactivated or exhibits reduced activity in the presence of a different ratio of the same surfactants, or in the presence of any subgroup of surfactants, but in the absence of at least one of the surfactants included mixtures. In some cases, the protease is inactivated or exhibits reduced activity in the presence of one of the surfactants, in the absence of another surfactant that is part of the mixture. Similarly, in some cases, the presence of the first surfactant allows the use of an increased amount of the second surfactant, the same amount of the second surfactant in the absence of the first surfactant inactivating or decreasing its activity.

In addition to the above, the presence of a first type of surfactant (e.g., a non-ionic or anionic detergent) allows the use of a second type of detergent, the second type of detergent in the absence of a first type of surfactant inactivating or reducing its activity.

In these experiments, subtilisin Bacillus amyloliquefaciens BPN'-Y217L ("FNA"; Genencor), as well as variants of this enzyme, are used. At low temperatures (for example, at 16 ° C), the protease is active in a composition containing a relatively low amount of AE and a higher amount of AES or LAS, or both. In comparison, a protease has lower activity in a composition containing only AES, only LAS and, in particular, only AE. Therefore, the preferred composition is not a composition containing only one surfactant in which the protease is the most active, but rather a composition that contains at least two, preferably all three surfactants, any of which can inactivate or reduce protease activity if used alone at a sufficient level. A specific ratio of AES> LAS> AE gives the best result for a given protease at low temperature.

Conversely, at high temperatures (for example, at 32 ° C), the protease is active in a composition containing only AES, or AES in combination with LAS, and a relatively low amount of AE. Thus, resistance to AES, increasing with increasing temperature, makes the inclusion of other surfactants in the composition less important.

Some other tests are also performed. Protease activity after incubation is determined using an AAPF-pNA substrate. In each detergent composition, the melting point of the enzyme, T. pl. Finally, four compositions were selected, tested using a tergotometer, and the correlation with the results of the analysis was determined in a 96-well format (see Example 22).

Definitions

Unless otherwise indicated, in the implementation of the present invention, you can use traditional methods commonly used in molecular biology, protein engineering, microbiology and methods for producing recombinant DNA, which are within the competence of specialists in this field. Such methods are known to those skilled in the art and are described in numerous manuals and reference books (see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual", Second Edition (Cold Spring Harbor), [1989]); and Ausubel et al., "Current Protocols in Molecular Biology" [1987]). All patents, patent applications, articles and publications mentioned in this document both above and below are expressly incorporated into the present invention by reference.

Unless otherwise specified, all technical and scientific terms used in this document have traditional meanings known to ordinary specialists in the field to which this invention belongs. Definitions of these terms can be found in the various available dictionaries known to those skilled in the art. Preferred methods and materials are described herein, however, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Accordingly, the terms defined below are more fully described with reference to the Description as a whole. In addition, in this document the singular includes a reference to the plural, unless the context clearly indicates otherwise. Numeric intervals are inclusive for numbers defining an interval. Unless otherwise indicated, nucleic acid sequences are described from left to right in the direction from the 5'-end to the 3'-end; amino acid sequences are described from left to right in the direction from the amino terminus to the carboxy terminus, respectively. It should be understood that the present invention is not limited to the specific methods, schemes, and reagents described, since they may vary depending on the environment in which they are used by specialists in this field.

Unless otherwise indicated, in the implementation of the present invention using traditional methods of protein purification, molecular biology, microbiology, recombinant DNA and protein sequencing, known to specialists in this field.

In addition, the headers of the present description are not a limitation of various aspects or embodiments of the invention, and may be incorporated by reference into the description as a whole. Accordingly, the terms defined below can be more fully described with reference to the description as a whole. However, in order to facilitate understanding of the invention, definitions of a number of terms are given below.

As used herein, the terms “protease” and “proteolytic activity” refer to a protein or peptide having the ability to hydrolyze peptides or substrates containing peptide bonds. There are many ways to measure proteolytic activity (Kalisz, "Microbial Proteinases," In: Fiechter (ed.), Advances in Biochemical Engineering / Biotechnology, [1988]). For example, proteolytic activity can be determined using comparative assays to measure the ability of the corresponding protease to hydrolyze a commercial substrate. Examples of substrates used to analyze protease or proteolytic activity include, but are not limited to, dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625) and bovine keratin (ICN Biomedical 902111 ) Methods for colorimetric assays using these substrates are well known in the art (see, for example, WO 99/34011; and US Patent No. 6376450, which are incorporated herein by reference). The concentration of the active enzyme in the fractions collected during gradient elution can be determined using pNA analysis (see, for example, Del Mar et al., Anal. Biochem. 99: 316-320 [1979]). This assay measures the release rate of p-nitroaniline by enzymatic hydrolysis of a soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPF-pNA). The rate of formation of the yellow product as a result of the hydrolysis reaction, measured at 410 nm using a spectrophotometer, is proportional to the concentration of the active enzyme. Total protein concentration can be determined by measuring absorbance at 280 nm. The purity of the enzyme is expressed as the ratio of active enzyme / total protein.

As used herein, the "genus Bacillus " includes all kinds of the "Bacillus gene" known to those skilled in the art, which include, without limitation, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus , B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus and B. thuringiensis . It has been shown that the genus Bacillus continues to undergo taxonomic reorganization. So, it is understood that this genus contains reclassified species, including, without limitation, organisms such as B. stearothermophilus , which is now called " Geobacillus stearothermophilus ". The production of stable endospores in the presence of oxygen is considered to be a defining feature of the genus Bacillus , although this characteristic also applies to organisms with the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibibillus, Salibibillus, Paenibibillus, Salibibillus, Paenibibillus, Salibibillus , Paibibillus .

The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to polymeric forms of nucleotides of any length that contain either ribonucleotides or deoxyribonucleotides. These terms include, without limitation, single-stranded, double-stranded or tri-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrids or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, unnatural or derivatized nucleotide bases. Non-limiting examples of polynucleotides are: genes, gene fragments, chromosomal fragments, EST, exons, introns, mRNAs, tRNAs, rRNAs, ribozymes, cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA with any sequence, any R , nucleotide probes and primers. In some embodiments, polynucleotides contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups, such as fluorribose and thioate, and nucleotide branches. In alternative embodiments, the nucleotide sequence is interrupted by non-nucleotide components.

As used herein, the terms “DNA construct” and “transforming DNA” are used interchangeably and refer to DNA used to introduce sequences into a host cell or organism. DNA can be obtained in vitro by PCR or by any other suitable method (s) known to those skilled in the art. In particularly preferred embodiments, the DNA construct contains a sequence of interest (e.g., an exogenous sequence). In some embodiments, such a sequence is operably linked to other elements, such as regulatory elements (e.g., promoters, etc.). The DNA construct may also contain a selectable marker. In addition, it may contain an exogenous sequence flanked by homologous boxes. In another embodiment, the transforming DNA contains other non-homologous sequences attached to the ends (for example, regular sequences or flanking sequences). In some embodiments, the ends of the exogenous sequence are closed so that the transforming DNA forms a closed cycle. Transforming sequences may be wild-type, mutant, or modified. In some embodiments, the DNA construct contains sequences homologous to the sequences present on the chromosome of the host cell. In other embodiments, the DNA construct contains non-homologous sequences. The obtained in vitro DNA construct can be used to: 1) insert heterologous sequences into the target target sequence of the host cell, and / or 2) mutagenesis of the chromosome region of the host cell (i.e., replace the endogenous sequence with a heterologous sequence), 3) removing the target genome; and / or 4) introducing a replicable plasmid into the host.

As used herein, the terms “expression cassette” and “expression wind” refer to nucleic acid constructs obtained by recombinant or synthetic methods that contain a number of specific nucleic acid elements that transcribe a specific nucleic acid in a target cell. The recombinant expression cassette can be inserted into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, a recombinant expression cassette, as part of an expression vector, contains, among other sequences, a nucleotide sequence and a promoter to be transcribed. In preferred embodiments, the expression vectors have the ability to introduce heterologous DNA fragments into the host cell and express them. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of suitable expression vectors is within the purview of specialists in this field. The terms “expression cassette” and “DNA construct”, as well as their grammatical equivalents, are used interchangeably. The selection of suitable expression vectors is within the purview of specialists in this field.

As used herein, the term “vector” refers to a polynucleotide construct designed to introduce nucleic acids into cells of one or more types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, cassettes, and the like. In some embodiments, the polynucleotide construct comprises a DNA sequence encoding a protease (e.g., a protease precursor or mature protease) that is operably linked to a suitable pro-sequence (e.g., a secretory sequence, etc.) capable of expressing the DNA in a suitable host.

As used herein, the term "plasmid" refers to a double-stranded (ds) DNA construct used as a cloning vector that forms an extrachromosomal self-replicating genetic element in some eukaryotes or prokaryotes, or integrates into the host chromosome.

As used herein, the term “introduction” as applied to a nucleotide sequence refers to any method suitable for transferring a nucleotide sequence into a cell. Such routes of administration include, but are not limited to, protoplast fusion, transfection, transformation, conjugation and transduction (see, for example, Ferrari et al., "Genetics," in Hardwood et al, (eds.), Bacillus, Plenum Publishing Corp. ., pages 57-72, [1989]).

As used herein, the terms “transformed” and “stably transformed” refer to a cell that contains a non-natural (heterologous) polynucleotide sequence that is integrated into the genome of the cell, or that exists as an episomal plasmid, and that lasts for at least two generations.

A nucleic acid is "functionally linked" if it is placed in a functional relationship to another nucleotide sequence. For example, DNA encoding a secretory leader sequence (i.e., a signal peptide) is operably linked to DNA encoding a polypeptide if it is expressed as a precursor protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence, if its location provides translation. As a rule, the term "functionally linked" means that the linked DNA sequences are adjacent, and in the case of a secretory leader sequence, they are adjacent and are in the same reading phase. However, enhancers may not be contiguous. Binding is carried out by ligation at traditional restriction sites. If such sites are absent, synthetic oligonucleotide adapters or linkers are used, in accordance with normal practice.

As used herein, the term “gene” refers to a polynucleotide (eg, a DNA segment) that encodes a polypeptide and contains regions preceding and accompanying the coding regions, as well as sequences (introns) inserted between individual coding segments (exons).

As used herein, the term “homologous genes” refers to a pair of genes from different, but usually related, species that correspond to each other and are identical or have a high degree of similarity. The term encompasses genes that are separated during speciation (i.e., the development of new species) (e.g., orthological genes), as well as genes that are separated by genetic duplication (e.g., paralogous genes).

In this document, proteins are defined as having the same “stacking” if they contain the same primary secondary structures with the same configuration and the same topological bonds. Different proteins with the same folding often contain peripheral elements of the secondary structure and sections of bends, which differ in size and conformation. In some cases, these distinct areas may be half the structure. Proteins that belong to the same stacking category do not necessarily have a common evolutionary origin (for example, structural similarity due to the physical and chemical properties of proteins contributes to certain folding order and chain topology).

As used herein, the term “homology” refers to the similarity or identity of a sequence, preferably identity. Homology is determined using standard methods known in the art (see, for example, Smith and Waterman, Adv. Appl. Math., 2: 482 [1981]; Needleman and Wunsch, J. MoI. Biol., 48: 443 [ 1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 [1988]; programs such as GAP, BESTFIT, FASTA and TFASTA from Wisconsin Genetics software (Genetics Computer Group, Madison, WI); and Devereux et al., Nucl. Acid Res., 12: 387-395 [1984]).

As used herein, a “similar sequence" is a sequence related to a gene whose function is almost identical to that of the BPN 'protease gene. Similar sequences are determined using known sequence alignment methods. The BLAST alignment method is widely used, although, as described above and below, there are other ways to align sequences.

One example of a suitable algorithm is PILEUP. PILEUP aligns several sequences from a group of related sequences using a progressive two-point method. This algorithm can also draw a tree showing the relationships in the clusters, which is used for alignment. PILEUP uses the simplification of the Feng and Doolittle progressive alignment method (Feng and Doolittle, J. MoI. Evol., 35: 351-360 [1987]). This method is similar to that described by Higgins and Sharp (Higgins and Sharp, CABIOS 5: 151-153 [1989]). PILEUP uses parameters that include a default gap weight of 3.00, a default gap length of 0.10, and weighted-end gaps.

Another example of a suitable algorithm is BLAST described by Altschul et al., (Altschul et al., J. MoI. Biol., 215: 403-410, [1990]; and Karlin et al., Proc. Natl. Acad. Sci. USA 90: 5873-5787 [1993]). A preferred BLAST program is WU-BLAST-2 (see Altschul et al., Meth. Enzymol., 266: 460-480 [1996]). WU-BLAST-2 uses several search parameters, most of which have default values. The adjustable values are assigned the following values: overlap interval = 1, overlap fraction = 0.125, word threshold (T) = 11. The parameters HSP S and HSP S2 have dynamic values and are set by the program itself depending on the composition of a particular sequence and the content of a specific database, in comparison with which the analysis of the sequence of interest is carried out. However, the values can be adjusted to increase sensitivity. The value of% amino acid sequence identity is determined by dividing the number of identical identical residues by the total number of residues in the "longer" sequence of aligned regions. A “longer” sequence has the largest number of actual residuals in the alignment section (the gaps introduced by the WU-Blast-2 program to minimize points during alignment are ignored).

Thus, “percent (%) nucleotide sequence identity” is defined as the percentage of nucleotide residues in a candidate sequence identical to the nucleotide residues of the original sequence (i.e., the sequence of interest). In a preferred method, the default parameter set BLASTN module WU-BLAST-2 is used with the overlap interval and overlap fraction set to 1 and 0.125, respectively.

As used herein, the term “recombinant” refers to cells or vectors modified by introducing a heterologous nucleotide sequence, or to cells derived from such a modified cell. For example, recombinant cells express genes that are not found in identical form in the natural (non-recombinant) cell precursor, or express native genes that are expressed abnormally, at a reduced level in the non-recombinant cell precursor, or are not expressed at all as a result of intentional human intervention. "Recombination", "recombination" and preparation of a "recombinant" nucleic acid typically involves the assembly of two or more nucleic acid fragments to produce a chimeric gene.

In some preferred embodiments, mutant DNA sequences are obtained by site-saturating mutagenesis of at least one codon. In another preferred embodiment, site-saturating mutagenesis is carried out at two or more codons. In a further embodiment, the homology of the mutant DNA sequences with respect to the wild-type sequence is more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, more than about 95%, or more than about 98%. In alternative embodiments, mutant DNA is obtained in vivo using any known mutagenesis method, such as, for example, irradiation, the use of nitrosoguanidine and the like. The target DNA sequence is then isolated and used in the methods of the present invention.

As used herein, the terms "amplification" and "gene amplification" refer to a disproportionate DNA replication process that provides a higher copy number of the amplified gene than is originally present in the genome. In some embodiments, cell selection by growing in the presence of a drug (such as an inhibitory enzyme inhibitor) leads to the amplification of either the endogenous gene encoding the gene product necessary for growth in the presence of the drug, or to the amplification of exogenous (i.e., introduced) sequences encoding the specified gene product, or both.

Amplification is a special case of nucleic acid replication involving a specific matrix. It should be distinguished from replication on a non-specific matrix (i.e., replication, which is matrix-dependent but not dependent on a specific matrix). The specificity of the matrix in this description is distinguished from the accuracy of reproduction during replication (i.e., synthesis of the correct polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. The specificity of the matrix is often called the specificity of the “target”. Target sequences are “targets” in the sense that they need to be separated from other nucleic acids. Amplification methods are designed primarily for such separation.

As used herein, the term “primer” refers to an oligonucleotide, either natural, for example, isolated from a restriction hydrolyzate, or synthetically prepared that is able to function as a point of synthesis initiation under conditions suitable for inducing the synthesis of a primer extension product complementary to the nucleotide chain (i.e. i.e., in the presence of nucleotides and an inducing reagent such as DNA polymerase, at suitable temperatures and pH). For maximum amplification efficiency, it is preferable to use a single-stranded primer, however, it can also be double-stranded. If the primer is double-stranded, it is first treated to separate the chains, and then used to obtain extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to initiate the synthesis of extension products in the presence of an inducing agent. The exact length of the primers depends on many factors, including temperature, source of primer, and application of the method.

As used herein, the term “probe” refers to an oligonucleotide (ie, a nucleotide sequence), either natural, for example, isolated from a restriction hydrolyzate, or synthesized by a recombinant method or PCR amplification method that is capable of hybridizing with another of interest oligonucleotide. The probe may be single-stranded or double-stranded. Probes can be used to detect, identify and isolate specific gene sequences. It is understood that the present invention uses probes labeled with some kind of "reporter molecule", which allows them to be detected in any detection system, including, without limitation, enzyme (for example, ELISA, as well as enzymatic histochemical analyzes), fluorescence, radioactive and luminescent system. It is believed that the present invention is not limited to any particular detection system or tag.

As used herein, the term “target” as applied to a polymerase chain reaction refers to a portion of a nucleic acid that binds to primers used for the polymerase chain reaction. Thus, the "target" can be separated from other nucleotide sequences. A “segment” is a portion of a nucleic acid in a target sequence.

As used herein, the term "polymerase chain reaction" ("PCR") refers to the methods described in US Pat. Nos. 4,683,195 to 4,683,202 and 4,965,188, incorporated herein by reference, which are methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. Said method for amplifying a target sequence involves introducing a large excess of two oligonucleotide primers into the DNA mixture containing the target target sequence, after which the mixture is subjected to thermal cycling sequences in the presence of DNA polymerase. Two primers are complementary to the corresponding chains of the double stranded target sequence. In order to amplify, the mixture is denatured, after which the primers are annealed with complementary sequences from the target molecule. After annealing, the primers are extended using polymerase to give a new pair of complementary chains. The stages of denaturation, annealing of primers and extension by polymerase can be repeated many times (i.e., denaturation, annealing and extension of the chain comprise one “cycle”; there can be many such “cycles”) to obtain a high concentration of the amplified segment of the target sequence - the target. The length of the amplified segment of the target target sequence is determined by the positions of the primers with respect to each other and, therefore, is an adjustable parameter. Since this process involves repeating steps, the method is called a "polymerase chain reaction" (hereinafter "PCR"). Since the target amplifiable segments of the target sequence become dominant (in terms of concentration) in the mixture, they are called "PCR amplified."

As used herein, the term “amplification reagents” refers to the reagents (deoxyribonucleotide triphosphates, buffer, etc.) necessary for amplification, with the exception of primers, the nucleotide matrix, and the enzyme used for amplification. As a rule, reagents for amplification together with other components of the reaction are placed in a reaction vessel (test tube, microplate well, etc.).

As used herein, the term "RT-PCR" refers to the replication and amplification of RNA sequences. In this method, PCR is carried out by the reverse transcription method, most often using a single enzyme, thermostable polymerase, as described in US patent No. 5322770, incorporated herein by reference. In the RT-PCR process, the RNA matrix is converted into cDNA using the reverse transcriptase activity of the polymerase and then amplified using the polymerizing activity of the polymerase (i.e., as in other PCR methods).

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cuts double-stranded DNA at or near a specific portion of a nucleotide sequence.

The term "restriction site" refers to a nucleotide sequence recognized and cleaved by a specific restriction endonuclease, and is usually the insertion site of DNA fragments. In some embodiments, restriction sites are inserted by recombinant methods into a selectable marker and at the 5 ′ and 3 ′ ends of the DNA construct.

The term “homologous recombination” refers to the exchange of fragments of two DNA molecules or paired chromosomes over regions of identical or almost identical nucleotide sequences. In a preferred embodiment, chromosomal integration is homologous recombination.

As used herein, the term “amino acid” refers to peptide or protein sequences or fragments thereof. The terms "protein", "peptide" and "polypeptide" are used interchangeably.

As used herein, the terms “protein of interest” and “polypeptide of interest” refer to the target and / or test protein / polypeptide. In some embodiments, the protein of interest is expressed within the cell, while in other embodiments it is a secreted polypeptide. In particularly preferred embodiments, said enzymes include the serine proteases of the present invention. In some embodiments, the protein of interest is a secreted polypeptide hybridized to a signal peptide (i.e., the secreted protein has an amino-terminal extension). Almost all secreted proteins contain an amino-terminal extension, which plays an important role in directing the precursor proteins towards the membrane and moving them across the membrane. This extension can be removed proteolytically by signal peptidase during transfer through the membrane or immediately after this transfer.

