DE102022205594A1 - PERFORMANCE-IMPROVED AND STORAGE-STABLE PROTEASE VARIANTS - Google Patents

Abstract

The invention relates to proteases which have proteolytic activity and comprise an amino acid sequence which is at least 70% identical to the amino acid sequence specified in SEQ ID NO:1 over its entire length, the protease, in each case based on the numbering according to SEQ ID NO:1 , at (i) the positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I and (ii) at least one and increasingly preferably two, three, four, five or six of the positions corresponding to positions 74 , 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256 correspond to at least one and increasingly preferably two, three, four, five or six further amino acid substitution (s). ), which consists of N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V /W, A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, are selected from the existing group, as well as their production and use. Such proteases show very good cleaning performance.

Description

The invention lies in the field of enzyme technology. The invention relates to proteases whose amino acid sequence has been changed, particularly with a view to use in detergents and cleaning agents, in particular with regard to liquid detergents and cleaning agents, in order to improve their cleaning performance, and to the nucleic acids encoding them and their production. The invention further relates to the uses of these proteases and processes in which they are used, as well as agents containing them, in particular detergents and cleaning agents, in particular liquid detergents and cleaning agents.

Proteases are among the most technically important enzymes of all. For detergents and cleaning agents, they are the longest established enzymes and are contained in practically all modern, high-performance detergents and cleaning agents. They break down protein-containing dirt on the items being cleaned. Particularly important here are proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62), which are serine proteases due to the catalytically active amino acids. They act as nonspecific endopeptidases and hydrolyze any acid amide bonds that lie inside peptides or proteins. Their pH optimum is usually in the significantly alkaline range. An overview of this family can be found in the article “ Subtilases: Subtilisin-like Proteases” by R. Siezen, pages 75-95 in “Subtilisin enzymes”, edited by R. Bott and C. Betzel, New York, 1996 . Subtilases are produced naturally by microorganisms. Of these, the subtilisins formed and secreted by Bacillus species should be mentioned in particular as the most important group within the subtilases.

Examples of the subtilisin-type proteases that are preferably used in detergents and cleaning agents are the subtilisins BPN' and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, subtilisin DY and the enzymes thermitase, proteinase K and the proteases TW3 and TW7, which can be assigned to the subtilases but no longer to the subtilisins in the narrower sense, as well as variants of the proteases mentioned which have an amino acid sequence that is changed compared to the original protease. Proteases are modified in a targeted or random manner using methods known from the prior art and are thus optimized, for example, for use in detergents and cleaning agents. These include point, deletion or insertion mutagenesis or fusion with other proteins or parts of proteins. Correspondingly optimized variants are known for most of the proteases known from the prior art. In e.g WO 95/23221 , WO 92/21760 WO 2013/060621 and EP 3660151 Variants of the alkaline protease from Bacillus lentus DSM 5483 are disclosed which are suitable for use in detergents or cleaning agents. Furthermore, in e.g WO 2011/032988 , WO 2016/096714 and EP 3044302 Detergents and cleaning agents are disclosed which contain variants of the alkaline protease from Bacillus lentus DSM 5483.

In general, only selected proteases are suitable for use in liquid surfactant-containing preparations. Many proteases do not show sufficient catalytic performance in such preparations. For the use of proteases in detergents and cleaning agents, high catalytic activity under conditions such as those encountered during a washing cycle and high storage stability are particularly desirable.

Consequently, protease- and surfactant-containing liquid formulations from the prior art have the disadvantage that the proteases they contain do not have satisfactory proteolytic activity and/or are insufficient under standard washing conditions (e.g. in a temperature range of 20° C. to 40° C.). are storage-stable and the formulations therefore do not show optimal cleaning performance on protease-sensitive soils.

Surprisingly, it has now been found that a protease of the alkaline protease type from Bacillus lentus DSM 5483 or a sufficiently similar protease (based on the sequence identity), which, based on the numbering according to SEQ ID NO: 1 (i), is at the positions corresponding to positions 3, 4 and 199, having amino acid substitutions 3T, 4l and 1991, and (ii) at least one and increasingly preferably at two, three, four, five or six of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256 correspond to at least one and increasingly preferably two, three, four, five or six amino acid substitution (s) from the from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/ W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, are selected from the existing group its washing performance is visibly improved compared to the wild-type form (SEQ ID NO:1) or an initial variant, and is therefore particularly suitable for use in detergents or cleaning agents.

The invention therefore relates to a protease comprising an amino acid sequence which has proteolytic activity and has an amino acid sequence which is at least 70% identical to the amino acid sequence specified in SEQ ID NO:1 over its entire length, and, in each case based on the numbering according to SEQ ID NO:1, (i) at positions corresponding to positions 3, 4, and 199 having amino acid substitutions 3T, 4l and 1991, and (ii) at least one and increasingly preferably at two, three, four, five or six of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, at least one and increasingly preferably two, three , four, five or six amino acid substitution(s) consisting of N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/ L/S/T, Y203K/V/W, A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, existing group are selected, having.

A further subject of the invention is a method for producing a protease as defined above, comprising (i) introducing three amino acid substitutions 3T, 4I and 199I at the positions which correspond to positions 3, 4 and 199 based on the numbering according to SEQ ID NO:1 , and (ii) introducing at least one amino acid substitution at at least one of the positions which, based on the numbering according to SEQ ID NO:1, are positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185 , 200, 203, 209, 212 or 256, into a starting molecule that has an amino acid sequence that has at least 70% sequence identity with the amino acid sequence specified in SEQ ID NO:1 over its entire length.

A protease within the meaning of the present patent application therefore includes both the protease as such and a protease produced using a method according to the invention. All statements on the protease therefore refer both to the protease as such and to the proteases produced using appropriate processes.

Further aspects of the invention relate to the nucleic acids encoding these proteases, non-human host cells containing proteases according to the invention or nucleic acids, as well as agents comprising proteases according to the invention, in particular detergents and cleaning agents, washing and cleaning processes, and uses of the proteases according to the invention in detergents or cleaning agents for removing protein-containing soiling.

These and other aspects, features and advantages of the invention will become apparent to those skilled in the art from reading the following detailed description and claims. Any feature from one aspect of the invention can be used in any other aspect of the invention. Furthermore, it is to be understood that the examples contained herein are intended to describe and illustrate the invention, but not to limit it and in particular the invention is not limited to these examples.

DEFINITIONS

Unless otherwise stated, all percentages are percentages by weight (wt.%).

Numerical ranges specified in the format “from x to y” include the stated values. When multiple preferred numerical ranges are specified in this format, it is understood that any ranges resulting from the combination of the various endpoints will also be captured.

“At least one,” as used herein, means one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more.

The term “detergent and cleaning agent” or “detergent or cleaning agent” as used herein is synonymous with the term “agent” and refers to a composition for cleaning textiles and/or hard surfaces, in particular tableware, as in the description explained.

“About,” “approximately,” or “approximately,” as used herein in reference to a numerical value, refers to the corresponding numerical value ±10%, preferably ±5%.

“Substantially free of” means that the composition or agent contains less than 2% by weight, preferably less than 1% by weight, more preferably less than 0.5% by weight and particularly preferably less than 0. 1% by weight of the corresponding substance, based on the total weight of the composition/agent.

“Liquid” as used herein includes liquids and gels as well as pasty compositions. It is preferred that the liquid compositions be flowable and pourable at room temperature, but it is also possible that they have a yield point. A substance, for example a composition or an agent, is solid according to the definition of the invention if it is in the solid state at 25 ° C and 1013 mbar. A substance, e.g. a composition or an agent, is liquid according to the definition of the invention if it is in the liquid state at 25 ° C and 1013 mbar. Liquid also includes gel form.

“Variant,” as used herein, refers to natural or engineered variations of a native protease that has an amino acid sequence modified from the reference form.

DETAILED DESCRIPTION

The present invention is based on the inventors' surprising finding that amino acid substitutions at the positions described herein result in improved cleaning performance of this modified protease in detergents and cleaning agents.

In preferred embodiments, the change(s) according to the invention lead (i) to positions corresponding to positions 3, 4 and 199 based on the numbering according to SEQ ID NO:1, and (ii) to at least one of the positions based on the numbering according to SEQ ID NO:1 correspond to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, to an improved cleaning performance of these modified protease in detergents and cleaning agents on at least one protease-sensitive soil. Proteases according to the invention consequently enable improved removal of at least one, preferably several, protease-sensitive stains on textiles and/or hard surfaces, for example dishes.