It is said that a polynucleotide “encodes” an RNA or polypeptide if, in its native state, or after manipulation using methods known to those skilled in the art, it can undergo transcription and / or translation to form RNA, polypeptide or fragments thereof. It is believed that the antisense strand of such a nucleic acid also encodes a sequence. As is known in the art, DNA can be transcribed by RNA polymerase to produce RNA, and RNA can undergo reverse transcription to produce DNA. Thus, DNA can encode RNA, and vice versa.

The terms “host strain” or “host cell” refer to a suitable host of an expression vector containing the DNA of the present invention.

An enzyme is "expressed at an increased level" in a host cell if its expression level in that cell exceeds the expression level in the corresponding wild-type cell.

The terms “protein” and “polypeptide” are used interchangeably herein. The 3-letter code approved by the Joint Commission on Biochemical Nomenclature (JCBN) is used to refer to amino acids in this document. In addition, it should be understood that the polypeptide can be encoded by multiple nucleotide sequences due to the degeneracy of the genetic code.

A "sequence" is an amino acid sequence between a signal sequence and a mature protease, which is necessary for secretion of the protease. Cleavage of the prosequence leads to the formation of a mature active protease.

The term "signal sequence" or "signal peptide" refers to any sequence of nucleotides and / or amino acids that may be involved in the secretion of mature forms of the protein or their precursors. This definition of a signal sequence is functional and applies to all amino acid sequences encoded by the N-terminal fragment of a protein gene that is involved in the secretion of the protein. Signal sequences often, but not always, attach to the N-terminal fragment of the protein or to the N-terminal fragment of the protein precursor. The signal sequence may be endogenous or exogenous. The signal sequence may be a sequence usually associated with a particular protein (eg, a protease), or it may be the product of a gene encoding another secreted protein. An exogenous signal sequence example includes the first seven amino acid residues of the Bacillus subtilis subtilisin signal sequence attached to the residue of the Bacillus lentus subtilisin signal sequence (ATCC 21536).

The term "hybrid signal sequence" refers to signal sequences that contain a fragment derived from the owner of an expression vector that is hybridized with the signal sequence of the expressed gene. In some embodiments, synthetic sequences are used.

The term “mature” form of a protein or peptide refers to the final functional form of a protein or peptide. For example, the mature form of the protease of the present invention contains at least an amino acid sequence identical to the sequence of residues at positions 1-275 of SEQ ID NO: 2.

The term “precursor” of a protein or peptide refers to a mature form of the protein containing a pro-sequence operably linked to the amino or carboxy terminus of the protein. The precursor may also contain a “signal” sequence operably linked to the amino terminus of the pro sequence. The precursor may also contain other polynucleotides that are involved in post-translational activity (for example, polynucleotides, after cleavage of which a mature form of the protein or peptide is formed).

The term "natural enzyme" refers to an enzyme having an unmodified amino acid sequence identical to that present in nature. Natural enzymes include native enzymes, enzymes that are naturally expressed or are present in a particular microorganism.

The terms “derived from” and “derived from” refer not only to a protease that is produced or can be produced by a test strain of an organism, but also to a protease encoded by a DNA sequence isolated from such a strain and produced by a host organism containing such a DNA sequence. In addition, this term refers to a protease that is encoded by a DNA sequence obtained synthetically and / or from cDNA, and which has the distinctive characteristics of the protease in question.

A “derivative”, within the scope of this definition, usually preserves the proteolytic activity characteristic of the wild-type form, the native or the original form, to the extent that it allows the derivative to be used for the same purposes as the wild-type form, the native or the original form. Functional derivatives of a serine protease encompass the natural, synthetic, or recombinant peptides or peptide fragments that have the basic characteristics of the serine protease of the present invention.

The term “functional derivative” refers to a nucleic acid derivative that has the functional characteristics of a nucleic acid encoding a serine protease. Functional nucleic acid derivatives encoding the serine protease of the present invention include natural, synthetic or recombinant nucleic acids or fragments thereof, and encode the serine protease of the present invention. The wild-type nucleic acid encoding the serine proteases of the present invention includes natural alleles and homologs known in the art resulting from the degeneracy of the genetic code.

The term "identical" as applied to two nucleotide or polypeptide sequences refers to the residues of two sequences that are recognized to be the same after sequence alignment to obtain maximum match using one of the sequence comparison or analysis algorithms.

The term “optimal alignment” refers to alignment that gives the maximum percentage of matches.

"Percentage of sequence identity", "percentage of identity of amino acid sequences", "percentage of identity of gene sequences" and / or "percentage of identity of nucleotide / polynucleotide sequences" as applied to two amino acid, polynucleotide and / or gene sequences (as appropriate), refers to the percentage of identical residues in two sequences with optimal sequence alignment. So, 80% amino acid sequence identity means that 80% of the amino acids in the two polypeptide sequences are identical with optimal alignment.

The phrase “substantially identical” as used with respect to two nucleic acids or polypeptides means that the polynucleotide or polypeptide is at least about 70%, preferably at least about 75%, preferably at least about 80%, preferably at least about 85%, preferably at least about 90%, preferably at least about 95%, preferably at least about 97%, preferably at least about 98% and preferably at least D, approximately 99% identical to the compared sequence, where the comparison is carried out using programs or algorithms (such as BLAST, ALIGN, CLUSTAL) using standard parameters. The ability of the first polypeptide and the second polypeptide to undergo cross-immunological reactions indicates that the two polypeptides are almost identical. Typically, polypeptides that differ in conservative amino acid substitutions are able to enter into cross-immunological reactions. Thus, the polypeptide is almost identical to the second polypeptide, for example, if these polypeptides differ only by conservative substitution. Another sign that the two nucleotide sequences are almost identical is the ability of the two molecules to hybridize with each other under stringent conditions (for example, in the range of conditions from medium to high stringency).

The term “equivalent” as used herein refers to serine protease enzymes encoded by a polynucleotide capable of hybridizing under medium to high stringency conditions with a polynucleotide having the sequence of SEQ ID NO 1. For example, an equivalent mature serine protease has a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91% at least about 9 2%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98% and / or at least about 99% identical to the amino acid sequence of the mature BPN ′ SEQ ID NO: 2 mature serine protease.

The term "isolated" or "purified" refers to a substance removed from the original environment (for example, from the natural environment, if it occurs in nature). For example, it is said that a substance is “purified” if it is present in a particular mixture at a higher or lower concentration than in nature or in the wild-type organism. For example, a natural polynucleotide or polypeptide present in a living organism is not isolated, however, the same polynucleotide or polypeptide separated from some or all of the substances that exist together with it in the natural system is called isolated. In some embodiments, such polynucleotides are part of the vector and / or such polynucleotides or polypeptides are included in the composition, and yet are isolated, since the vector or composition is not part of their natural environment. In preferred embodiments, the nucleic acid or protein is said to be purified if they give essentially one band in gel electrophoresis or blotting.

The term "isolated" as applied to a DNA sequence means that the DNA sequence has been removed from the natural genetic environment, therefore, does not contain other extraneous or undesired coding sequences, and is in the form suitable for use in a recombinant protein production system. Such isolated molecules are molecules that are separate from their natural environment and include cDNA and genomic clones. The isolated DNA molecules of the present invention do not contain other genes with which they are usually associated, however, they may contain naturally occurring 5'- and 3'-untranslated regions, such as promoters and terminators. Methods for identifying associated sites are known to those skilled in the art (see, for example, Dynan and Tijan, Nature 316: 774-78 [1985]). An alternative to the term "isolated DNA sequence" may be the expression "cloned DNA sequence".

The term "isolated" as applied to a protein means that the protein is in conditions different from its natural environment. In a preferred form, the isolated protein is substantially free of other proteins, especially other homologous proteins. The isolated protein has a purity of more than about 10%, preferably more than about 20%, more preferably more than about 30% according to SDS-PAGE. Other aspects of the present invention encompass protein in a highly purified form (i.e., purity is more than about 40%, more than about 60%, more than about 80%, more than about 90%, more than about 95%, more than approximately 97% and even more than approximately 99%) according to SDS-PAGE.

As used herein, the term “combinatorial mutagenesis” refers to methods for producing libraries of variants of the parent sequence. Variants that are part of these libraries contain one or more mutations selected from a previously defined set of mutations. In addition, these methods allow the introduction of random mutations that are not members of a previously defined set of mutations. In some embodiments, the methods include methods described in US patent application No. 09/699250, filed October 26, 2000, which is incorporated into this description by reference. In alternative embodiments, combinatorial mutagenesis methods encompass the use of commercially available kits (e.g., QuikChange® Multisite, Stratagene, San Diego, CA).

As used herein, the term “library of mutants” refers to a population of cells that are identical across the main part of the genome but contain different homologues of one or more genes. Such libraries can be used, for example, to identify genes or operons with improved properties.

As used herein, the term “parent gene” refers to a gene of interest that encodes a protein of interest that is to be improved and / or modified using the methods of the present invention.

As used herein, the term “multiple sequence alignment” (“MSA”) refers to sequence alignment of several homologs of a source gene using an algorithm (eg, Clustal W).

As used herein, the terms “consensus sequence” and “canonical sequence” refer to an archetypal amino acid sequence with which all variants of a particular protein or sequence of interest are compared. The terms also refer to a sequence that contains the nucleotides most commonly found in a DNA sequence of interest. For each position of the gene, the consensus sequence provides the amino acid that is most often found in this position according to the results of MSA.

As used herein, the term “consensus mutation” refers to the difference in the sequence of the original gene and the consensus sequence. Consensus mutations are identified by comparing the sequences of the parent gene and the consensus sequence by MSA. In some embodiments, consensus mutations are introduced into the original gene to make it more similar to the consensus sequence. Consensus mutations also include amino acid changes. leading to a change in the amino acid in the original gene to an amino acid that is more common in this position according to MSA results compared to the frequency of occurrence of this amino acid in the original gene. Thus, the term consensus mutation includes all single amino acid changes corresponding to the replacement of the amino acid of the original gene with an amino acid that is more common according to MSA.

As used herein, the term “original hit” refers to a variant that is identified by screening a library with combinatorial consensus mutagenesis. In preferred embodiments, the parent hits have improved functional characteristics compared to the parent gene.

As used herein, the term "improved hit" refers to a variant that is identified by screening an improved library with combinatorial consensus mutagenesis.

In this document, the terms “improving mutation” and “mutation that improves functional characteristics” refer to a mutation that, after introduction into the original gene, leads to an improvement in its functional characteristics. In some preferred embodiments, said mutations are identified by sequencing hits that were identified in the method at the screening step. In most embodiments, the mutations that are more likely to be found in hits are more likely to be improving mutations than mutations in a non-screened library with combinatorial consensus mutagenesis.

As used herein, the term “improved library with combinatorial consensus mutagenesis” refers to a CCM library that is created and constructed based on the screening and / or sequencing of products obtained in a previous CCM mutagenesis and screening cycle. In some embodiments, an improved CCM library is obtained based on the sequence of the starting material obtained in the previous CCM cycle. In other embodiments, when constructing an improved CCM library, mutations that are often observed in the starting products obtained in previous mutagenesis and screening cycles are preferred. In some preferred embodiments, when receiving such libraries, primers that encode mutations that degrade the performance are neglected or increase the concentration of primers that encode mutations that enhance the performance with respect to other primers that are used to obtain the previous CCM libraries.

As used herein, the term “degrading mutations” refers to mutations in the combinatorial consensus mutagenesis library that are less common in products obtained after screening than in combinatorial consensus mutagenesis library that has not been screened. In preferred embodiments, the screening process removes and / or reduces the occurrence of variants that contain “mutations that degrade performance”.

As used herein, the term “functional assay” refers to an assay by which protein activity is determined. In particularly preferred embodiments, the term refers to assay systems in which a protein is assayed for its ability to function in its usual role. For example, in the case of enzymes, functional analysis includes determining the effectiveness of the enzyme in catalysing the reaction.

As used herein, the term “target property” refers to the property of the original gene that needs to be changed. It is assumed that the present invention is not limited to any specific target property. However, in some preferred embodiments, the target property is the stability of the gene product (for example, resistance to denaturation, proteolysis or other destructive factors), although in other embodiments, the level of production in the host is subject to change. It is contemplated that virtually any property of the parent gene can be used in the present invention.

The term “property” or its grammatical equivalents as applied to nucleic acid in this document refers to any characteristic features or characteristics of a nucleic acid that can be selected or detected. These properties include, without limitation, a property that affects binding to a polypeptide, a property that appears in a cell containing a specific nucleic acid, a property that affects gene transcription (e.g., promoter length, promoter recognition, promoter regulation, enhancer function) a property that affects RNA processing (e.g., RNA splicing, RNA stability, RNA conformation, and post-transcriptional modification), a property that affects translation (e.g., mRNA level, mRNA regulation, mRNA binding to p bosomalnymi proteins, post-translational modification). For example, the binding site of a transcription factor, polymerase, regulatory factor, etc. in a nucleic acid can be changed to obtain desired characteristics or to identify undesirable characteristics.

The term “property” or its grammatical equivalents as applied to a polypeptide (including proteins) in this document refers to any characteristic features or characteristics of the polypeptide that can be selected or detected. These properties include, without limitation, oxidation resistance, substrate specificity, catalytic activity, thermal stability, alkali resistance, activity profile at different pH values, proteolytic degradation resistance, K _M , k _cat , k _cat / k ratio _M , protein folding, ability to induce an immune response, ability to bind to a ligand, ability to bind to a receptor, ability to secrete, ability to present on the cell surface, ability to oligomerize, the ability to participate in signal transmission, the ability to stimulate cell proliferation, the ability to inhibit cell proliferation, the ability to induce apoptosis, the ability to undergo modification due to phosphorylation or glycosylation, and / or the ability to treat a disease, etc.

As used herein, the term “screening” has the meaning traditionally used in the art and generally refers to a multi-stage process. In the first step, a mutant nucleic acid or a variant polypeptide encoded by it is prepared. In a second step, the property of a mutant nucleic acid or variant polypeptide is determined. In a third step, a specific property is compared with the property of the corresponding starting nucleic acid, with the property of the corresponding natural polypeptide, or with the property of the starting material (e.g., the starting sequence) used to produce the mutant nucleic acid.

Specialists in this field know that the choice of screening procedure used to obtain a nucleic acid or protein with a modified property depends on the properties of the starting material, the modification of which leads to the creation of a mutant nucleic acid, which is supposed to facilitate. Therefore, one skilled in the art can determine that the invention is not limited to any particular property to be screened, and that only illustrative examples are listed in the following property description. Methods of screening for specific properties are widely described in this field. For example, you can measure the binding, pH, specificity, etc. before and after the introduction of the mutation, where the replacement determines the change. High throughput screenings are preferably used, allowing for the simultaneous analysis of several samples, which include, without limitation, analyzes using chips, phage displays and several substrates and / or indicators.

As used herein, some embodiments of screenings include the steps of enriching a population of variants with the variants of interest. Examples of these embodiments include the selection of variants providing primary growth to the host organism, as well as methods of phage displays or other displays in which individual variants from a population of variants are captured based on binding or catalytic properties. In a preferred embodiment, the library of variants is exposed (heat, protease, denaturation), after which variants remaining intact are identified by screening, or the library is enriched with these variants by selection. This term is intended to encompass all suitable selection methods. Of course, it is intended that the present invention is not limited to any particular screening method.

As used herein, the term “directional randomization” refers to a method for producing a plurality of sequences in which one or two positions are randomized. In some embodiments, randomization is complete (i.e., all four nucleotides, A, T, G, and C, may occur in a randomized position). In alternative embodiments, nucleotide randomization is limited to a subset of four nucleotides. Directed randomization can be applied to one or more codons of a sequence encoding one or more proteins of interest. Upon expression, the resulting libraries produce populations of proteins in which one or more amino acid positions may contain a mixture of all 20 amino acids, or a subgroup of amino acids, depending on the randomization scheme of a particular codon. In some embodiments, individual members of a population resulting from directed randomization differ in the number of amino acids due to directed or random insertion or deletion of codons. In other embodiments, the resulting protein populations comprise synthetic amino acids. In some preferred embodiments, most of the members of the population resulting from targeted randomization are more homologous to the consensus sequence than the original gene. In some embodiments, the sequence encodes one or more proteins of interest. In alternative embodiments, proteins perform different biological functions. In some preferred embodiments, the exogenous sequence comprises at least one selectable marker. Such a sequence may encode one or more proteins of interest. It can perform other biological functions. Often, an exogenous sequence may contain a gene responsible for antibiotic resistance as a selectable marker.

The terms “modified sequence” and “modified genes” are used interchangeably herein and refer to a sequence that differs from the native nucleic acid sequence by deletion, insertion or termination of the sequence. In some preferred embodiments, the expression product of the modified sequence is a truncated protein (for example, if the modification is a deletion or sequence termination). In some particularly preferred embodiments, the truncated protein retains biological activity. In alternative embodiments, the expression product of the modified sequence is an elongated protein (for example, if the modification includes insertion into a nucleic acid sequence). In some embodiments, the consequence of the insertion is the expression of a truncated protein (for example, if the insertion leads to the formation of a stop codon). Thus, insertion can lead to the expression of both shortened and elongated protein.

As used herein, the terms “mutant sequence” and “mutant gene” are used interchangeably and refer to a sequence that comprises a change of at least one codon present in the sequence of a wild-type host cell. The expression product of the mutant sequence is a protein whose amino acid sequence differs from the wild-type sequence. The expression product may have altered functional characteristics (for example, increased enzymatic activity).

The terms “mutagenic primer” or “mutagenic oligonucleotide” (used interchangeably herein) refer to oligonucleotide formulations that correspond to and are capable of hybridizing to a matrix sequence fragment. The mutagenic primer does not exactly match the nucleotide matrix, and mismatch, or mismatch, can be used to introduce the target mutation into the nucleic acid library. As used herein, the terms "non-mutagenic primer" or "non-mutagenic oligonucleotide" refer to oligonucleotide compositions that exactly match the nucleotide matrix. In one embodiment of the present invention, only mutagenic primers are used. In another preferred embodiment of the invention, the primers are designed so that at least one region corresponds to an oligonucleotide mixture containing both a mutagenic primer and a non-mutagenic primer. By adding a mixture of mutagenic primers and non-mutagenic primers corresponding to at least one of the mutagenic primers, a nucleic acid library can be obtained in which a number of combinatorial mutational models are presented. For example, if you want some members of the library of mutant nucleic acids to maintain the original sequence in separate positions, and other members would have mutations in these areas, non-mutagenic primers provide a certain level of non-mutant members of the library of nucleic acids for this residue. The methods of this invention use mutagenic and non-mutagenic oligonucleotides, the length of which is usually 10-50 bases, more preferably about 15-45 bases. However, to obtain the desired mutagenesis result, primers may be required whose length is either less than 10 bases or more than 50 bases. Mutagenic and non-mutagenic primers do not have to be the same length, but they must overlap at the site of mutation addition. Primers can be added in a predetermined ratio in accordance with the present invention. For example, a library containing a significant level of some specific mutation and a lower amount of another mutation in one site, or in different sites, can be obtained by regulating the number of primers added. Alternatively, by adding smaller or larger amounts of non-mutagenic primers, the frequency of occurrence of the corresponding mutation (s) in the mutant nucleic acid library can be controlled.

As used herein, the phrase “adjacent mutations” refers to mutations carried by a single oligonucleotide primer. For example, regardless of whether adjacent mutations are near or close to each other, they are introduced into the final mutant nucleotide matrix using a single primer.

As used herein, the phrase “non-contiguous mutations” refers to mutations that are located on different oligonucleotide primers. For example, non-adjacent mutations are introduced adjacent to the final mutant nucleotide matrix using separately prepared oligonucleotide primers.

The terms “wild-type sequence” or “wild-type gene”, used interchangeably herein, refer to a native sequence or sequence that is naturally present in the host cell. In some embodiments, the term wild-type sequence refers to a sequence of interest that is used as starting material in the preparation of a recombinant protein. The wild-type sequence can encode either homologous or heterologous protein. A homologous protein is a protein that a host cell produces without external intervention. A heterologous protein is a protein that a host cell does not produce without external intervention.

Unless otherwise indicated, the numbering of amino acid positions corresponds to the numbering used for the mature subtilisin sequence of Bacillus amyloliquefaciens SEQ ID NO: 2. However, the invention is not limited to a mutation of said specific subtilisin, but encompasses precursor proteases containing amino acid residues at positions "equivalent" to the specific identified subtilisin residues of Bacillus amyloliquefaciens.

As used herein, the terms “modification” and “mutation” refer to any change or transformation of an amino acid sequence. The term is intended to encompass substitutions, deletions, insertions and / or substitution of amino acid side chains in an amino acid sequence of interest (e.g., subtilisin sequence). It is also intended that this term encompasses chemical modification of an amino acid sequence of interest (e.g., subtilisin sequence).

As used herein, the term “antibodies” refers to immunoglobulins. Antibodies include, without limitation, immunoglobulins directly derived from species from which it is desirable to obtain antibodies. In addition, the present invention encompasses modified antibodies. The term also refers to antibody fragments that retain the ability to bind the epitope to which an intact antibody binds, and include fragments of polyclonal antibodies, monoclonal antibodies, chimeric antibodies, anti-idiotypic (anti-ID) antibodies. Antibody fragments include, but are not limited to, hypervariable regions (CDRs), variable regions of single chain fragments (scFvs), variable regions of heavy chains (VH), variable regions of light chains (VL). Polyclonal and monoclonal antibodies are also within the scope of the present invention. Preferably, the antibodies are monoclonal antibodies.

The term "oxidation resistant" refers to the proteases of the present invention that retain a certain amount of enzymatic activity for a given period of time under conditions prevailing during the course of proteolysis, hydrolysis, cleaning, or another process of the present invention, for example, when exposed to bleaching or oxidizing agents, or when brought into contact with the specified means. In some embodiments, the proteases retain at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% of proteolytic activity after contact with a bleaching or oxidizing agent for a certain period of time, comprising, for example, at least about 1 minute, about 3 minutes, about 5 minutes, about 8 minutes, about 12 minutes, about 16 minutes, about 20 minutes, etc. In some embodiments, stability is measured using the methods described in the Examples.

The term "resistant to chelating agent" refers to the proteases of the present invention, which retain a certain amount of enzymatic activity for a given period of time under conditions prevailing during the proteolysis, hydrolysis, cleaning or other process of the present invention, for example, when exposed to a chelating agent, or when contacting with a chelating agent. In some embodiments, the proteases retain at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% of proteolytic activity after contacting with a chelating agent for a certain period of time, for example, at least about 10 minutes, about 20 minutes, about 40 minutes, about 60 minutes, about 100 minutes, etc. In some embodiments, resistance to a chelating agent is measured using the methods described in the Examples.

The terms "thermally stable" and "thermostable" refer to the proteases of the present invention, which retain a certain amount of enzymatic activity after exposure to a certain temperature for a given period of time under conditions prevailing during the course of proteolysis, hydrolysis, cleaning, or another process of the present invention, for example, when temperature change. A change in temperature includes an increase or decrease in temperature. In some embodiments, the proteases retain at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% of proteolytic activity after exposure to a changed temperature for a certain period of time, for example, at least about 60 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes etc. In some embodiments, thermal stability is measured using the methods described in the Examples.

The term “enhanced stability” refers to a protease resistant to oxidation, chelating agents, temperature and / or pH, which retains a higher value of proteolytic activity over time than other serine proteases (eg, subtilisin proteases) and / or wild-type enzymes .

The term "reduced stability" refers to a protease that is resistant to oxidation, the action of a chelating agent, temperature and / or pH, which retains a higher value of proteolytic activity over time than other serine proteases (eg subtilisin proteases) and / or wild-type enzymes .

The term "cleaning activity" refers to the cleaning characteristics of the protease under conditions prevailing during the course of proteolysis, hydrolysis, cleaning or other process of the present invention. In some embodiments, the cleaning characteristics are determined using various brushing assays, including enzyme-sensitive stains, such as grass, blood, milk or egg white stains, by various chromatographic, spectrophotometric or other quantitative methods after exposure to stains under standard washing conditions. Examples of assays include, without limitation, the assays described in WO 99/34011 and US Pat. No. 6,605,458 (which are incorporated herein by reference), as well as the methods described in the Examples.

The term “purification effective amount” as applied to a protease refers to an amount of a protease previously described that provides the desired level of enzymatic activity in a particular cleaning composition. The indicated effective amount that can be easily determined by an ordinary person skilled in the art depends on many factors, such as the specific type of protease used, the purpose of the cleaning, the specific composition of the cleaning composition, the desired form of the composition, liquid or dry (e.g. granular, whetstones), and other

The term “auxiliary cleaning agents” as used herein refers to all liquid, solid or gaseous substances selected for a particular type of desired cleaning composition and product form (such as liquid, granular, powdery, in the form of bars, paste-like, in the form of a spray, tablet, gel or foam composition), wherein said substances are preferably compatible with the protease enzyme used in the composition. In some embodiments, the granular compositions are in a "compact" form, and in other embodiments, the liquid compositions are in a "concentrated" form.

The term “improved performance” as applied to cleaning activity refers to increased or improved cleaning activity for stains sensitive to a particular enzyme, such as stains from eggs, milk, grass or blood, which is determined visually after a standard cleaning cycle and / or several cleaning cycles.

The term “degraded performance” as applied to cleaning activity refers to reduced or degraded cleaning activity with respect to stains sensitive to a particular enzyme, such as stains from eggs, milk, grass or blood, which is determined visually after a standard cleaning cycle.

The term “comparable performance” refers to a cleaning activity that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least at least about 95% of the cleaning activity of the comparable subtilisin protease (e.g., a commercially available protease), including, without limitation, OPTIMASE ^™ protease (Genencor), PURAFECT ^™ protease products (Genencor), SAVINASE ^™ protease (Novozymes), BPN ' options (see, for example, US Patent No. Re 34606), RELASE ^™ , DURAZYME ^™ , EVERLASE ^™ , KANNASE ^™ protease (N ovozymes), MAXACAL ^™ , MAXAPEM ^™ , PROPERASE ^™ proteases (Genencor; see also US Patent No. Re 34606 and US Patent Nos. 5,700,676; 5,955,340; 6,312,936; and 6,482,628), and variant B. lentus protease products (e.g. WO 92/21760, WO 95/23221 and / or WO 97/07770). Examples of subtilisin protease variants include, but are not limited to, variants containing substitutions or deletions at residual positions equivalent to positions 76, 101, 103, 104, 120, 159, 167, 170, 194, 195, 217, 232, 235, 236 , 245, 248 and / or 252 BPN '. Cleaning characteristics can be determined by comparing the proteases of the present invention with subtilisin proteases using various cleaning assays using enzyme-sensitive stains, such as grass, blood or milk stains, and detection by conventional spectrophotometric or analytical methods after a cleaning cycle under standard conditions.

As used herein, “fabric cleaning compositions” include hand and machine washable laundry detergent compositions, including additional laundry detergent compositions and compositions suitable for soaking and / or pre-treating soiled fabrics (eg, clothes, bedding, and other textile materials).

As used herein, the term “compositions for cleaning non-tissue products” refers to compositions for cleaning non-textile (ie, non-fabric) surfaces, including, without limitation, detergent dishwashing compositions, oral care compositions, compositions for cleaning dentures and compositions for personal hygiene.

The "compact" form of the cleaning compositions of the present invention is best characterized by the density and, in terms of composition, the amount of inorganic salt used as a filler. Inorganic salts used as a filler are traditional ingredients of powdered detergent compositions. In traditional detergent compositions, salts used as a filler are present in large quantities, usually comprising from about 17 to 35%, relative to the total weight of the composition. In compact compositions, on the contrary, the filler salt is present in an amount not exceeding about 15% of the total weight of the composition. In some embodiments, the filler salt is present in an amount not exceeding about 10%, or more preferably about 5%, based on the weight of the composition. In some embodiments, inorganic filler salts are selected from sulfates and chlorides of alkali and alkaline earth metals. A preferred filler salt is sodium sulfate.

As used herein, the term “surfactant” refers to any compound that is known in the art as having surfactant properties. Surfactants typically include anionic, cationic, nonionic and zwitterionic compounds, which are further described herein.

As used herein, the terms “contacting” and “exposure” refers to placing a surfactant and a lipolytic enzyme in sufficient proximity to an oily stain or oily contamination to provide at least a reduction in the amount of stain or contamination by the enzyme and a surfactant by producing fatty acids that are solubilized in a surfactant. Contacting can take place in a washing machine, a sewer, on a body surface, and the like.

experimental part

The present invention is described in more detail in the following examples, which are not intended to limit the scope of the invention defined in the claims. The following examples are provided to illustrate, but not to limit, the claimed invention.

The following abbreviations are used in the description of the experimental part below: (parts per million); M (molar); mm (millimolar); μM (micromolar); nM (nanomolar); mole (moth); mmol (millimoles); micromoles (micromoles); nmol (nanomoles); g (grams); mg (milligrams); mcg (micrograms); pg (picograms); l (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); units (units); V (volt); MM (molecular weight); sec (seconds); min (minutes); h (hours); ° C (degrees Celsius); QS (sufficient amount); ND (not finished); NA (not applicable); rpm (revolutions per minute); H ₂ O (water); dH ₂ O (deionized water); (HCl (hydrochloric acid); ac (amino acid); bp (base pairs); so (thousand base pairs); cD (kilodaltons); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single-stranded DNA); dsDNA (double-stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); MgCl ₂ (magnesium chloride); NaCl (sodium chloride); mass / vol (mass to volume ratio); vol / vol ( volume ratio); g (gravity); OD (optical density); Dulbecco's phosphate-buffered solution (DPBS); SOC (2% bactotrypton, 0.5% bacterial yeast extract, 10 mM NaCl, 2.5 mM KCl); Terrific broth (TB; 12 g / L bactotriptone, 24 g / L glycerol, 2.31 g / L KH ₂ PO ₄ and 12.54 g / L K ₂ HPO ₄ ); OD ₂₈₀ (optical density at 280 nm); OD ₆₀₀ (optical density at 600 nm); A ₄₀₅ (absorption at 405 nm); Vmax (maximum initial reaction rate catalyzed by the enzyme); PAGE (polyacrylamide gel electrophoresis) ; PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PBST (PBS + 0.25% TWEEN® 20); PEG (polyethylene glycol); PCR (polymerase chain reaction); RT-PCR (reverse transcription PCR); SDS (sodium dodecyl sulfate); tris (tris (hydroxymethyl) aminomethane); HEPES (N- [2-hydroxyethyl] piperazine-N- [2-ethanesulfonic acid]); HBS (HEPES buffered saline); Tris-HCl (Tris [hydroxymethyl] aminomethane hydrochloride); tricin (N- [tris (hydroxymethyl) methyl] glycine); CHES (2- (N-cyclohexylamino) ethanesulfonic acid); TAPS (3 - {[tris- (hydroxymethyl) methyl] amino} propanesulfonic acid); CAPS (3- (cyclohexylamino) propanesulfonic acid; DMSO (dimethyl sulfoxide); DTT (1,4-dithio-DL-threitol); SA (synapinic acid (s, 5-dimethoxy-4-hydroxycinnamic acid); TCA (trichloroacetic acid) ; Glut and GSH (reduced glutathione); GSSG (oxidized glutathione); TCEP (tris [2-carboxyethyl] phosphine); Ci (curie); mCi (millicure); μCi (microcurie); HPLC (high performance liquid chromatography); RP HPLC (reverse phase high performance liquid chromatography); TLC (thin layer chromatography); MALDI-TOF (time-of-flight laser desorption ionization using using a matrix); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms (mesyl); Et (ethyl), Me (methyl); Taq (Thermus aquaticus DNA polymerase); Klenov fragment (large fragment (Klenova ) DNA polymerase I); EGTA (ethylene glycol bis (β-aminoethyl ether) N, N, N ', N'-tetraacetic acid); EDTA (ethylenediaminetetraacetic acid); bla (β-lactamase or ampicillin resistance gene); HDL (high density lipoprotein); MJ Research (MJ Research, Reno, NV); Baseclear (Baseclear BV, Inc., Leiden, the Netherlands); PerSeptive (PerSeptive Biosystems, Framingham, MA); ThermoFinnigan (ThermoFinnigan, San Jose, CA); Argo (Argo BioAnalytica, Morris Plains, NJ); Seitz EKS (SeitzSchenk Filtersystems GmbH, Bad Kreuznach, Germany); Pall (Pall Corp., East Hills, NY); Spectrum (Spectrum Laboratories, Dominguez Rancho, CA); Molecular Structure (Molecular Structure Corp., Woodlands, TX); Accelrys (Accelrys, Inc., San Diego, CA); Chemical Computing (Chemical Computing Corp., Montreal, Canada); New Brunswick (New Brunswick Scientific, Co., Edison, NJ); CFT (Center for Test Materials, Vlaardingen, the Netherlands); Test Fabrics (Test Fabrics, Inc., West Pittiston, PA), Procter & Gamble (Procter & Gamble, Inc., Cincinnati, OH); GE Healthcare (GE Healthcare, Chalfont St. Giles, United Kingdom); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme (Megazyme International Ireland Ltd., Bray Business Park, Bray, Co., Wicklow, Ireland); Finnzymes (Finnzymes Oy, Espoo, Finland); Kelco (CP Kelco, Wilmington, DE); Corning (Corning Life Sciences, Corning, NY); (NEN (NEN Life Science Products, Boston, MA); Pharma AS (Pharma AS, Oslo, Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis, Lewisville, TX); ATCC (American Type Culture Collection, Rockville, MD); Gibco / BRL (Gibco / BRL, Grand Island, NY); Sigma (Sigma Chemical Co, St. Louis, MO); Pharmacia (Pharmacia Biotech, Piscataway, NJ); NCBI (National Center for Biotechnology Information); Applied Biosystems (Applied Biosystems, Foster City, CA); BD Biosciences and / or Clontech (BD Biosciences CLONTECH Laboratories, Palo Alto, CA); Operon Technologies (Operon Technologies, Inc., Alameda, CA); MWG Biotech (MWG Biotech, High Point, NC); Oligos Etc (Oligos Etc. Inc, Wilsonville, OR); Bachem (Bachem Bioscience, Inc., King of Prussia, PA); Difco (Difco Laboratories, Detroit, MI); Mediatech ( Mediatech, Herndon, VA; Santa Cruz (Santa Cruz Biotechnology, Inc, Santa Cruz, CA); Oxoid (Oxoid Inc, Ogdensburg, NY); Worthington (Worthington Biochemical Corp, Freehold, NJ); GIBCO BRL or Gibco BRL (Life Technologies, Inc, Gaithersburg, MD); Millipore (Millipore, Billerica, MA); Bio-Rad (Bio-Rad, Hercules, CA); Invitrogen (Invitrogen Corp, San Diego, CA); NEB (New England Biolabs, Ipswich, MA); Sigma (Sigma Chemical Co, St. Louis, MO); Pierce (Pierce Biotechnology, Rockford, IL); Takara (Takara Bio Inc. Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science (EM Science, Gibbstown, NJ); Qiagen (Qiagen, Inc, Valencia, CA); Biodesign (Biodesign Intl., Saco, Maine); Aptagen (Aptagen, Inc., Herndon, VA); Sorvall (Sorvall brand, from Kendro Laboratory Products, Asheville, NC); United States Testing (United States Testing Co, Hoboken, NJ); Molecular Devices (Molecular Devices, Corp, Sunnyvale, CA); R&D Systems (R&D Systems, Minneapolis, MN); Stratagene (Stratagene Cloning Systems, La Jolla, CA); Marsh (Marsh Biosciences, Rochester, NY); Geneart (Geneart GmbH, Regensburg, Germany); DNA2.0 (DNA2.0, Menlo Park, CA); Gene Oracle (Gene Oracle, Mountain View, CA); Bio-Tek (Bio-Tek Instruments, Winooski, VT); Biacore (Biacore, Inc, Piscataway, NJ); PeproTech (PeproTech, Rocky Hill, NJ); SynPep (SynPep, Dublin, CA); New Objective (New Objective brand; Scientific Instrument Services, Inc., Ringoes, NJ); Waters (Waters, Inc., Milford, MA); Matrix Science (Matrix Science, Boston, MA); Dionex (Dionex, Corp., Sunnyvale, CA); Monsanto (Monsanto Co, St. Louis, MO); Wintershall (Wintershall AG, Kassel, Germany); BASF (BASF Co, Florham Park, NJ); Huntsman (Huntsman Petrochemical Corp, Salt Lake City, UT); Enichem (Enichem Iberica, Barcelona, Spain); Fruka Chemie AG (Fluka Chemie AG, Buchs, Switzerland); Gist-Brocades (Gist-Brocades, NV, Delft, the Netherlands); Dow Corning (Dow Corning Corp, Midland, MI); and Microsoft (Microsoft, Inc., Redmond, WA).

EXAMPLE 1

Analyzes

In the following examples, different assays are used as described below for ease of reading. All deviations from the following methods are indicated in the examples.

A. Determination of protein content by TCA using 96-well microtiter plates

Analysis of BPN '(e.g., standard protease) and BPN' variants is carried out using filtered culture supernatant from microtiter plates after growing for 3-4 days at 33 ° C and shaking at 230 rpm in a humid atmosphere. A new 96-well flat-bottom microtiter plate (MTP) was used for analysis. First, 100 μl / well of 0.25 N HCl are placed in each well. Then add 50 μl of filtered culture broth. The scattering / absorption of light is determined at 405 nm (use the 5-second mixing mode in a tablet reader) to provide a “control” reading. For analysis, 100 μl / well of 15% (w / v) trichloroacetic acid (TCA) was added to the plates and incubated for 5 to 30 minutes at room temperature. Then, scattering / absorption of light is determined at 405 nm (use the 5-second mixing mode in a tablet reader).

An analysis of GG36 (e.g., a standard protease) and its variants is carried out using filtered culture supernatant from microtiter plates after growing for about 3 days at 37 ° C and shaking at 300 rpm in a humid atmosphere. In this assay, 100 μl of a 0.25 M HCl solution is added to each well of a 96-well flat-bottom microtiter plate. Aliquots of filtered culture supernatants (containing proteases) of 25 μl are then added to the wells. The scattering / absorption of light is determined at 405 nm (use the 5-second mixing mode in a tablet reader) to provide a “control” reading. After the measurement, 100 μl of a 30% (w / v) TCA solution was added to all wells, and microtiter plates were incubated for 5 to 15 minutes at room temperature. Finally, determine the final value of the scattering / absorption of light at 405 nm (using the 5-second mixing mode in a tablet reader).

Equipment used: Biomek FX Robot (Beckman Coulter) and reader for MTP SpectraMAX (type 340; Molecular Devices); Costar MTP (type 9017). Equipment used: Biomek FX Robot (Beckman Coulter) and reader for MTP SpectraMAX, type 340 (Molecular Devices); MTP type 9017 (Costar).

The calculations are carried out by subtracting the values of the control samples (TCA is absent) from the values of the tested samples containing TCA, to obtain the relative protein content in the samples. If desired, a standard curve can be obtained by calibrating the values of samples containing TCA using AAPF analyzes of clones with known transition coefficients. However, the curve for samples containing TCA is linear in the region corresponding to a protein concentration of 50 to 500 protein molecules per ml (ppm), and, therefore, can be used to determine the characteristics of the enzyme in order to select options with good characteristics. The increase in turbidity / light scattering in the samples correlates with the total amount of precipitated protein in the culture supernatant.