In preferred embodiments, the protease according to the invention has (i) at the positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I are selected from the existing group, and (ii) at at least one of the positions corresponding to positions 74 , 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, at least one amino acid substitution consisting of N74D/E/Q, A136Q, R143L/ W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W, N212S/T and L256D/E/Q , preferably S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, is selected from the existing group, wherein the combination of the three amino acid substitutions from group (i) and the at least one amino acid substitution from group (ii) to one Improved cleaning performance of this modified protease in detergents and cleaning agents leads to at least one protease-sensitive stain.

In preferred embodiments, the protease according to the invention has (i) at positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I are selected from the existing group, (ii) at least one and increasingly preferably two, three, four , five or six of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, at least one and increasingly preferably two, three, four, five or six amino acid substitution(s) consisting of N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A /L/S/T, Y203K/V/W, A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, existing group is selected , and (iii) at the position corresponding to position 99 does not contain the amino acid Glu (E) or Asp (D).

In preferred embodiments, the protease according to the invention has (i) at positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I are selected from the existing group, (ii) at least one and increasingly preferably two, three, four , five or six of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, at least one and increasingly preferably two, three, four, five or six amino acid substitution(s) consisting of N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A /L/S/T, Y203K/V/W, A209K/W, N212S/T and L256D/E/Q, preferred S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, is selected from the existing group, and (iii) at the position corresponding to position 99, the amino acid Arg (R).

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, and (ii) at least one and increasingly preferably two, three, four, five or six of the positions corresponding to positions 74, 136, 143, 154, 160 , 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256 correspond to at least one and increasingly preferably two, three, four, five or six amino acid substitution (s) derived from N74D/E /Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W, N212S/ T and L256D/E/Q, preferably S154D, S160G, A181 D, F183R, Q185R, Q200L, Y203W, A209K and L256E, are selected from the existing group.

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, and (ii) at least one of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256 Amino acid substitution selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E.

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, and (ii) at least two of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least two Amino acid substitutions selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E.

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, and (ii) at least three of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least three Amino acid substitutions selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E.

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, and (ii) at least four of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, has at least four amino acid substitutions selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E.

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, and (ii) at least five of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least five Amino acid substitutions selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E.

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, and (ii) at least six of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least six Amino acid substitutions selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E.

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least one and increasingly preferably two, three, four, five or six of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256 correspond to at least one and increasingly preferably two, three, four, five or six amino acid substitution (s) from the N74D / E / Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181 D, F183R, Q185R, Q200L, Y203W, A209K and L256E, are selected from the existing group, and (iii) at the position corresponding to position 99, not the amino acid Glu (E) or Asp (D).

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least one of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least one amino acid substitution , which are selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, not the amino acid Glu (E) or Asp (D).

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least two of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least two amino acid substitutions selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99 the amino acid Glu (E) or Asp (D).

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least three of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least three amino acid substitutions , which are selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, not the amino acid Glu (E) or Asp (D).

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least four of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least four amino acid substitutions , which are selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, not the amino acid Glu (E) or Asp (D).

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least five of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least five amino acid substitutions , selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, not the amino acid Glu (E) or Asp (D).

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least six of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least six amino acid substitutions , which are selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, not the amino acid Glu (E) or Asp (D).

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least one and increasingly preferably two, three, four, five or six of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, at least one and increasingly preferably two, three, four, five or six amino acid substitution (en) from the N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K /V/W, A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181 D, F183R, Q185R, Q200L, Y203W, A209K and L256E, existing group are selected, and (iii) at the Position corresponding to position 99 has the amino acid Arg (R).

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least one of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least one amino acid substitution , selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, the amino acid Arg (R). .

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least two of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least two amino acid substitutions , selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, the amino acid Arg (R). .

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least three of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least three amino acid substitutions , selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, the amino acid Arg (R). .

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least four of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least four amino acid substitutions , selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, the amino acid Arg (R). .

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% are identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I, (ii) at least five of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least five amino acid substitutions selected from S154D, S160G, A181D, F183R , Q185R, Q200L, Y203W, A209K and L256E, are selected from the existing group, and (iii) has the amino acid Arg (R) at the position corresponding to position 99.