B. AAPF Assay of Protease in 96-well Microplate Microtiter Plates

To determine the protease activity of the present invention and its variants, the hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide (suc-AAPF-pNA) is analyzed. The following reagent solutions are used: 100 mM Tris / HCl, pH 8.6, containing 0.005% TWEEN®-80 (Tris dilution buffer); 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl ₂ and 0.005% TWEEN®-80 (Tris / Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (stock solution of suc-AAPF-pNA) (Sigma: S-7388). To obtain the suc-AAPF-pNA working solution, 1 ml of the suc-AAPF-pNA stock solution was added to 100 ml of Tris / Ca-buffer and mixed thoroughly for at least 10 seconds. The analysis is carried out by adding 10 μl of a dilute protease solution to each well, after which 190 μl of suc-AAPF-pNA working solution with a concentration of 1 mg / ml is immediately added. The solutions are mixed for 5 seconds and then read the change in absorption in the kinetic mode (20 readings for 5 minutes) at 410 nm in an MTP reader at 25 ° C. Protease activity is expressed as absorption units (activity = ΔOD ^. Min ^-1 ml ^-1 ).

C. Surfactant and Chelating Resistance Assays

Resistance to LAS and LAS / EDTA is determined after incubation of the analyzed protease with LAS and LAS / EDTA, respectively, as a function of the residual activity determined using the AAPF analysis.

Resistance to LAS, a technique

Reagents:

Dodecylbenzenesulfonate, sodium salt (= LAS): Sigma D-2525

TWEEN®-80: Sigma P-8074

Tris buffer (free acid): Sigma T-1378; 6.35 g is dissolved in approximately 960 ml of water; The pH was adjusted to 8.2 using 4N HCl. The final concentration of tris is 52.5 mm.

Stock LAS solution: 10.5% solution of LAS in water MQ is obtained (= 10.5 g per 100 ml MQ)

Tris buffer: 100 mM / pH 8.6 (100 mM Tris / 0.005% Tween®-80)

Tris-Ca buffer, pH 8.6 (100 mM Tris / 10 mM CaCl ₂ / 0.005% Tween®-80)

Equipment:

Flat Bottom MTP (Costar No. 9017)

Biomek fx

ASYS Multichannel Pipette

Reader for MTP Spectramax

IEMS Incubator / Shaker

Incubator / Shaker Innova 4330

Biohit multichannel pipette

Shaker BMG Thermostar

Obtain a 0.063% solution of LAS in 52.5 mm Tris-buffer pH 8.2. A working solution of suc-AAPF-pNA is prepared by adding 1 ml of a stock solution of suc-AAPF-pNA at a concentration of 100 mg / ml (in DMSO) to 100 ml (100 mmol) of Tris buffer, pH 8.6. To dilute the supernatants, dilution buffer was added to the flat-bottomed plates, then an aliquot of the supernatant was added and mixed thoroughly. The dilution ratio depends on the concentration of protease controls in culture plates (AAPF activity). The desired protein concentration is 80 ppm.

Ten μl of the diluted supernatant is added to 190 μl of 0.063% LAS buffer / well. The MTP is film-coated, shaken for several seconds, placed in an incubator (Innova 4230) and incubated at 45 ° C in the case of BPN 'or GG36 for 60 minutes, shaking at 200 rpm. Initial activity (t = 10 minutes) was determined after 10 minutes of incubation by transferring 10 μl of the mixture to each well of a new MTP containing 190 μl of suc-AAPF-pNA working solution. The resulting solutions were thoroughly mixed and the AAPF activity was measured using an MTP reader (20 readings for 5 minutes at 25 ° C).

To determine the final activity (t = 60 minutes), after 60 minutes of incubation, another 10 μl of solution was taken from the incubation plate. AAPF activity is determined using the method described above. The stability of the samples is determined by calculating the ratio of residual and initial AAPF activity according to the following formula:

Residual activity (%) = [t-60 value] * 100 / [t-10 value].

Determination of resistance to LAS / EDTA

The stability of the protease variants in the presence of a typical anionic surfactant (LAS = linear alkylbenzenesulfonate, sodium dodecylbenzenesulfonate - DOBS) and EDTA disodium salt is measured after incubation under certain conditions, the residual activity is determined using AAPF analysis. Sodium salt of dodecylbenzenesulfonate (DOBS, Sigma No. D-2525), TWEEN®-80 (Sigma No. P-8074), di-sodium EDTA (Siegfried Handel No. 164599-02), HEPES (Sigma No. H-7523), control buffer: 50 mM HEPES (11.9 g / l) + 0.005% TWEEN®-80, pH 8.0, test buffer: 50 mM HEPES (11.9 g / l), 0.1 % (w / v) DOBS (1 g / l), 10 mM EDTA (3.36 g / l), pH 8.0, culture supernatants containing a standard protease and protease variants with a protein content of 200-400 μg / ml. Equipment: MTP with V- or U-shaped bottom as dilution plates (Greiner 651101 and 650161, respectively), MTP with F-shaped bottom (Corning 9017) for control buffer and LAS / EDTA buffer, as well as tablets for suc- AAPF-pNA, Biomek FX (Beckman Coulter), reader for MTP Spectramax Plus 384 (Molecular Devices), incubator / shaker iEMS (1 mm amplitude) (Thermo Electron Corporation), adhesive film: Nunc (236366).

The iEMS incubator / shaker (Thermo / Labsystems) is set at 29 ° C. Cultural supernatants are diluted in tablets containing control buffer to a concentration of ~ 25 ppm. (tablet of the main dilution). 20 μl of the sample from the main dilution plate is added to tablets containing 180 μl of control buffer to obtain a final incubation concentration of 2.5 ppm. The contents are mixed, kept at room temperature, and AAPF analysis is performed on the same plate. 20 μl of the sample from the main dilution plate is also added to tablets containing 180 μl of test buffer (50 mM HEPES (11.9 g / l), 0.1% (w / v) DOBS (1 g / l), 10 mM EDTA (3.36 g / l), pH 8.0). The solutions are mixed and immediately placed in an iEMS shaker, where they are shaken at 29 ° C for 30 minutes at 400 rpm. After incubation for 30 minutes, the test plate is subjected to AAPF analysis. The stability of the samples is determined by calculating the ratio of residual and initial AAPF activity according to the following formula:

Residual activity (%) = [mOD.min ^-1 experienced] * 100 / [mOD.min ^-1 control].

D. Analyzes of cleaning performance

The ability of protease variants to remove stains is determined in commercially available detergents. Commercial detergent compositions are subjected to inactivation by heating to destroy the enzymatic activity of all protein components while maintaining the properties of non-enzymatic components. This method can be used to obtain commercial detergents suitable for testing enzyme variants of the present invention.

Tissue microsamples:

Microscopic samples of round tissue with a diameter of ¼ ″ are ordered and obtained from the CFT. One or two micro-samples are placed vertically in each well of a 96-well MTP so that the entire surface is exposed (i.e., they are not placed on the bottom of the well).

BMI analysis of tissue microsamples

Micro samples containing blood, milk, and ink (BMI) 0.25 inches in diameter are obtained from the CFT. Before cutting samples, the fabric (EMPA 116) is washed with water. One microsample is placed vertically in each well of a 96-well microtiter plate so that the entire surface of the sample is exposed (i.e., not placed on the bottom of the well). The desired detergent solution is prepared according to the method described herein. After equilibrating the thermomixer at 25 ° C, 190 μl of detergent solution is added to each well of the MTP containing the micro samples. 10 μl of a diluted enzyme solution is added to the resulting mixture so that the final concentration of the enzyme is 1 μg / ml (determined by BCA analysis). MTP is covered with a film and placed in an incubator for 30 minutes, shaking at 1400 rpm. After incubation under appropriate conditions, 100 μl of the solution from each well is transferred to fresh MTP. A new MTP containing 100 μl of solution / well was read at 405 nm using a SpectraMax MTP reader. The analysis also uses empty controls and a control that contains two microsamples and a detergent, but does not contain an enzyme.

Baked egg microsample analysis

This assay uses 96-well plates containing baked chicken egg yolks as a substrate. Chicken egg yolks are separated from proteins, released from the membrane pouch and diluted with 20% (v / w) Milli-Q water. The diluted yolk is stirred for 15 minutes at room temperature using a magnetic stirrer. Five μl is carefully transferred using an 8-channel pipette into the center of each well of a 96-well V-shaped plate (Costar # 3894). The plates are heated at 90 ° C for 1 hour and then cooled to room temperature. Plates containing baked egg yolk as a substrate are stored at room temperature and used within one week of receipt. Detergents for dishwashers are prepared using any of the methods described herein and preheated to 50 ° C. An aliquot of 190 μl detergent was added to each well of a 96-well plate using an 8-channel pipette. Then, 10 μl of the diluted enzyme was added to each well using a 96-channel pipetting device. The tablet is carefully covered with sticky foil and incubated at 50 ° C with shaking for 30 minutes. Then 120 μl of the reaction mixture was transferred to a new 96-well flat-bottomed plate and the absorption / scattering of light was determined at 405 nm. The absorption / scattering of light at 405 nm is proportional to the removal of the egg yolk.

Analysis using microsamples of egg yolk

Dishwasher detergents are prepared using any of the methods described herein. The equipment used is a New Brunswick Innova 4230 shaker / incubator and an MTP SpectraMAX reader (type 340). MTP is obtained from Costar (type 9017). An old egg yolk with stained samples (CS-38) is obtained from the Center for testing materials. Before cutting 0.25 inch round microsamples, the fabric is washed with water. One microsample was placed in each well of a 96-well microtiter plate. The test detergent is balanced at 50 ° C. 190 μl of detergent solution is added to each MTP well containing microsamples. To the resulting mixture was added 10 μl of a diluted enzyme solution. MTP is covered with sticky foil and placed in an incubator for 30 minutes with shaking. After incubation, 100 μl of the solution from each well is transferred to fresh MTP. This MTP is read at 405 nm with a SpectraMax MTP reader. The analysis also uses empty controls and controls that contain microsamples and detergent but do not contain an enzyme.

Fixing samples "3K"

This type of microsample is pretreated using the fixation method described below. In a 10-liter beaker, 8 liters of distilled water and 80 ml of a 30% hydrogen peroxide solution are thoroughly mixed with a ladle. Forty pieces of EMPA 116 samples were fan-shaped before being added to the solution to ensure uniform fixation. Using a ladle, the samples were forced to circulate in the solution for only 30 minutes, continuously for the first five minutes and periodically for the remaining 25 minutes. After the fixation process is complete, the solution is discarded, and the samples are washed 6 times using approximately 6 liters of distilled water for one rinse. After washing, the samples are dried on paper towels. The air-dried samples are then composted using a ¼ "ring stamp on a displacement press. Finally, one micro-sample is vertically placed in each well of a 96-well MTP so that the entire surface is exposed (i.e., do not lay on the bottom of the well).

Soaked Sample

This type of microsample is soaked in deionized water for 20 minutes at ambient temperature. After soaking, the samples are placed on paper towels and dried. The air-dried samples are then composted using a ¼ "ring stamp on a displacement press. Finally, two micro-samples are vertically placed in each well of a 96-well MTP so that the entire surface is exposed (i.e., do not lay on the bottom of the well).

Detergents

Inactivation by heating of liquid washing detergents in North America (NA) and Western Europe (WE) for use under severe conditions (HDL) is carried out by placing a pre-weighed liquid detergent (in a glass bottle) in a water bath at 95 ° C, where it is kept for 2 hours. The incubation time for heating inactivation of the granular washing detergents of North America (NA) and Japan (JPN) for use under severe conditions (HDG) is 8 hours, and for HDG detergents in Western Europe (WE), the incubation time is 5 hours. The incubation time for heating inactivation of detergents for dishwashers (ADW) NA and WE is 8 hours. Detergents are obtained from local supermarkets. Treated and untreated detergents are analyzed for 5 minutes, dissolving the detergent to accurately determine the percentage of decontamination. Enzyme activity is tested using AAPF analysis.

Working solutions are prepared from heat inactivated stock solutions. All detergents are commercially available. To recreate the desired conditions, appropriate amounts of substances that provide water hardness (6 or 12 grains per gallon) and buffer (table 1-1) are added to the detergent solutions. The solutions are mixed on a vortex or by inverting the bottles.

Table 1-1 Washing and washing conditions Region The form Dose Detergent * Buffer Gpg pH T (° C) Washing detergents (liquid and granular for severe conditions) NA HDL 0.78 g / l P&G TIDE® 2X 5 mM HEPES 6 8.0 twenty WE HDL 5.0 g / l Henkel persil 5 mM HEPES 12 8.2 40 WE HDG 8.0 g / l P&G Ariel 2 mm Na ₂ CO ₃ 12 10.5 40 Jpn HDG 0.7 g / l P&G TIDE® 2 mm Na ₂ CO ₃ 6 10.0 twenty NA HDG 1.0 g / l P&G TIDE® 2 mm Na ₂ CO ₃ 6 10.0 twenty Detergents for dishwashers WE ADW 3.0 g / l RB Calgonit 2 mm Na ₂ CO ₃ 21 10.0 40 NA ADW 3.0 g / l P&G Cascade 2 mm Na ₂ CO ₃ 9 10.0 40 * Abbreviations: Procter & Gamble (P&G) and Reckitt Benckiser (RB).

The ability of a standard serine protease and its variants to remove stains from tissue micro-samples is determined in MTP format using a commercially available detergent (Calgonit 5 ml). The following reagents are used: 5 mM HEPES buffer, pH 8.0 or 5 mM MOPS, pH 7, 3: 1 Ca: Mg to medium hardness of water. (CaCl ₂ : MgCl ₂ · 6H ₂ O); a stock solution with a hardness of 15,000 grains per gallon (gpg) is diluted to 6 gpg, 2 BMI samples (blood / milk / ink) per plate: EMPA-116 BMI cotton samples treated with CFT: pre-rinsed and cut 2 samples per well, and inactivated by heating the ready-to-use TIDE® 2X Cold detergent with a confirmed lack of protease activity.

Table 1-2 Detergent Working Solutions Detergent Pace.
(ºС) Detergent,
g / l pH Buffer gpg Protease TIDE® 2X Cold 16 0.98 8 5 mM HEPES 6 BPN ' TIDE® 2X Cold 32 0.98 8 5 mM HEPES 6 GG36,
BPN ' TIDE® 2X Cold 16 0.98 7 5 mm MOPS 6 BPN '

The incubator is set to the desired temperature (16 ° C or 32 ° C). 10 μl of samples from a basic dilution plate containing ~ 10 ppm the enzyme is added to the tablets containing 2 samples of BMI and 190 μl of the working solutions of the detergent described above. The volume is adjusted so as to obtain a final concentration of options in analytical tablets of 0.5 ppm. The plates are immediately transferred to iEMS incubators and incubated for 30 minutes, shaking at 1400 rpm at a predetermined temperature. After incubation, 100 μl of the supernatant was transferred to a new 96-well plate and absorbance was measured at 405 nm and / or 600 nm using an MTP reader. Control assays that contain 1 or 2 microsamples and detergent but do not contain protease samples are also used in this assay. Measurement at 405 nm gives a higher absorption value and indicates the removal of pigment, while measurement at 600 nm allows to obtain data on turbidity and purification.

The calculation of the ability to remove stains used in all methods of analysis of microsamples:

The absorption value is adjusted taking into account the control sample (substrate in the absence of an enzyme) and a measure of hydrolytic activity is obtained. For each sample (option) calculate the indicator of functioning. The performance indicator indicates the activity of the variant (actual value) compared with the standard enzyme (theoretical value) at the same protein concentration. In addition, theoretical values can be calculated using the parameters of the Langmuir equation for a standard enzyme.

Enzymes and equipment

Samples of the standard serine protease and its variants are obtained from filtered culture broths grown in MTP plates. Equipment used: Biomek FX Robot (Beckman Coulter), reader for MTP SpectraMAX (type 340; Molecular Devices), incubator / shaker iEMS (Thermo / Labsystems); MTP with an F-shaped bottom (Costar, type 9017, used to read reaction plates after incubation); and MTP with a V-shaped bottom (Greiner 651101, used for pre-dilution of the supernatant). In this assay, proteases hydrolyze the substrate and release pigment and insoluble particles. Therefore, the degree of turbidity is an indicator of enzyme activity.

E. Relative specific activity of proteases and their variants

To characterize protease variants, the relative specific activity is calculated using suc-AAPF-pNA as a substrate, which allows you to compare the variants with a standard wild-type protease and classify them. The specific activity of the suc-AAPF-pNA substrate is determined by dividing the proteolytic activity by the values of the TCA results obtained for each sample using the assays described above. Using the indicated values, the relative specific activity (specific activity of the variant / specific activity of the standard protease) is calculated.

F. Dishwashing Effectiveness

The effectiveness of protease variants is tested under different conditions of automatic dishwashing. The detergent compositions for washing dishes are shown in the following tables. These detergents are supplied by wfk Testmaterials (www.testgewebe.de/en/products/detergents/) and are labeled with wfk Testmaterials. These detergents are obtained from the supplier without enzymes in order to analyze protease variants.

Table 1-3 Phosphate-free detergent IEC-60436 WFK type B (pH = 10.4 at 3 g / l) Component Mass % Sodium Citrate Dehydrate 30,0 Maleic acid / acrylic acid copolymer sodium salt 12.0 Sodium Perborate Monohydrate 5,0 TAED 2.0 Sodium Disilicate: Protil A (Cognis) 25.0 Linear fatty alcohol ethoxylate 2.0 Anhydrous Sodium Carbonate up to 100

Table 1-4 Phosphate Detergent:
IEC-60436 WFK Type C (pH = 10.5 at 3 g / L) Component Mass % Sodium Tripolyphosphate 23.0 Sodium Citrate Dehydrate 22.3 Maleic acid / acrylic acid copolymer sodium salt 4.0 Sodium Perborate Monohydrate 6.0 TAED 2.0 Sodium Disilicate: Protil A (Cognis) 5,0 Linear fatty alcohol ethoxylate 2.0 Anhydrous Sodium Carbonate up to 100

Methods for obtaining spots of all types (egg yolk, minced meat and egg, as well as an egg with milk) are described below. Before applying certain types of impurities, the test items are thoroughly washed. This must be done, since the remains of some stubborn stains may remain on the dishes after previous tests. Before the first use in the test, new dishes are also thoroughly washed three times.

Getting Egg Yolk Stains on Stainless Steel

Stainless steel trays used in these experiments (10 x 15 cm; sanded on one side) are thoroughly washed at 95 ° C in a laboratory dishwasher using a highly alkaline commercial detergent (e.g. ECOLAB® detergent; Henkel) to obtain clean and fat-free baking trays . Before the first use, the burrs are removed from these sheets. Before contamination with egg yolk, the baking sheets are dried for 30 minutes at 80 ° C in a heat oven. Surfaces to be sanded do not touch before contamination. In addition, water stains or lint are not allowed on surfaces. Before contamination, chilled baking sheets are weighed.

Egg yolks are obtained by separating approximately 10-11 eggs (200 g of egg yolk) from proteins. The yolks are mixed with a fork in a glass beaker until a homogeneous suspension of the yolk is obtained. Then the yolks are filtered (through a sieve with holes of about 0.5 mm) to remove large particles and all fragments of the eggshell.

Using a flat brush (2.5 "), apply 2.0 ± 0.1 g of egg yolk suspension as uniformly as possible over 140 cm ^{2 of the} polished sides of all stainless steel baking sheets, leaving an unpolluted edge approximately 1 cm wide ( if necessary use adhesive tape.) Contaminated baking sheets are dried in a horizontal position (to prevent droplets from forming on the edges of the baking sheets) at room temperature for 4 hours (max. 24 hours).

To denature the egg yolk proteins, the baking sheets are immersed for 30 seconds in boiling demineralized water (if necessary, use a fastening device). Then the baking sheets are again dried for 30 minutes at 80 ° C. After drying and cooling, the trays are weighed. After weighing, the baking sheets are left for at least 24 hours (20 ° C, 40-60% relative humidity) and then they are subjected to a washing test. For testing use only baking sheets that meet the testing requirements, that is, containing 1000 ± 100 mg / 140 cm ² (egg yolk after denaturation). After washing tests, the trays are dried for 30 minutes at 80 ° C in a heat cabinet, cooled and weighed again. The percentage of cleaning efficiency is determined by dividing the mg of egg yolk released during washing by mg of denatured applied egg yolk and multiplying by 100.

Getting stains of minced meat and eggs on porcelain plates

In these experiments, dessert plates (Arzberg, 19 cm in diameter, white, glazed porcelain) are used, corresponding to EN 50242, form 1495, No. 0219. A total of 225 g of lean pork and beef (at a ratio of 50:50) is finely chopped and kept chilled. The mixture is passed twice through a meat grinder. Maintain a temperature not exceeding 35 ° C. Then 225 g of minced meat is mixed with 75 g of eggs (protein and yolk are mixed together). Then the drug is frozen and kept for up to three months at -18 ° C before use. If pork is missing, use 100% beef, as they are interchangeable.

A mixture of chopped meat and eggs (300 g) is warmed to room temperature and mixed with 80 ml of demineralized water. The mixture is then homogenized for 2 minutes using a hand-held kitchen blender. Using a fork, distribute 3 g of the chopped meat / egg / water mixture on each white porcelain plate, leaving an uncontaminated strip at the edge of the plate about 2 cm wide. The applied amount is about 11.8 ± 0.5 mg / cm ² . The plates are dried for approximately 2 hours at 120 ° C in a preheated heat oven. After cooling, the plates are ready for use.

After testing the washing of dishes, a solution of ninhydrin (1% in ethanol) is sprayed onto the plates to better identify the residues of chopped meat protein. To initiate a color reaction, the plates are heated for 10 min at 80 ° C in a heat cabinet. Determining the effectiveness of washing is determined by visual observation of the color reactions of the residues of minced meat in accordance with the IKW photographic catalog (IKW - The German Cosmetic, Toiletry, Perfumery and Detergent Association).

Getting egg / milk stains on stainless steel

The stainless steel trays used in these experiments (10 x 15 cm; sanded on one side) are thoroughly washed at 95 ° C in a laboratory dishwasher using a highly alkaline commercial detergent to remove grease and clean the trays. Baking sheets are ground in a dry state with a cellulose cloth. Surfaces to be sanded do not touch before contamination. In addition, water stains or lint are not allowed on surfaces. Before contamination, the baking sheets are placed for 30 minutes in a heat cabinet at 80 ° C. After cooling, the baking sheets are weighed.

The yolks and proteins of whole raw eggs (3-4 eggs; approximately 160 g / egg) are placed in a bowl and beat with an egg whisk. Then, 50 ml of skim milk is added to the mixture (1.5% fat, ultrahigh-temperature, homogenized). Milk and eggs are mixed without foam. Using a flat brush, uniformly distribute 1.0 ± 0.1 g of the egg / milk mixture onto the polished side of the stainless steel baking sheets, using a balance to check the distribution. A strip with a width of approximately 1.0 cm is left on the short sides of the baking sheets. Contaminated baking sheets are dried in a horizontal position (to prevent droplets from forming on the edges of the baking sheets) at room temperature for 4 hours (max. 24 hours).

Then the baking sheets are immersed for 30 seconds in boiling demineralized water (if necessary, use a mounting device). Then the baking sheets are again dried for 30 minutes at 80 ° C. After drying and cooling, the trays are weighed. After weighing, the trays are left to stand for at least 24 hours (20 ° C, 40-60% relative humidity) and then they are subjected to a washing test. For testing use only baking sheets that meet the requirements of the test, that is, containing 190 ± 10 mg of egg yolk / milk.

After washing tests, the baking sheets are dried for 30 minutes at 80ºC in a heat cabinet, cooled and weighed again. The percentage of cleaning efficiency is determined by dividing mg of the egg / milk mixture released during washing by mg of the applied egg / milk mixture and multiplying by 100.

Washing equipment and washing conditions

Washing tests are carried out in a dishwasher (Miele, model G690SC) equipped with dirty dishes and dirty stainless steel trays obtained using the methods described above. Use a certain amount of detergent. Tests are carried out at a temperature of 50 ° C. Water hardness is 21 ° GH (German hardness). As described above, after washing, dishes contaminated with minced meat are visually evaluated using a photographic rating scale from 0 to 10, where “0” corresponds to a completely dirty plate and “10” corresponds to a clean plate. These values are an indicator of the ability of an enzyme-containing detergent to remove stains or dirt (SR). Washed stainless steel plates contaminated with egg yolk or an egg yolk / milk mixture are analyzed gravimetrically to determine the amount of stain remaining after washing. The protease variant is tested at a level of 0 to 30 mg / active protein per wash cycle.

G. Inhibition analysis using eglin C

As described herein, the concentration and specific activity of a serine protease are determined by titration with an inhibitor. Eglin C from the leech Hirudo medicinalis is a strong binding inhibitor of subtilisins and ASP protease (Heinz et al., Biochemistry, 31: 8755-66, 1992), therefore, it can be used to measure the concentration of the enzyme, which, in turn, is used to calculation of specific activity. In short, the degree of inhibition of the enzyme is determined by the action of several known concentrations of eglin C. Based on the information obtained, the concentration of eglin C, which is necessary for complete inhibition, is calculated. It corresponds to the concentration of the enzyme in the sample.

Protease activity is measured using the above analysis using chromogenic AAPF. The eglin C gene is synthesized and expressed in E. coli using standard methods. It has the same properties and inhibitory activity as eglin C obtained from Sigma. The concentration of the eglin C stock solution is determined by measuring the inhibition of a Bacillus lentus subtilisin sample with known specific activity. Then a calibrated sample of eglin C is used to determine the concentration and specific activity of subtilisin variants. The indicated values are used to obtain 96-well plates with stock solutions of the enzyme, where all variants are diluted to the usual concentration.

H. Performance indicator

The performance indicator indicates the activity of the variant (actual value) compared with the standard protease (theoretical value) at the same protein concentration. In addition, theoretical values can be calculated using the binding curve parameters (i.e., the Langmuir equation) for the standard protease. If the functioning index (PI) is greater than 1 (PI> 1), the variant is considered better than the standard enzyme (for example, wild-type), if PI is 1 (PI = 1), this indicates that the variant has the same characteristics. like a standard enzyme, and if PI is less than 1 (PI <1), it is believed that the variant functions worse than the standard enzyme. Thus, PI allows you to identify the best options, as well as less desirable options for use in certain conditions.

EXAMPLE 2

Obtaining BPN 'protease in B. subtilis

This example describes the experiments used to obtain BPN 'protease in B. subtilis. Transformation of B. subtilis with the plasmid pHPLT-BPN 'is carried out using methods known in the art (see, for example, WO 02/14490, incorporated herein by reference). The following is a DNA sequence (aprE-BPN 'fusion leader sequence, pro-BPN' DNA sequence and mature BPN 'from B. amyloliquefaciens) encoding a BPN' precursor protein:

In the above sequence, the DNA sequence encoding the mature protease is in bold, the standard font denotes the leader sequence (aprE-hybrid leader BPN '), and pro-sequences (BPN') are underlined. The amino acid sequence (aprE-BPN 'hybrid leader, pro-BPN' and mature BPN ') of the BPN' precursor protein is shown below. The mature BPN 'protease sequence is highlighted in bold and underlined.

Construction of expression vector BPN '(pHPLT-BPN')

PCR fragments were obtained under standard conditions in the presence of TGO polymerase (Roche Diagnostics) using pJH101term. Plasmid pJH101 corresponds to pJH101 (Ferrari et al., J Bacteriol, 154: 1513-5 [1983]), where the following fragment is inserted into the EcoRI / BamHI site:

This fragment includes the BPN 'gene and a hybrid signal sequence (containing the first eight amino acids of the B. subtilis aprE signal sequence and B. amyloliquefaciens BPN' signal sequence, starting from the 9th amino acid). This fragment is used as a template for the PCR reaction, which is carried out using the following primers: AK04-14: GtcctctgttaacTTACTGAGCTGCCGCCTGTAC (SEQ ID NO: 4), which is annealed at the 3 ′ end of the mature BPN ′ gene by introducing the HpaI region below the translation stop codon ; and AK04-21.1: TTATGCGAGgctagcaaaagagagagggtaaagagtgagaagc (SEQ ID NO: 5), which is annealed at the 5'-end of the BPN 'signal sequence by introducing the NheI site at the 5'-end.

The PCR fragment obtained by this reaction is purified using a Qiagen PCR purification kit under standard conditions. The purified fragment is digested with restriction enzymes NheI and HpaI under standard conditions and then ligated into a NheI / HpaI digested vector that contains the LAT promoter (pHPLT-VAAc1 described in US2006 / 0014265 and shown in Fig. 3A).

The pHPLT-BPN 'ligation mixture is then used to transform B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE: :( xy1R, pxy1A-comK), as described in WO 02/14490, incorporated herein by reference Selective cultivation of B. subtilis transformants carrying the pHPLT-BPN 'vector (see FIG. 3B) is carried out by shaking in flasks containing 25 ml of MBD medium (medium of a specific composition based on MOPS) in the presence of 20 mg / l neomycin. Incubation of transformed cells yields a secreted BPN 'protease with proteolytic activity. using 10% NuPage Novex bis-tris gels (Invitrogen®, catalog number NP0301BOX) To obtain samples for analysis, 2 volumes of the supernatant are mixed with 1 volume of 1M HCl, 1 volume of 4X LDS sample buffer (Invitrogen®, catalog number NP0007) and 1% PMSF (20 mg / ml), after which the mixture is heated for 10 minutes at 70 ° C. Then 25 μl of each sample is loaded onto the gel along with 10 μl of SeeBlue and 2 pre-stained protein standards (Invitrogen®, Catalog No. LC5925). The results clearly demonstrate that the BPN 'cloning method described in this example provides active BPN' produced by B. subtilis.

EXAMPLE 3

Getting BPN 'Site-Defining Libraries and Multiple Mutation Libraries

This example describes the construction of BPN 'variants.

Getting BPN 'Site Definition Library (SEL)

The pHPLT-BPN 'vector containing the BPN' expression cassette is used as template DNA. This vector contains a unique BgKI restriction site, which is used to construct SEL. To obtain libraries using primers synthesized by Invitrogen® (desalted, at 50 nmol) and are listed in table 7-1.

To obtain SEL BPN ', three PCR amplifications are performed: two mutagenic PCRs are performed to introduce the mutant codon of interest into the DNA sequence encoding mature BPN', and a third PCR is performed to hybridize the two mutagenic PCR products to obtain a pHPLT- expression vector BPN 'containing the target mutant codon in the sequence encoding mature BPN'. The mutagenesis method is based on a codon-specific strategy. In this method, all possible mutations are obtained simultaneously in a specific DNA triplet using direct and reverse oligonucleotide primers from 25 to 45 nucleotides in length containing a specific synthesized DNA triplet sequence (in NNS N = A, C, T or G, and S = C or G), which corresponds to the sequence of the codon to be changed. This allows for random inclusion of nucleotides in a particular codon of a sequence encoding mature BPN '. The number given in the name of the primer (table 3-1) corresponds to the position of a particular codon of the sequence encoding mature BPN '. The other two primers used to construct SEL contain a BglII restriction site along with a pHPLT-BPN 'DNA sequence fragment flanking the BglII restriction site.

The construction of each SEL begins with two primary PCR amplifications using a pHPLT-BglII-FW primer and a specific reverse mutagenic primer BPN ', and a subsequent amplification of PCR using a pHPLT-BglII-RV primer and a specific direct mutagenic BPN' primer (direct and reverse mutagenic primers present equivalent positions of the mature BPN 'codon). The introduction of mutations into the mature BPN 'sequence is carried out using Finnzymes Phusion high-precision DNA polymerase (catalog number F-530L). All PCR amplifications are carried out in accordance with the Finnzymes method using polymerase. Use the following PCR conditions:

Primary PCR 1:

primer pHPLT-BglII-FW and BPN 'specific reverse mutagenic primer - 1 μl (10 μM);

Primary PCR 2:

primer pHPLT-BglII-RV and BPN 'specific direct mutagenic primer - 1 μl (10 μM); in an environment containing

5X Phusion HF buffer 10 μl 10 mM dNTP mix 1 μl Phusion DNA Polymerase 0.75 μl (2 u / μl) DMSO, 100% 1 μl pHPLT-BPN 'template DNA 1 μl (0.1-1 ng / μl) distilled, autoclaved water up to 50 μl

PCR was performed using a PTC-200 Peltier MJ Research thermal cycler (location) using the following program: 30 seconds 98 ° C, 30X (10 seconds 98 ° C, 20 seconds 55 ° C, 1 minute 72 ° C) and 5 min 72 ° C. As a result of the reactions, two fragments of about 2 to 3 kb in length are obtained, which overlap about 30 nucleotide bases near the mature BPN 'codon of interest. Hybridization is carried out in the third reaction, using the two above fragments, as well as direct and reverse primers of BglII. The PCR hybridization reaction is carried out as follows:

primer pHPLT-BglII-FW and primer pHPLT-BglII-RV - 1 μl (10 μM)

5X Phusion HF buffer 10 μl 10 mM dNTP mix 1 μl Phusion DNA Polymerase 0.75 μl (2 u / μl) DMSO, 100% 1 μl primary PCR reaction mixture 1 1 μl reaction mixture of primary PCR 2 1 μl distilled, autoclaved water up to 50 μl

The following program is used for hybridization PCR: 30 seconds 98 ° C, 30X (10 seconds 98 ° C, 20 seconds 55 ° C, 2:05 minutes 72 ° C) and 5 min 72 ° C, and the PTC-200 Peltier MJ Research thermal cycler ®.

Amplified linear fragment of 4.8 t.o. purified using a Qiagen® Qiaquick PCR product purification kit (catalog no. 28106) and digested with the restriction enzyme BglII to give sticky ends on both sides of the hybrid fragment. The reaction mixtures used for cleavage contain:

- 35 μl of purified linear DNA fragment

- 4 μl React® 3 buffer (Invitrogen®)

- 1 μl BglII, 10 units / ml (Invitrogen®)

Reaction conditions: 1 hour, 30 ° C.

As a result of ligation of the cleaved and purified BglII fragment, cyclic and multimeric DNA products containing the desired mutation are obtained, which are then directly transferred to competent Bacillus subtilis cells:

- 30 μl of purified BglII-cleaved DNA fragment

- 8 μl of T4 DNA ligase buffer (Invitrogen® catalog number 46300-018)

- 1 μl of T4 DNA ligase, 1 u / μl (Invitrogen® catalog number 15224-017)

Reaction conditions: 16-20 hours, 16ºC

The ligation mixture is used to transform B. subtilis (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE: :( xy1R, pxy1A-comK), as described in WO 02/14490, incorporated herein by reference. For each library 96 individual colonies were selected and grown in MOPS medium containing neomycin and 1.25 g / l yeast extract, followed by sequence analysis (BaseClear) and screening. The library components range from 1 to 275. Each number represents a mature bpn 'codon randomly mutated Each library contains a maxi max 19 BPN 'options.

(Table 3-1, see end of description)

Getting BPN 'Multiple Mutation Library

BPN 'synthetic multiple mutation libraries (or combinatorial libraries) are produced by Geneart, Baseclear, and Bioke. Each synthetic library of multiple BPN 'mutations contains a mixture of BPN' genes in which two or more selected mature sequence codons are randomly replaced by specific DNA sequences. For example: the BPN 'combinatorial library can be designed so that the mature sequence codon 22 is replaced by DNA triplets encoding Thr, Gln, Val or Tyr, the codon 26 is replaced by DNA triplets encoding Val, Gln, Asn or Tyr, the codon 31 is replaced by triplets DNA encoding Ile, His, Tyr or Asn, and codon 48 will be replaced by DNA triplets encoding Ala, Glu, His or Asp. In this example, the combinatorial library contains a maximum of 256 combinatorial BPN 'variants. However, a typical BPN 'multiple mutation library may contain up to several thousand unique variants of the BPN' gene. Multiple mutation library components containing AvaI and HindIII terminal regions (see, for example, wild-type BPN ') are digested with AvaI and HindIII, purified by gel chromatography and cloned in pHPLT vector by ligase reaction using Invitrogen® T4 DNA ligase ( Catalog number 15224-025) in accordance with the manufacturer's recommendations for general cloning using sticky ends. Wild type AvaI / HindIII BPN 'fragment:

To carry out the transformation using the ligation reaction mixture, the DNA library (mixture of BPN 'library components is cloned into pHPLT) was amplified using the TempliPhi kit (Amersham, Catalog No. 25-6400). To amplify, 1 μl of the ligation reaction mixture was mixed with 5 μl of sample buffer from the TempliPhi kit and heated for 3 minutes at 95 ° C to denature the DNA. The reaction mixture is cooled by placing on ice for 2 minutes, and then centrifuged for a short period of time. Then 5 μl of reaction buffer and 0.2 μl of phi29 polymerase from the TempliPhi kit are added and the reaction mixtures are incubated at 30 ° C in an MJ Research PCR apparatus for 4 hours. The phi29 enzyme is inactivated by heating by incubation at 65 ° C for 10 minutes.

To carry out the transformation using libraries, 0.1 μl of the product of the amplification reaction of TempliPhi was mixed with 500 μl of competent B. subtilis cells (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE: :( xy1R, pxy1A-comK) followed by vigorous shaking with 37 ° C for 1 hour .After this time, aliquots of 100 and 500 μl were placed on HI agar plates containing 20 ppm neomycin and 0.5% skim milk. The following table lists some of the individual substitutions, used in the present invention.

(Table 3-2, see end of description)

Obtaining crude samples of enzymes containing BPN 'variants

Variant BPN 'proteins are obtained by growing B. subtilis transformants in 96-well MTP at 37 ° C for 68 hours in MBD medium (medium with a defined composition based on MOPS). The MBD medium is prepared essentially according to a method known in the art (see Neidhardt et al., J. Bacteriol., 119: 736-747 [1974]), except that NH ₄ Cl ₂ , FeSO ₄ and CaCl _{2 are} not included in the basic medium, 3 mM K ₂ HPO _{4 are used,} and the main medium is supplemented with 60 mM urea, 75 g / l glucose and 1% soyton. Micronutrients are used as a 100X stock solution, one liter of which contains 400 mg of FeSO ₄ · 7H ₂ O, 100 mg of MnSO ₄ · H ₂ O, 100 mg of ZnSO ₄ · 7H ₂ O, 50 mg of CuCl ₂ · 2H ₂ O, 100 mg CoCl ₂ · 6H ₂ O, 100 mg NaMoO ₄ · 2H ₂ O, 100 mg Na ₂ B ₄ O ₇ · 10H ₂ O, 10 ml 1M CaCl ₂ and 10 ml 0.5 M sodium citrate.

EXAMPLE 4

Resistance of BPN 'options to LAS

This example describes experiments to evaluate the stability of different BPN 'variants with one substitution and multiple BPN' variants in the presence of LAS. LAS resistance is measured by determining AAPF activity before and after incubation in the presence of LAS at elevated temperature (45 ° C) using the methods described in Example 1. The following tables show the results as relative stability values, which reflect the stability of the variants BPN 'compared to wild-type BPN' enzyme. Namely, relative stability is the ratio of the residual activity of the variant to the residual activity of the wild-type enzyme. A value greater than one indicates increased stability in the presence of LAS.

Table 4-1 shows the relative stability of BPN 'variants compared to wild-type BPN'. In developing the present invention, it has been found that many BPN 'variants clearly have higher stability in the presence of LAS than wild-type BPN'. In particular, a LAS resistance analysis of 92 BPN 'sites shows that 40 sites (43%) are unfavorable (harmful) and 52 sites (57%) are beneficial (beneficial). In addition, out of 1508 tested options, 96 (6%) are excellent (increased resistance), 196 (13%) are neutral (same resistance), and 1216 (81%) are poor (reduced resistance).

(Table 4-1, see end of description)

(Table 4-2, see end of description)

EXAMPLE 5

Specific Activity of BPN 'Options

This example describes experiments to determine the relative specific activity of BPN 'and BPN' variants. AAPF specific activity was measured using the methods described in Example 1. Table 5-1 shows the results as relative specific activity values that reflect the activity of BPN 'variants compared to wild-type BPN' activity.

Namely, the relative specific activity is the ratio of the specific activity of the variant to the specific activity of the wild-type enzyme. A value greater than one (PI> 1) indicates increased specific activity for the AAPF substrate.

EXAMPLE 6

Comparative analysis of data obtained for BPN 'options

This example describes the results of experiments conducted to determine protein expression, stain removal, LAS resistance, and AAPF activity (testing for properties of interest) BPN 'and BPN' variants. The results are obtained using the methods described in Example 1. As described herein, the functionality of BPN 'variants is quantified as a performance indicator (PI), which is the ratio of the effectiveness of the variant to the efficiency of the starting protein. The following PI grading is used herein: enhancing mutations (PI> 1.0); neutral mutations (PI> 0.5); harmless mutations (PI> 0.05); and harmful mutations (PI <0.05). “Combinable mutations” are mutations leading to variants that have a functional index of = 0.5 for at least one property, and> 0.05 for all properties. Combination mutations are mutations that can be combined to produce proteins with suitable performance indicators for one or more desired properties. The positions in which mutations occur are classified as follows: non-limiting positions that contain ≥20% mutations that are neutral for at least one property; and restrictive provisions that contain <20% mutations that are neutral for activity and stability.

These data can be used for genetic engineering of any subtilisin. Even if the subtilisin to be genetically engineered contains in a certain position an amino acid other than the BPN 'subtilisin amino acid, such information can be used to identify at least one substitution that changes the desired properties by identifying the best substitution, including substitution wild-type BPN amino acid.

Table 6-1 shows the performance index (Pi) for 5004 BPN 'subtilisin variants at 275 positions. Functional indicators less than or equal to 0.05 are taken as 0.05 and are shown in bold italics. In addition, when determining stability, if the indicator of functioning in relation to activity in the stability analysis is less than or equal to 0.05, the associated indicator of functioning in relation to stability is taken as 0.05.

(Table 6-1, see end of description)

Combined mutations :

Table 6-2 shows BPN 'subtilisin variants that contain combinable mutations at 275 positions (2907). These options have a performance index of ≥0.5 for at least one property, and> 0.05 for both properties.

(Table 6-2, see end of description)

EXAMPLE 7

Comparative analysis of data obtained for variant proteases

This example compares the results of experiments conducted to determine cleaning ability, resistance to LAS, AAPF activity and protein content (testing for properties of interest) BPN ', GG36 (GCI-P036) and variant proteases. As described herein, the functionality of variant proteases is quantified as an indicator of functioning (PI), which is the ratio of the effectiveness of a variant to the effectiveness of a standard protease. The following PI classification is used in this document: enhancing mutations (PI> 1.0); neutral mutations (PI> 0.5); harmless mutations (PI> 0.05); and harmful mutations (PI≤0.05).

Productive plots are plots containing at least one enhancing mutation for any property. Productive, non-limiting regions are regions containing ≥20% neutral mutations (PI> 0.5) and at least one upward mutation ((PI> 1.0) for any test property (other than protein expression). Table 7-1 below, the results obtained for variants that satisfy the definition of productive, non-limiting regions are shown as a percentage (%) of tested variants that satisfy the definition of an improvement mutation (PI> 1).

Highly productive regions are regions that contain ≥20% enhancing mutations (PI> 1) for at least one property other than protein expression (TCA analysis). In Table 7-2 below, the results obtained for variants that satisfy the definition of highly productive regions are shown as a percentage (%) of tested variants that satisfy the definition of an improvement mutation (PI> 1).

Boundary sites are sites that contain less than 20% mutations that are neutral with respect to activity and stability. In Table 7-3 below, the results obtained for variants that satisfy the definition of limiting regions are shown as a percentage (%) of the tested variants that satisfy the definition of neutral mutation (PI> 0.5).

In short, during the development of the present invention, it has been found that 10 positions of the mature portion of two standard subtilisins constitute limiting provisions with respect to activity and stability. Therefore, the remaining 265 positions of the mature portion of the two standard subtilisins are non-limiting (≥20% neutral mutations) in terms of activity and stability.

EXAMPLE 8

Obtaining a protease in B. subtilis

This example describes experiments conducted to obtain different proteases in B. subtilis. In particular, methods for transforming B. subtilis with expression vectors carrying GG36 and BPN'-Y217L are described. The transformation is carried out using methods known in the art (see, for example, WO 02/14490).

Obtaining protease GG36

This example describes the experiments conducted to obtain GG36 (also called B. lentus subtilisin) in B. subtilis. The expression plasmid pAC-GG36ci contains the improved codon GG36 gene, hybridized at the eighth codon with the aprE signal sequence, and controlled by the aprE consensus promoter and BPN 'transcription terminator. In the sequence below, the aprE consensus promoter is shown in bold italics, the standard font indicates the signal sequence, the underscore indicates the sequence, the bold DNA that encodes the mature GG36 protease, and the underline in italics indicates the BPN 'terminator. The site encoding the mature GG36 protease is flanked by the KpnI and XhoI restriction sites, providing cloning:

The following is the amino acid sequence of the GG36 precursor protein. In this sequence, the mature GG36 protease is shown in bold:

The amino acid sequence of the mature GG36 protease (SEQ ID NO: 562) is used as the basis for the preparation of the variant libraries described here:

Plasmid pAC-GG36ci contains the following elements: pUB110 = DNA fragment from plasmid pUB110 (McKenzie et al., Plasmid 15: 93-103 [1986]), pBR322 = DNA fragment from plasmid pBR322 (Bolivar et al., Gene 2: 95- 113 [1977]), pC194 = DNA fragment from the plasmid pC194 (Horinouchi et al., J Bacteriol, 150: 815-825 [1982]). The plasmid has the following characteristics: Ori for B. subtilis = pUB110 replication start point, CAT = pC194 chloramphenicol resistance gene, pMB1 origin = pBR322 replication start point, bla = pBR322 beta-lactamase, Short aprE promoter = consensus trinscriptor promoter, Signal Peptide = signal peptide, ProPeptide = GG36 subregion, GG36ci Mature Peptide = mature GG36 (replaced by coding regions for each variant expressed in this study), BPN 'terminator = BPN' subtilisin transcription terminator.

Preparation of BPN'-Y217L protease (PURAFECT® PRIME subtilisin)

This example describes experiments conducted to produce B. amyloliquefaciens BPN'-Y217L subtilisin (commercially available as PURAFECT® PRIME subtilisin; also called "FNA") in B. subtilis. The expression plasmid pAC-FN Are contains the BPN'-Y217L gene hybridized at the eighth codon with the aprE signal sequence and is controlled by the aprE consensus promoter and BPN 'transcription terminator. In the sequence below, the aprE consensus promoter is shown in bold italics, the standard font indicates the signal sequence, the underscore indicates the sequence, the bold DNA that encodes the mature BPN'-Y217L protease, and the underline in italics indicates the BPN 'terminator. The plot encoding the mature protease BPN'-Y217L contains restriction sites KpnI and XhoI, providing cloning:

The following is the amino acid sequence of the BPN'-Y217L precursor protein. In this sequence, the mature BPN'-Y217L protease is shown in bold:

The amino acid sequence of the mature BPN'-Y217L protease is used as the basis for the preparation of the variant libraries described here:

Plasmid pAC-FNAre contains the following elements: pUB110 = DNA fragment from plasmid pUB110 (McKenzie et al., Plasmid 15: 93-103, [1986]), pBR322 = DNA fragment from plasmid pBR322 (Bolivar et al., Gene 2:95 -113 [1977]), pC194 = DNA fragment from the plasmid pC194 (Horinouchi et cil, J. Bacteriol 150: 815-825 [1982]). The plasmid has the following characteristics: Ori for B. subtilis = pUB110 replication start point, CAT = pC194 chloramphenicol resistance gene, pMB1 origin = pBR322 replication start point, bla = pBR322 beta-lactamase, Short aprE promoter = consensus trinscriptor promoter, Signal Peptide = signal peptide, ProPeptide = lane BPN'-Y217L, BPN'-Y217L Mature Peptide = mature BPN'-Y217L (replaced by coding regions for each variant expressed in this study), BPN 'terminator = BPN' subtilisin transcription terminator.

EXAMPLE 9

Expression of enzyme variants

This example describes methods for expressing various recombinant enzymes in transformed B. subtilis.

Subtilisin - 2 ml scale

B. subtilis clones containing BPN'-Y217L or GG36 expression vectors are replicated using a steel 96-well replicator from stock solutions in glycerol in 96-well culture plates (BD, 353075) containing 200 μl LB medium + 25 μg / ml Chloramphenicol is grown overnight at 37 ° C, 220 rpm in a humid atmosphere. After overnight cultivation, an aliquot of 200 μl was used to inoculate 2000 μl of medium with a specific composition of + 25 μg / ml chloramphenicol in 5 ml plastic culture tubes. The culture medium is a medium with a semi-defined composition based on MOPS buffer, containing urea as the main source of nitrogen, glucose as the main source of carbon, and supplemented with 1% soiton for healthy cell growth. The culture tubes are incubated at 37 ° C, 220 rpm, for 60 hours. After incubation, the culture broths are centrifuged at more than 8000 × RCF. The supernatant solution is decanted into 15 ml polypropylene conical tubes for storage. No further purification or concentration is carried out. Stock solutions of the supernatant in 40% propylene glycol (final concentration) were obtained to ensure long-term stability, and stored at 4 ° C.

EXAMPLE 10

Obtaining enzyme variants

This example describes the preparation of charging "ladders" of enzymes and combinatorial charged libraries.

Enzyme Charging Stairs

Several protein variants spanning a range of physical properties of interest are selected from existing libraries or obtained using directed mutagenesis techniques known in the art (see, for example, US Patent Applications Nos. 10/576331, 11/581102 and 11/583334) . Then, the determined set of probe proteins obtained is analyzed using a test of interest.

Typical variants of the charged staircase proteases shown in the following tables are analyzed using the methods described herein. In these tables, the change in charge is compared with a wild-type enzyme.

Table 10-1 Charged Staircase Options BPN'-Y217L Variant BPN'-Y217L (BPN 'numbering) Charge change S87D-N109D-S188D-S248D -four S87D-N109D-S188D -3 S87D-N109D -2 N109D -one (BPN'-Y217L) 0 N109R +1 S87R-N109R +2 S87R-N109R-S188R +3 S87R-N109R-S188R-S248R +4

Table 10-2 GG36 Charged Stair Options Option GG36 (numbering GG36) Option GG36 (BPN 'Numbering) Charge change S85D-Q107D-S182D-N242D S87D-Q109D-S188D-N248D -four S85D-Q107D-S182D S87D-Q109D-S188D -3 S85D-Q107D S87D-Q109D -2 Q107D Q109D -one (Gg36) (Gg36) 0 Q107R Q109R +1 S85R-Q107R S87R-Q109R +2 S85R-Q107R-S182R S87R-Q109R-S188R +3 S85R-Q107R-S182R-N242R S87R-Q109R-S188R-N248R +4

Combinatorial Charged Enzyme Libraries

Obtaining combinatorial charged subtilisin libraries of B. lentus (= GG36)

Plasmid pAC-GG36ci containing the improved codon GG36 gene is sent to DNA2.0 to generate combinatorial charged libraries (CCL). They are also sent a strain of Bacillus subtilis (genotype: ΔaprE, ΔnprE, ΔspoIIE, amyE :: xylRPxylAcomK-phleo) for transformation. Also in DNA2.0 Inc. order positional libraries for each of the four sites of the GG36 protease shown in Table 10-3. Options are obtained as stock solutions in glycerol in 96-well plates.

The GG36 CCL is constructed by identifying four uniformly distributed, exposed on the surface, uncharged polar amino acid residues outside the active site. Such residues are Ser-85, Gln-107, Ser-182 and Asn-242 (residues 87, 109, 188 and 248 according to BPN 'numbering). A combinatorial 81-member library (from G-1 to G-81) is obtained by compiling all combinations of the three possibilities for each site: wild-type amino acids, arginine, or aspartic acid.

Obtaining CCL subtilisin B. amyloliquefaciens BPN'-Y217L

Plasmid pAC-FNAre containing the BPN'-Y217L gene is sent to DNA2.0 Inc. (Menlo Park, CA) for CCL. They are also sent a strain of Bacillus subtilis (genotype: ΔaprE, ΔnprE, ΔspoIIE, amyE :: xylRPxylAcomK-phleo) for transformation. At DNA2.0 Inc. order positional libraries for each of the four BPN'-Y217L protease sites shown in Table 10-4. Options are obtained as stock solutions in glycerol in 96-well plates.

A combinatorial charged subtilisin library BPN'-Y217L is constructed by identifying four uniformly distributed, exposed on the surface of uncharged polar amino acid residues outside the active center. Such residues are Ser-87, Asn-109, Ser-188 and Ser-248. A combinatorial 81-member library (F-1 to F-81) is obtained by compiling all combinations of three possibilities for each site: wild-type amino acids, arginine, or aspartic acid.

EXAMPLE 11

The effectiveness of the option BPN'-Y217L and subtilisin GG36

This example describes the testing of BPN'-Y217L and GG36 variants using analyzes of BMI and baked egg microbes in detergents representing different geographical regions of the market (for example, differing in pH, T and / or water hardness), for use in washing and dishwashers as described in example 1.

The above observations apply to other serine proteases, such as subtilisins BPN'-Y217L and GG36. For example, FIGS. 2A and 2B show a charge of BPN'-Y217L and GG36, respectively, optimal for efficient cleaning in North America washing conditions using the ready-made, heat-inactivated TIDE® 2X detergent. The left Y axes demonstrate the cleaning efficiency of the microsamples, where a higher number indicates better BMI spot removal. The right Y axes show a performance indicator defined as the cleaning efficiency of options (filled characters) compared to the original molecule (unpainted characters). The horizontal lines represent the performance indicator for standard deviations of 2 or 3 above the noise level of this analysis. The BPN'-Y217L Combinatorial Charged Library (CCL) demonstrates that the optimal charge is observed at zero charge changes compared to the original BPN'-Y217L, while the CCL GG36 demonstrates that the optimal charge is observed when two negative charges differ from the original GG36.

Figa, 3B, 4A, 4B, 5A and 5B demonstrate that the location of the optimal charge depends on the solution medium, which determines the composition of the detergent, pH, temperature and ionic strength, which depends on the hardness of the water and the concentration of detergent. For example, the optimum charge for CCL BPN'-Y217L shifts strongly from zero for washing conditions in North America to more positive values in Western Europe and Japan. Moreover, the optimal charge is observed for liquid and granular (powder) compositions of washing detergent. Similarly, the optimum charge is observed for CCL of both BPN'-Y217L and GG36, in dishwasher detergent (ADW) when using (for example, Reckitt Benckiser Calgonite 40 ° C, 12 gpg, pH 10) baked egg as a substrate.

In developing the present invention, it has been shown that the cleaning effectiveness of charged protease variants (e.g., GG36, BPN'-Y217L, etc.) in different detergents is largely dependent on the pH and conductivity of the working solution. Final conductivity is a measure of ionic strength and depends on the hardness of the water, the concentration and composition of the detergent. For example, there is a correlation between the cleaning efficacy of GG36 and BPN'-Y217L for baked egg stains in ADW detergents in Europe and North America at a pH of 10.6 and a conductivity of 3.0 mS / cm. In particular, the cleaning efficiency of the charged variants correlates well under the condition of the same pH and conductivity. This fact allows us to extrapolate the results of screening the enzyme's efficiency using a specific detergent to another detergent with the same pH and conductivity. Similarly, it is possible to extrapolate the results of screening for enzyme efficacy in a buffer with matching pH and conductivity values to a detergent having similar operating pH and conductivity values.

Table 11-1 Options Total charge PI washing detergent NA PI washing detergent WE PI dishwashing detergent for baked eggs Gg36 0 1,000 1,000 1,000 G-2 -one 0.950 0.939 1,450 G-3 one 0.578 0.759 1,231 G-4 -one 1,219 1,539 1,467 G-5 -2 1,261 1,194 1,508 G-6 0 0.936 0,999 1,563 G-7 one 0.568 0.834 0.712 G-8 0 0,043 0.151 -0.033 G-9 2 0.350 0.601 0.708 G-10 -one 1,266 1,089 1,022 G-11 -2 1,280 1,209 0.788 G-12 0 0.810 1,074 0.977 G-13 -2 1,317 1,411 1,300 G-14 -3 0,080 0.144 -0.007 G-15 -one 0.917 1,254 1.393 G-16 0 0.750 1,081 0.742 G-17 -one 0.815 0.894 0,909 G-18 one 0.675 0.931 0.867 G-19 one 0.713 0.856 1,310 G-20 0 0,071 0.129 -0.015 G-21 2 0.434 0.834 1,098 G-22 0 0.782 1.014 1,447 G-23 -one 0.964 0.939 1,396 G-24 one 0.466 0.729 1,368 G-25 2 0.322 0.744 0.638 G-26 one 0.517 0.984 0.694 G-27 3 0,303 1,074 0.971 G-28 -one 1,126 1,141 1,023 G-29 -2 1,126 0,991 1,037 G-30 0 0.945 1,149 1.006 G-31 -2 1,331 1,149 1,412 G-32 -3 1,345 0,999 1,303 G-33 -one 0.950 1,036 1,420 G-34 0 0.671 0,999 0.673 G-35 -one 0.694 1,021 1,026 G-36 one 0.415 0.774 0.704 G-37 -2 1,410 1,554 -0.011 G-38 -3 0.457 0.759 1,081 G-39 -one 0.936 1,186 0.940 G-40 -3 0,043 0.106 -0.006 G-41 -four 1,163 0.496 0.988 G-42 -2 1.359 1,276 1,165 G-43 -one 0.782 1,119 0.740 G-44 -2 0.926 1,051 0.748 G-45 0 0.503 0.961 0.619 G-46 0 0.759 0.916 1,035 G-47 -one 0.871 1,051 1,057 G-48 one 0.452 0.864 1,060 G-49 -one 0.885 0,909 1,239 G-50 -2 0.912 0,909 1,613 G-51 0 0.638 1.006 1,723 G-52 one 0.396 0,909 0.820 G-53 0 0.568 0,909 0.806 G-54 2 0.345 0.766 0.641 G-55 one 0.689 1,036 1,230 G-56 0 0.675 1,134 0.818 G-57 2 0.452 0.766 0.922 G-58 0 1,024 1,216 1,444 G-59 -one 1,131 1,306 1,473 G-60 one 0.699 0.946 1,520 G-61 2 0.457 0.886 0.680 G-62 one 0.759 1,059 1,169 G-63 3 0.327 0.669 0.687 G-64 0 0.847 1,119 1.001 G-65 -one 0.601 0.879 1.014 G-66 one 1.001 1,261 1,042 G-67 -one 1,196 1,411 1,489 G-68 -2 1,131 1,179 1,163 G-69 0 0.768 0,999 1,488 G-70 one 0.647 1,809 0.229 G-71 0 0.620 1,081 0.631 G-72 2 0.364 0.819 0.634 G-73 2 0.387 0.729 0,997 G-74 one 0.638 0.939 1.105 G-75 3 0.657 0.856 1,081 G-76 one 0,071 0.136 -0.018 G-77 0 0.866 0.969 1,400 G-78 2 0.434 0.789 1,175 G-79 3 0.327 0.789 0.874 G-80 2 0.355 0.781 0.833 G-81 four 0.229 0.466 0.653

Table 11-2 Options Total charge PI washing detergent NA PI washing detergent WE PI washing detergent JPN PI dishwashing detergent for baked eggs BPN'-Y217L 0 1,000 1,000 1,000 1,000 F-2 -one 0.828 0.794 1,020 1,572 F-3 one 0.866 1,687 1,712 1,322 F-4 -one 0.814 0.868 0.810 1,211 F-5 -2 0.753 0.988 0.458 1,395 F-6 0 1,032 1,479 1,529 1,273 F-7 one 0.805 1,792 1.359 0.969 F-8 0 0,909 1,360 1,576 0.855 F-9 2 0.705 1,360 1,529 0.777 F-10 -one 1.102 0.749 0,607 1,073 F-11 -2 0.904 0,600 0.410 1.004 F-12 0 1,221 1,911 1,583 1,030 F-13 -2 0.838 0.690 0.234 0.832 F-14 -3 0.549 0.705 0.139 0.665 F-15 -one 1,093 1,107 0.654 1,063 F-16 0 1,268 1,524 1,258 0.571 F-17 -one 0.819 0.764 0.722 0.710 F-18 one 1,098 1,226 1,705 0.742 F-19 one 1.003 1,196 1,590 1,298 F-20 0 0.875 1,047 1,542 1,328 F-21 2 0.866 1,568 1,976 1,327 F-22 0 1.017 1,211 1,569 1,736 F-23 -one 0.771 1,241 0.973 1,335 F-24 one 0.970 2,045 1,942 1,668 F-25 2 0.738 1,315 1,664 1.015 F-26 one 0.937 1,434 1,624 0.919 F-27 3 0.535 1,122 1,990 1,283 F-28 -one 0.743 0.854 0.776 0.946 F-29 -2 0.691 0.749 0.451 0.707 F-30 0 1,183 1,464 1,549 0.979 F-31 -2 0.738 0.720 0.519 1,185 F-32 -3 0.497 0.645 0.376 0.908 F-33 -one 1,107 1,062 1,332 0.989 F-34 0 0.809 1,241 1.393 0.640 F-35 -one 0.672 0.824 0.844 0.928 F-36 one 0.823 1,211 1,420 1,130 F-37 -2 0.880 0.720 0.227 0.678 F-38 -3 0.597 0.779 0.139 0.865 F-39 -one 0.890 1.017 0.661 0.760 F-40 -3 0.469 0.660 0.132 0.568 F-41 -four 0.322 0.541 0,064 0.464 F-42 -2 1,022 0.720 0.247 0.948 F-43 -one 0.852 1,092 0.980 0.605 F-44 -2 0.530 0.794 0.369 0.804 F-45 0 0.980 1,419 1,122 0.730 F-46 0 0.819 1.002 1,380 0,999 F-47 -one 0.984 0.928 1.102 1,172 F-48 one 0.956 1,419 2,010 1,377 F-49 -one 0.913 0.898 0.925 1,263 F-50 -2 0.743 0.779 0.756 1,084 F-51 0 1.008 1,300 1,481 1,588 F-52 one 0,800 1,360 1,481 0.851 F-53 0 0.705 1,077 1,271 0.792 F-54 2 0.677 1,270 1,468 1,172 F-55 one 1,050 1,538 1,515 0,996 F-56 0 1,112 1,122 1,088 0.721 F-57 2 0.866 1,330 2,173 1.014 F-58 0 1,036 1,107 1,183 1,341 F-59 -one 1,117 1,077 0.898 0.878 F-60 one 0.928 1,449 1,976 1,483 F-61 2 0.942 1,687 1,542 0.785 F-62 one 1.017 1,494 1,651 1.010 F-63 3 0.630 1,613 1,522 0.910 F-64 0 1,131 1,241 0.912 0.907 F-65 -one 0,994 0.958 1,136 0.582 F-66 one 1,216 1,464 2,308 0.985 F-67 -one 1,126 0.854 0.485 0.855 F-68 -2 0.748 0.705 0.261 0.794 F-69 0 1,249 1,479 1,041 0.925 F-70 one 1,050 1,911 1,631 0.878 F-71 0 1,136 1,687 1,298 0,998 F-72 2 0.871 2,893 1,949 0.850 F-73 2 0,998 1,717 1,990 1,276 F-74 one 1.102 1,509 2,281 1,186 F-75 3 0.672 1,687 2,044 1.351 F-76 one 0.946 1,449 1,807 1,647 F-77 0 1,050 1,509 1,793 1,287 F-78 2 0.975 1,256 2,207 1,786 F-79 3 0.606 1,285 1,508 0.954 F-80 2 0.795 2,655 1,800 0.896 F-81 four 0.611 1,419 1,759 0.965

EXAMPLE 12

Resistance to LAS and chelating agents

This example describes the determination of the relationship between protein charge and stability in a reaction medium containing one or both of an anionic surfactant and a chelating agent. To determine protease activity in the experimental and control samples, analysis was performed using suc-AAPF-pNA. To determine alpha-amylase activity in the experimental and control samples, analysis is performed using BODIPY-starch. Residual amounts of LAS and EDTA on the experimental plates do not affect the results of analyzes using suc-AAPF-pNA or BODIPY-starch.

Resistance to LAS / EDTA

As reagents used: control buffer: 50 mm HEPES, 0.005% Tween-80, pH 8.0; and experimental buffer: 50 mM HEPES, 0.1% (w / v) LAS (sodium salt of dodecylbenzenesulfonate, Sigma D-2525), 10 mM EDTA, pH 8.0. Enzyme variants (20 ppm) were diluted 1:20 in a 96-well non-binding, flat-bottomed plate containing either a control or experimental buffer and mixed. The control plate was incubated at room temperature, and the test plate was immediately incubated at 37 ° C for 30-60 minutes (depending on the stability of the test enzyme). After incubation, enzyme activity is measured using a proteolytic activity assay using suc-AAPF-pNA. The fraction of remaining or residual activity is determined by dividing the reaction rate of the test sample by the reaction rate of the control sample. The starting enzymes and variants were found to be stable for 60 minutes in the control buffer.

Figure 6 shows the dependence of resistance to LAS / EDTA on the change in the total charge compared to the original BPN'-Y217L, for a library containing 80 options. This library is obtained and constructed using the methods described in example 2, so as to compare several total charges with the original molecule BPN'-Y217L. As can be seen from the figure, an increase in the number of negative charges (to -4) compared with the original BPN'-Y217L improves the combined resistance to LAS / chelating agent. This is an example of optimizing the physical properties of a protein, in this case, the total charge, leading to improved stability of the protein in a complex washing detergent environment.

EXAMPLE 13

The ability of variants from the library of multiple mutations (MML) BPN 'remove stains

Multiple BPN 'mutation libraries (or combinatorial libraries) produce Geneart and DNA2.0, as described above, using BPN' as the source protein. As stated earlier, these companies also produce variant BPN 'proteins. The protein concentration in the culture supernatants is determined by deposition using TCA, as described in example 1. The ability of the options to remove stains during washing is analyzed on EMPA 116 samples (BMI stains, CFT) at pH 8/16 ° C, pH 7/16 ° C and pH 8/32 ° C using the methods described in example 1 using the following modifications. As a detergent, heat inactivated TIDE® 2X Cold (Procter & Gamble) is used. Commercial detergent formulations are heat inactivated to disrupt the endogenous enzymatic activity of all protein components while maintaining the properties of the non-enzymatic components. Inactivation by heating detergents is carried out by placing a pre-weighed liquid detergent (in a glass bottle) in a water bath at 95 ° C, where it is kept for 2 hours. Detergent is obtained from local supermarkets. Treated and untreated detergents are analyzed for 5 minutes, dissolving the detergent to accurately determine the percentage of decontamination. Enzyme activity is tested using AAPF analysis. The functionality of BPN 'variants is quantified as an indicator of performance (Pi) (i.e., the ratio of the characteristics of the variant to the initial BPN'). The results are shown in table 13-1. BPN 'variants with a Pi value greater than or equal to 0.5, according to the results of one or more analyzes of the ability to remove BMI spots and / or deposition of TCA are characterized by improved properties and / or increased expression. Functional indicators less than or equal to 0.05 are assigned to 0.05 and are highlighted in bold italics in the table. For each option with a functioning index less than or equal to 0.05, according to the results of the TCA analysis of the protein, all values refer to 0.05. In this table, "ND" means "not defined."

Table 13-1 Pi values of expression-tested BPN 'variants (TCA) and stain removal ability (BMI pH 8/16 ° C, BMI pH 7/16 ° C and BMI pH 8/32 ° C) Option Code TCA BMI
pH 8/16 ° C BMI
pH 7 / 16ºC BMI
pH 8 / 32ºC Original BPN ' 1.00 1.00 1.00 1.00 BPN'Y217L 1.01 1.12 1,08 1,08 A92g 0.52 1,04 Nd Nd A92G-G100T 0.58 0.37 Nd Nd A92G-G128A 0.49 0.83 Nd Nd A92G-G97A 0.77 1.14 Nd Nd A92G-I111V 0.6 1,02 Nd Nd A92G-L126A 0.68 1.14 Nd Nd A92G-L96T 0.68 0.69 Nd Nd A92G-M124V 0.55 1,07 Nd Nd A92G-N123G 0.46 0.42 Nd Nd A92G-V227T 0.52 0.54 Nd Nd A92G-W106F 0.44 0.4 Nd Nd A92G-Y104N 0.65 0.41 Nd Nd A92G-Y167A 0.44 0.56 Nd Nd A92G-Y217Q 0.56 1,2 Nd Nd BPN'-Y217L 1.01 1.12 1,08 1,08 G100t 0.71 1.14 Nd Nd G100T-G128A 0.46 0.14 Nd Nd G100T-I111V 0.46 0.77 Nd Nd G100T-L126A 0.59 0.1 Nd Nd G100T-M124V 0.57 0.05 Nd Nd G100T-N123G 0.56 0.05 Nd Nd G100T-V227T 0.37 0.42 Nd Nd G100T-W106F 0.62 0.05 Nd Nd G100T-Y104N 0.68 0.05 Nd Nd G100T-Y167A 0.63 0.28 Nd Nd G100T-Y217Q 0.7 1,11 Nd Nd G128a 1,5 1.17 1,11 1.14 G128A-V227T 0.48 0.86 Nd Nd G128A-Y167A 0.57 0.78 Nd Nd G128A-Y217Q 1.19 1.38 1.12 1.16 G97a 1.37 1.12 1,03 1,1 G97A-G100T 0.6 0.99 Nd Nd G97A-G128A 1.17 1.19 1.06 1,02 G97A-I111V one 1.22 Nd Nd G97A-L126A 1.36 1.24 1,1 1,11 G97A-M124V 1.33 1.29 1.12 1.12 G97A-N123G 0.91 1.22 1.19 1.12 G97A-V227T 0.71 1,07 Nd Nd G97A-W106F 0.69 1,1 Nd Nd G97A-Y104N 0.69 1,1 Nd Nd G97A-Y167A 0.66 1,02 Nd Nd G97A-Y217Q 1.42 1.27 1,08 1.14 I111V 1,07 1,04 Nd Nd I111V-G128A 1.13 1,1 Nd Nd I111V-L126A 1.09 1,07 Nd Nd I111V-M124V 0.93 1.3 Nd Nd I111V-N123G 0.67 1.09 Nd Nd I111V-V227T 0.56 1.01 Nd Nd I111V-Y167A 0.66 0.97 Nd Nd I111V-Y217Q 1.06 1.28 Nd Nd L126A 1,56 1.16 1,1 1,08 L126A-G128A 0.76 0.83 0.9 0.92 L126A-V227T 0.73 0.8 Nd Nd L126A-Y167A 0.73 0.76 Nd Nd L126A-Y217Q 1.7 1.28 1,11 1,11 L96T 0.84 1.17 1,04 1.13 L96T-G100T 0.53 0.14 Nd Nd L96T-G128A 0.59 0.77 0.77 0.84 L96T-G97A 0.81 1.24 1,11 1.14 L96T-I111V 0.49 0.94 Nd Nd L96T-L126A 0.76 0.43 0.37 0.6 L96T-M124V 0.87 1.12 0.93 1.01 L96T-N123G 0.58 0.3 0.22 0.56 L96T-V227T 0.5 0.3 Nd Nd L96T-W106F 0.57 0.41 Nd Nd L96T-Y104N 0.43 0.05 Nd Nd L96T-Y167A 0.36 0.63 Nd Nd L96T-Y217Q 0.79 1.28 one 1.12 M124V 1,61 1.22 1.12 1.15 M124V-G128A 0.96 1.17 1,11 1.12 M124V-L126A 1.74 1.13 1.12 1,11 M124V-V227T 0.65 0.96 Nd Nd M124V-Y167A 0.56 1.17 Nd Nd M124V-Y217Q 1.47 1.37 1.09 1.19 N123G one 1.16 1.12 1,03 N123G-G128A 0.5 0.75 0.88 0.85 N123G-L126A 0.72 0.62 0.48 0.65 N123G-M124V 0.59 0.6 0.54 0.66 N123G-V227T 0.65 0.05 Nd Nd N123G-Y167A 0.59 0.34 Nd Nd N123G-Y217Q 0.82 1.25 1.18 1.13 N62Q 1,63 1.29 1.19 1.06 N62Q-A92G 0.59 1.17 Nd Nd N62Q-G100T 0.57 0.96 Nd Nd N62Q-G128A 1.09 1.21 1.15 1.12 N62Q-G97A 1,52 1.32 1.22 1.17 N62Q-I111V 1.4 1.25 Nd Nd N62Q-L126A 1.46 1,07 0.99 1,03 N62Q-L96T 0.73 1,02 0.97 0.99 N62Q-M124V 1.29 1.23 1.06 1.15 N62Q-N123G 0.57 1.06 0.97 one N62Q-S89Y 0.6 1.17 Nd Nd N62Q-V227T 0.57 1.06 Nd Nd N62Q-V68A 1.86 1,08 Nd Nd N62Q-Y104N 0.59 0.66 Nd Nd N62Q-Y167A 0.79 1.15 Nd Nd N62Q-Y217Q 1.35 1.28 1.13 1.21 P52l 0.5 0.59 Nd Nd P52L-A92G 0.69 0.2 Nd Nd P52L-G100T 0.59 0.39 Nd Nd P52L-G128A 0.55 0.51 Nd Nd P52L-G97A 0.54 0.78 Nd Nd P52L-I111V 0.43 0.68 Nd Nd P52L-L126A 0.69 0.6 Nd Nd P52L-L96T 0.67 0.34 Nd Nd P52L-M124V 0.56 0.72 Nd Nd P52L-N123G 0.48 0.34 Nd Nd P52L-N62Q 0.54 0.77 Nd Nd P52L-S89Y 0.51 0.42 Nd Nd P52L-V227T 0.38 0.27 Nd Nd P52L-V68A 0.6 1,2 Nd Nd P52L-W106F 0.43 0.52 Nd Nd P52L-Y104N 0.64 0.32 Nd Nd P52L-Y167A 0.38 0.4 Nd Nd P52L-Y217Q 0.34 0.77 Nd Nd S89y 0.94 1,07 Nd Nd S89Y-A92G 0.5 0.9 Nd Nd S89Y-G100T 0.65 0.67 Nd Nd S89Y-G128A 0.98 1,1 Nd Nd S89Y-G97A 1.26 1.06 Nd Nd S89Y-I111V 0.83 1,07 Nd Nd S89Y-L126A 0.93 1.17 Nd Nd S89Y-L96T 0.77 0.84 Nd Nd S89Y-M124V 1,1 1.31 Nd Nd S89Y-N123G 0.47 0.85 Nd Nd S89Y-V227T 0.66 0.87 Nd Nd S89Y-W106F 0.7 0.98 Nd Nd S89Y-Y104N 0.74 0.64 Nd Nd S89Y-Y167A 0.55 0.91 Nd Nd S89Y-Y217Q 1.32 1.17 Nd Nd V227T 0.65 1.09 Nd Nd V68a 2.02 1.33 Nd Nd V68A-A92G 0.81 1.42 Nd Nd V68A-G100T 0.87 0.31 Nd Nd V68A-G128A 1.16 1.28 Nd Nd V68A-G97A 2.12 1.33 Nd Nd V68A-I111V 2.06 1.31 Nd Nd V68A-L126A 1.66 0.58 Nd Nd V68A-L96T 0.68 0.86 Nd Nd V68A-M124V 1.25 0.96 Nd Nd V68A-N123G 0.92 0.69 Nd Nd V68A-S89Y 1.65 1.37 Nd Nd V68A-V227T one 1.35 Nd Nd V68A-W106F 0.7 1.29 Nd Nd V68A-Y104N 0.71 0.78 Nd Nd V68A-Y167A 0.87 1.19 Nd Nd V68A-Y217Q 2,34 1.32 Nd Nd W106f 0.78 1.17 Nd Nd W106F-G128A 0.68 0.86 Nd Nd W106F-I111V 0.61 1.06 Nd Nd W106F-L126A 0.67 0.82 Nd Nd W106F-M124V 0.56 0.96 Nd Nd W106F-N123G 0.47 0.29 Nd Nd W106F-V227T 0.45 0.76 Nd Nd W106F-Y167A 0.75 0.66 Nd Nd W106F-Y217Q 0.87 1.35 Nd Nd Y104N 0.88 1,03 Nd Nd Y104N-G128A 0.83 1.21 Nd Nd Y104N-I111V 0.6 0.94 Nd Nd Y104N-L126A 0.59 0.17 Nd Nd Y104N-M124V 0.51 0.43 Nd Nd Y104N-N123G 0.58 0.21 Nd Nd Y104N-V227T 0.4 0.32 Nd Nd Y104N-W106F 0.52 0.46 Nd Nd Y104N-Y167A 0.74 0.51 Nd Nd Y104N-Y217Q 0.68 1.27 Nd Nd Y167A 0.77 0.89 Nd Nd Y167A-V227T 0.45 0.43 Nd Nd Y167A-Y217Q 0.64 1.21 Nd Nd Y217Q 1.37 1.26 Nd Nd Y217Q-V227T 0.87 1,08 Nd Nd

EXAMPLE 14

The ability of double options BPN 'to remove stains

Using BPN 'as the starting protein, double variants at positions 217 and 222 (BPN' numbering) are obtained by hybridizing PCR. Variant BPN 'proteins are prepared using the methods described above. The protein concentration in the culture supernatants is determined by precipitation using TCA, as described in Example 1. The ability to remove stains during washing is analyzed on EMPA 116 samples (BMI stains, CFT) at pH 8/16 ° C in heat inactivated TIDE® 2X Cold (Procter & Gamble) using the methods described in Example 1. The functionality of the BPN 'variants is quantified as a performance indicator (Pi) (ie, the ratio of the variant characteristics to BPN'-Y217L). The results are shown in table 14-1. BPN 'variants with a Pi value greater than or equal to 0.5 according to BMI stain removal and / or TCA deposition assays are characterized by improved properties and / or increased expression.

Table 14-1 Ability to remove stains and expression of double variants of BPN ' Option BMI TCA Y217L-M222Q 0.88 1.27 Y217Q-M222Q 1,08 1.18 Y217V-M222Q 1,02 0.76

EXAMPLE 15

Obtaining ASP protease in B. subtilis

Methods for producing protease 69B4 (also referred to herein as “ASP”, “Asp”, “ASP protease” and “Asp protease”) in B. subtilis are described in US Patent Application No. 10/576331, incorporated herein by reference in fullness. In short, the following DNA sequence (synthetic ASP DNA sequence) containing a codon adapted for Bacillus species encodes an ASP precursor protein:

In this sequence, DNA that encodes a mature protease is shown in bold, the leader sequence is shown in standard font, and the N-terminal and C-terminal sequences are underlined. The mature enzyme serine protease obtained from strain Cellulomonas 69B4 (DSM 983316035) contains 189 amino acids in length, a catalytic triad consisting of His32, Asp56 and Ser137, as shown below (catalytic triad is highlighted in bold and underlined):

EXAMPLE 16

Obtaining Combinatorial ASP Mutants and Multiple Mutation Libraries

This example describes methods for producing combinatorial mutants and a library of multiple ASP mutations. Obtaining combinatorial ASP mutants is described in US patent application No. 10/576331.

Getting the library of multiple mutations

A library of multiple mutations is obtained in accordance with the instructions attached to the Stratagene QCMS kit, except for the primer concentration used in the reaction. Namely, 1 μl of methylated, purified pUC18-ASP plasmid (approximately 70 ng) was mixed with 15 μl of sterile distilled water, 1.5 μl of dNTP, 2.5 μl of 10 × buffer, 1 μl of enzyme mixture and 1.0 μl of mutant mixture primers (total 100 pmol of primers). A mixture of primers is prepared using 10 μl of each of the eighteen mutant primers (100 pmol / μl); adding 50 ng of each primer to the library, according to the recommendations of Stratagene, leads to a decrease in mutations in the previous mutagenesis cycle. Thus, the technique is modified in the current mutagenesis cycle by introducing a total of 100 pmol primers in each reaction. The reaction is carried out using the following cyclic conditions: 95ºC for 1 min, then 30 cycles, including 95ºC for 1 min, 55ºC for 1 min and 65ºC for 12 min, in the MJ Research PTC2-200 thermal cycler using thin-walled tubes for PCR with a volume of 0.2 ml. The reaction product is digested by incubation with 1 μl of DpnI from the QCMS kit at 37 ° C overnight. An additional 0.5 μl of DpnI was added, after which the reaction mixture was incubated for 1 hour.

Then the DNA library (mutated single-chain product pUC18-ASP) was introduced by electroporation into electrocompetent E. coli cells (Invitrogen®, catalog number C4040-52, One Shot® TOP10 Electrocomp ^TM E. coli, dam +), transformed cells were grown and subjected to selection on plates with agar containing 100 mg / L ampicillin, to obtain a library of multiple mutations in E. coli cells. Colonies (tens of thousands) were harvested and plasmid DNA was obtained using the Qiagen spin DNA minidrug kit (catalog number 27106) in accordance with the manufacturer's instructions. Minidrug DNA is eluted using 50 μl of Qiagen EB buffer included in the kit.

Minidrug DNA is digested with restriction enzymes PstI and HindIII DNA. A mixture of ASP library components (PstI × HindIII) was purified by gel chromatography and cloned into a 4154 base pair HindIII × PstI pHPLT vector fragment into which it was inserted by ligation using T4 Invitrogen® DNA ligase (Catalog No. 15224-025) as recommended by the manufacturer for cloning using sticky ends). In another strategy, the synthetic components of the ASP library are obtained from GeneArt. The obtained ASP library components are also cleaved with PstI and HindIII, purified and inserted into the HindIII × PstI pHPLT vector fragment of 4154 base pairs by ligase reaction.

To directly use the ligation reaction mixture to transform Bacillus cells, the DNA library (a mixture of ASP library components cloned into pHPLT) was amplified using the TempliPhi kit (Amersham, Catalog No. 25-6400). For this, 1 μl of the ligation reaction mixture was mixed with 5 μl of sample buffer from the TempliPhi kit and heated for 3 minutes at 95 ° C to denature the DNA. The reaction mixture was placed on ice, cooled for 2 minutes and centrifuged for a short period of time. Then, 5 μl of reaction buffer and 0.2 μl of phi29 polymerase from the TempliPhi kit are added, after which the reaction mixtures are incubated at 30 ° C in an MJ Research PCR apparatus for 4 hours. The phi29 enzyme is inactivated by heating in the reaction mixtures by incubation at 65 ° C for 10 min in a PCR device.

To transform Bacillus using libraries, 0.1 μl of TempliPhi amplification reaction product was mixed with 500 μl of competent B. subtilis cells (ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE:: (xy1R, pxy1A-comK) followed by vigorous shaking with 37 ° shaking for 1 hour, after which aliquots of 100 and 500 μl were placed on HI agar plates containing 20 ppm neomycin sulfate (Sigma, catalog No. N-1876; contains 732 μg neomycin per mg) and 0.5 % skim milk: Ninety-five clones are selected from the library for sequencing.

Mutagenesis proceeds favorably since only 14% of the clones have a sequence identical to the original (ASP containing R014I-A064K-T086K-T116E-R123F), and approximately 3% of the clones contain additional mutations. All other sequenced clones (72%) are mutants, and of these, approximately 94% are unique mutants. The library sequencing results are shown below in table 16-1.

Table 16-1
Options for ASP Containing R014I-A064K-T086K-T116E-R123F G54d N24A N24Q N24T N67S R127K R159F R159K R159K R159N R159N G78d R159F N24Q R35E N67S R159E R127K R159E R127K R159K R127K R159N R127Q R159K R35d R159E R35d R159K R35E R159K G54d R127K R159K G78d R127K R159K G78d R127K R159E G78d R127Q R159K N24A N67A R159K N24A N67S R159K N24E R35d G78d N24T N67S R159E N67L G78d R159K R35d G78d R159K N24A R35E G78d R159N N24D R35d G78d R159F N24E G54d G78d R159K N24E R35d G78d R127K R159N N24Q G54d G78d R159N N24Q N67L G78d R159E N24Q R35d R127K R159K N24T R35d G78d R159K N24T R35d G78d R159K N67S G78d R127K R159K R35d G78d R127K R159E R35d G78d R127K R159N R35d G78d R127Q R159K R35E G54d N67A R159F R35E N67S G78d R127Q N24A G54d N67S G78d R159F N24A R35d N67A G78d R159F N24Q R35d N67L G78d R159K N24Q R35d N67L G78d R159N N24Q R35d N67S R127K R159E N24Q R35E N67A R127K R159E N24Q R35E N67A G78d R159E N24T N67A G78d R127Q R159N N24T R35E N67A G78d R127Q R35E G54d N67S G78d R159K N24A G54d N67S G78d R127K R159K N24A R35E N67S G78d R127K R159K N24E R35E G54d N67S R127K R159N N24Q R35d N67S G78d R127K R159F N24T G54d N67S G78d R127Y R159E N24E R35E G54d N67S G78d R127K R159K

EXAMPLE 17

Correlation of harmful mutations in several properties

This example explains the principle that harmful mutations in one property correlate with harmful mutations in each other property, regardless of the correlation of properties. As indicated in this document, only a small number of provisions (5-10%) may contain mutations that adversely affect all properties. Such provisions determine protein folding and are conservative in terms of evolution. The significance of this phenomenon lies in the fact that, in contrast to the identification of enhancing mutations, which requires the use of, in essence, predictive screening for this property, the identification of mutations that presumably have a harmful effect on any property can be performed using any screening, including, without limitation, the methods described herein.

Variant enzymes are obtained using the methods described herein and in US patent applications Nos. 10/576331, 10/581014, 11/581102 and 11/583334, which are incorporated herein by reference in their entirety. The tables below show a pairwise comparison of a number of options with the masses. activity of more than 5% and with activity for each of the two properties less than 5%, and correlation coefficients for the two properties are indicated. The analytical systems used in this example are also offered for the above applications. Casein activity (CAS), keratin activity (KER), AAPF activity (AAPF), LAS resistance (LAS) and thermal stability in the case of ASP are used as properties; and the formation of peracids (PAF) and degradation of peracids (PAD) in the case of ACT.

Keratin Hydrolysis Analysis

The following solutions of chemicals and reagents are used in this analytical system:

Keratin ICN 902111 Detergent 1.6 g, the detergent is dissolved in 1000 ml of water (pH = 8.2), 0.6 ml of CaCl ₂ / MgCl ₂ , 10,000 gpg, and 1190 mg of HEPES are added to give a stiffness and ionic strength of 6 gpg and 5 mM buffer , respectively. The pH was adjusted to 8.2 with NaOH.
Picrylsulfonic Acid (TNBS)
Sigma P-2297 (5% solution in water) Reagent A 45.4 g of Na ₂ B ₄ O ₇ · 10H ₂ O (Merck 6308) and 15 ml of 4N NaOH are dissolved together to give a final volume of 1000 ml (if necessary, with heating) Reagent B 35.2 g of NaH ₂ PO ₄ · 1H ₂ O (Merck 6346) and 0.6 g of Na ₂ SO ₃ (Merck 6657) are dissolved together to give a final volume of 1000 ml.

Method:

Before incubation, keratin is sieved through a sieve of 100 μm in small portions. Then 10 g of keratin with a particle size <100 μm is mixed in a detergent solution for at least 20 minutes at room temperature, maintaining a pH of 8.2. Finally, the suspension is centrifuged for 20 minutes at room temperature (Sorvall, GSA rotor, 13000 rpm). Then the described procedure is repeated. Finally, the wet cake is suspended in the detergent to give a total volume of 200 ml and pipetting is carried out while stirring is continued. Before incubation, 200 μl of substrate per well is pipetted into the MTP using a Biohit multichannel pipette and a 1200 μl tip (6 200 μl portions that are dispensed as quickly as possible to avoid keratin settling on the tips). Then, 10 μl of filtered culture is added to the MTPs containing the substrate. The plates are covered with film, placed in an incubator and incubated at 20 ° C for 3 hours at 350 rpm (New Brunswick Innova 4330). After incubation, the plates are centrifuged for 3 minutes at 3000 rpm (Sigma 6K 15 centrifuge). About 15 minutes before removing the first plate from the incubator, a TNBS reagent is prepared by mixing 1 ml of TNBS solution and 50 ml of reagent A.

60 μl of TNBS reagent A mixture was added to each MTP well. From the incubation plates, 10 μl was transferred to MTP containing TNBS reagent A. The plates were film-coated and shaken for 20 minutes in a table shaker (BMG Thermostar) at room temperature and 500 rpm Finally, 200 μl of reagent B was added to the wells, mixed for 1 minute on a shaker and absorbance was measured at 405 nm using an MTP reader.

Calculation of keratin-hydrolyzing activity

The resulting absorption value is adjusted to an empty value (substrate in the absence of the enzyme). The resulting absorption value is a measure of hydrolytic activity. For each sample (option) calculate the indicator of functioning. The performance indicator indicates the activity of the variant (actual value) compared with the standard enzyme (theoretical value) at the same protein concentration. In addition, theoretical values can be calculated using the parameters of the Langmuir equation for a standard enzyme. If the functioning index (PI) is greater than 1 (PI> 1), the variant is considered better than the standard enzyme (for example, wild-type), if PI is 1 (PI = 1), this indicates that the variant has the same characteristics. like a standard enzyme, and if PI is less than 1 (PI <1), it is believed that the variant functions worse than the standard enzyme. Thus, PI allows you to identify the best options, as well as less desirable options for use in certain conditions.

Dimethyl Casein Hydrolysis Assay (96-well format)

The following solutions of chemicals and reagents are used in this analytical system:

Dimethyl Casein (DMC): Sigma C-9801 TWEEN®-80: Sigma P-8074 PIPES buffer (free acid): Sigma P-1851; 15.1 g are dissolved in approximately 960 ml of water; The pH was adjusted to 7.0 with 4N NaOH, 1 ml of 5% TWEEN®-80 was added and the volume was adjusted to 1000 ml. Final concentration PIPES and TWEEN®-80 are 50 mM and 0.005%, respectively. Picrylsulfonic acid (TNBS): Sigma P-2297 (5% solution in water) Reagent A: 45.4 g of Na ₂ B ₄ O ₇ · 10H ₂ O (Merck 6308) and 15 ml of 4N NaOH are dissolved together to a final volume of 1000 ml (if necessary, with heating) Reagent B: 35.2 g of NaH ₂ PO ₄ · 1H ₂ O (Merck 6346) and 0.6 g of Na ₂ SO ₃ (Merck 6657) are dissolved together to a final volume of 1000 ml.

Method:

To obtain a substrate, 4 g of DMC was dissolved in 400 ml of PIPES buffer. Filtered culture supernatants were diluted with PIPES buffer; the final concentration of controls in the culture plate is 20 ppm Then 10 μl of each diluted supernatant is added to the MTP wells containing 200 μl of substrate. The MTP tablet is coated with a film, shaken for several seconds, placed in a thermostat and kept at 37ºC for 2 hours without shaking.

About 15 minutes before removing the first plate from the thermostat, TNBS reagent is prepared by mixing 1 ml of TNBS solution and 50 ml of reagent A. 60 μl of TNBS-reagent A mixture is added to each MTP well. The incubation plates are shaken for several seconds and then 10 μl transferred to MTP containing a mixture of TNBS-reagent A. The plates are coated with a film and shaken for 20 minutes in a table shaker (BMG Thermostar) at room temperature and 500 rpm. Finally, 200 μl of reagent B was added to the wells, mixed for 1 minute on a shaker and absorbance was measured at 405 nm using an MTP reader.

Calculation of dimethylcasein hydrolyzing activity:

The resulting absorption value is adjusted to an empty value (substrate in the absence of the enzyme). The resulting absorption value is a measure of hydrolytic activity. (Conditional) The specific activity of the sample is calculated by dividing the absorption by a specific protein concentration.

Thermostability Analysis

This assay measures the hydrolysis of dimethyl casein (DMC) before and after heating a buffered culture supernatant. The same solutions of chemicals and reagents are used as in the DMC hydrolysis assay.

Method:

Filtered culture supernatants were diluted to 20 ppm. PIPES buffer (based on their concentration of controls in culture plates). Then, 50 μl of each diluted supernatant was placed in empty MTP wells. The MTP plate is incubated in an iEMS HT incubator / shaker (Thermo Labsystems) for 90 minutes at 60 ° C and 400 rpm. The tablets are cooled on ice for 5 minutes. Then 10 μl of the solution is added to a new MTP containing 200 μl of DMC substrate / well. This MTP is covered with a film, shaken for several seconds and placed in a thermostat, where it is kept at 37ºC for 2 hours without shaking. The same detection method is used as for the analysis of hydrolysis of DMC.

Thermostability calculation:

The residual activity of the sample is expressed as the ratio of the final absorption and the initial absorption, which are corrected for empty values.

As shown in the following tables, only properties such as CAS, KER, and AAPF in the case of ASP are correlated (correlation coefficients> 0.5). All other properties are not correlated (correlation coefficient <0.3). Despite the fact that the properties do not correlate, the likelihood that the mutation has a harmful effect on the two properties is much higher than that of a random coincidence. Table 17-1 shows the calculated ratios of the observed number of options and the number of options expected based on probability theory. Values greater than 1 indicate a positive correlation, and values less than 1 indicate a negative correlation.

Claims (15)

1. An isolated variant of Bacillus subtilisin, wherein said subtilisin variant is a mature form having subtilisin activity and containing a substitution at one or more positions, selected from: A1C, A1D, A1E, A1M, A1N, A1Q, A1S, Q2E, S3D, S3Q , Y6E, Y6W, S9C, S9E, S9G, S9P, S9T, Q10E, I11V, K12A, K12C, K12D, K12E, K12F, K12G, K12H, K12I, K12N, K12Q, K12S, K12T, K12V, K12W, K12Y, , P14D, P14E, P14G, P14I, P14L, P14N, P14S, P14T, P14V, A15C, A15D, A15E, A15P, L16I, S18E, S18H, S18I, S18M, S18Q, S18T, S18V, S18Y, Q19C, Q19D, , G20C, G20D, G20E, G20H, T22C, T22E, T22F, T22L, T22W, T22Y, N25E, N25F, N25W, K27G, K27P, I35A, I35C, I35S, I35T, I35V, S37E, S37R, P40C, P40E, , P40H, P40I, P40L, P40Q, P40R, P40W, K43A, K43C, K43D, K43E, K43G, K43L, K43M, K43R, K43S, K43W, M50I, M50K, P52K, P52R, S53 E, S53K, S53R, N56D, F58C, Q59C, Q59D, Q59E, Q59N, H67P, S78C, S78D, S78F, S78I, S78K, S78L, S78N, S78Q, S78R, S78W, S78Y, I79E, P86Y, S89C, S89D S89g, y91i, y91v, l96i, l96v, a98c, a98d, a98i, a98s, a98t, d99r, s101c, s101e, s101g, s101p, g102a, g102s, q103c, q103f, q103g, q103i Q103T, Q103W, S105A, S105C, S105D, S105E, S105K, S105L, W106A, W106C, W106E, W106F, W106G, W106H, W106I, W106L, W106M, W106R, W106S, W10, N10, N10, N10, N10 N109L, N109P, N109Q, N109R, N109V, N109W, N109Y, E112A, E112C, E112F, E112G, E112I, E112L, E112M, E112N, E112Q, E112S, E112T, E112V, E112W, E112Y, W113N, I115N, I115R, I115T, A116D, A116K, A116R, N118C, M124I, M124L, L126I, L126V, L126W, G128A, G128S, G131P, G131R, A133C, A133D, A133E, A133F, A133I, A133L, A13313, A13313, A13313, A13313, A13313, A13313, A13313, A13313, A13313, A13313, A13313, A13313, A13313 ,13313 ,13313 K136E, K136H, A137C, A137F, A137H, A137K, A137L, A137M, A137R, A137W, A137Y, V139C, K141C, K141D, K141E, K141F, K141G, K141H, K141I14, K14114, K14114 1W, K141Y, V143D, V143E, A144C, A144D, A144E, A144I, A144K, A144L, A144R, A144W, S145C, S145D, S145E, S145F, S145L, S145M, S145R, E158, 1515, 1515, 1515, 1515, 1515, 1515, 1515 S159D, S159Q, S162C, S162E, S162M, S162N, S162Q, G169A, K170A, P172E, P172R, A176C, S182C, S182D, S182Q, S183C, S183E, S183N, S183Q18, S183Q18818, Q18, Q18, Q188, Q188 S188P, P194C, P194E, Q206C, Q206D, Q206E, Q206L, Q206M, Q206W, Q206Y, N212C, N212E, K213A, K213C, K213D, K213E, K213H, K213I, K213L, K213K, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L, K213L A216C, A216D, A216E, Y217C, Y217D, Y217E, Y217K, Y217L, Y217M, Y217N, Y217Q, Y217R, N218C, N218S, N218T, S224C, P225A, P225C, P225G, P225I, P225G, P225G, P225G, P225G, P225G, P225G, P225G, P225G L235g, l235i, l235k, l235m, l235n, l235q, l235s, l235w, l235y, s236e, s236h, h238f, p239n, p239t, p239v, t244d, t244f, t244h, t244k N252E, T254A, T254C, T255C, T255D, T255E, K256A, K256C, K256D, K256E, K256F, K256H, K256I, K256L, K256M, K256N, K256P, K256Q, K2 56s, k256t, k256v, k256w, k256y, s260c, s260d, s260e, s260p, k265a, k265c, k265e, k265h, k265l, k265m, k265n, k265q, k265s, k265y T022M, T022S, T0026F, T0026G, I031E, I031F, I031H, I031N, I031S, I031T, A0045E, V072A, V072C, S087D, A092S, L096M, S101Y, Q103A, Q103E, Q10310, Q10310, Q10310, Q10310, Q10310, Q10310, Q10310 V147e, v147s, v149a, v149c, v149d, v149e, v149f, g166s, k170e, v203c, s101q, s101t, s101v, g166n, g166q, g166r, s101i, s101l G166C, G097D, V149P, where these positions correspond to the amino acid sequence of subtilisin BPN 'B. amyloliquefaciens presented in SEQ ID NO: 2. 2. A variant of subtilisin according to claim 1, wherein said substitution includes a combination of substitutions selected from: S101E-Y217L, K213I-Y217Q and K213I-Y217E. 3. The subtilisin variant of claim 1, wherein said substitution includes a combination selected from: S101N-K213I, K213L, K213N, K213I-Y217E, K213N-Y217Q, S101P-K213N, K213I-Y217Q, Y217Q, K213I, and S101 -Y217L. 4. A variant of subtilisin according to claim 1, wherein said substitution includes a combination selected from: Y217L and S101E-Y217L. 5. The selected nucleic acid encoding a variant of subtilisin according to claim 1. 6. An expression vector containing a nucleic acid according to claim 5. 7. A host cell capable of expressing the subtilisin variant of claim 1, comprising an expression vector of claim 6. 8. A cleaning composition containing a variant of subtilisin according to claim 1 in an effective amount. 9. The cleaning composition of claim 8, wherein said cleaning composition is a washing agent. 10. The cleaning composition of claim 9, wherein said detergent is a cold water detergent, a low pH detergent, or a compact detergent. 11. A method of obtaining a variant of subtilisin Bacillus, comprising the following stages:
a) transformation of the host cell with an expression vector containing a nucleic acid encoding a variant of subtilisin according to claim 1; and
b) culturing the transformed host cell under conditions suitable for obtaining a variant of subtilisin, to obtain a variant of subtilisin. 12. The method according to p. 11, further comprising the step of collecting the obtained variant subtilisin. 13. The method of claim 11, wherein said host cell is a Bacillus species. 14. The method of claim 13, wherein said Bacillus species is B. subtilis. 15. A cleaning method, which includes the step of bringing the surface and / or article containing the fabric into contact with a cleaning composition containing an isolated variant of subtilisin according to any one of claims. 1-4.

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