In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which is at least 70% and increasingly preferably at least 71%, 72%, 73% of the amino acid sequence specified in SEQ ID NO:1 over its entire length. 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96 .5%, 97%, 97.5%, 98%, 98.5% and 98.8% is identical, and, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions that represent the Positions 3, 4 and 199 correspond to the amino acid substitutions S3T, V4I and V199I, (ii) at least six of the positions corresponding to positions 154, 160, 181, 183, 185, 200, 203, 209 or 256, at least six amino acid substitutions , selected from the group consisting of S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, and (iii) at the position corresponding to position 99, the amino acid Arg (R). .

1. (a) S3T-V4I-V199I-Q200L-Y203W;
2. (b) S3T-V4I-V199I-Q200L-Y203W-S154D-L256E;
3. (c) S3T-V4I-V199I-Q200L-Y203W-A209K-S154D-L256E;
4. (d) S3T-V4I-V199I-Q200I-Y203Y-A209W-S154D-L256E;
5. (e) S3T-V4I-V199I-Q2001-Y203W-A209W-S154D-L256E;
6. (f) S3T-V4I-V199I-Q200L-Y203W-A209W-S154D-L256E;
7. (g) S3T-V4I-V199I-Q2001-Y203W A209K-S154D-L256E;
8. (h) S3T-V4I-V199I-Q2001-Y203Y-A209K-S154D-L256E;
9. (i) S3T-V41-V199I-Q200L-Y203W-A209K-S154D-L256E;

wobei die Nummerierung jeweils bezogen ist auf die Nummerierung gemäß SEQ ID NO:1.In particularly preferred embodiments, the protease comprises one of the following amino acid substitution combinations:

1. (a) S3T-V4I-V199I-Q200L-Y203W;
2. (b) S3T-V4I-V199I-Q200L-Y203W-S154D-L256E;
3. (c) S3T-V4I-V199I-Q200L-Y203W-A209K-S154D-L256E;
4. (d) S3T-V4I-V199I-Q200I-Y203Y-A209W-S154D-L256E;
5. (e) S3T-V4I-V199I-Q2001-Y203W-A209W-S154D-L256E;
6. (f) S3T-V4I-V199I-Q200L-Y203W-A209W-S154D-L256E;
7. (g) S3T-V4I-V199I-Q2001-Y203W A209K-S154D-L256E;
8. (h) S3T-V4I-V199I-Q2001-Y203Y-A209K-S154D-L256E;
9. (i) S3T-V41-V199I-Q200L-Y203W-A209K-S154D-L256E;

where the numbering is based on the numbering according to SEQ ID NO:1.

In preferred embodiments, the combinations of the amino acid substitutions according to the invention from group (i), (ii) and (iii) described herein lead to an improved cleaning performance of this modified protease in detergents and cleaning agents on at least one protease-sensitive soil. Proteases according to the invention consequently enable improved removal of at least one, preferably several, protease-sensitive stains on textiles and/or hard surfaces, for example dishes.

Proteases according to the invention have increased catalytic activity in detergents or cleaning agents. In various embodiments, the proteases according to the invention can have a proteolytic activity that is at least 101%, 102%, 103%, 104%, 105%, 106%, based on the wild type (SEQ ID NO:1) and/or an already performance-improved starting variant of the protease %, 107%, 108%, 109% or 110%. Such performance-improved proteases enable improved washing results on proteolytic-sensitive soils in different temperature ranges, in particular a temperature range from about 20 ° C to about 40 ° C.

Furthermore, preferred embodiments of proteases according to the invention have a particular stability in detergents or cleaning agents, for example to surfactants and/or bleaches and/or chelators, and/or to temperature influences, in particular to high temperatures, for example between 50 ° C and 65 ° C, in particular 60°C, and/or to acidic or alkaline conditions and/or to pH changes and/or to denaturing or oxidizing agents and/or to proteolytic degradation and/or to a change in redox ratios. Particularly preferred embodiments of the invention therefore provide improved performance and/or more temperature-stable protease variants. With other very special ones The preferred embodiments of the invention provide performance-improved and more temperature-stable protease variants. Such advantageous embodiments of proteases according to the invention consequently enable improved washing results on protease-sensitive soils in a wide temperature range.

In the context of the invention, cleaning performance is understood to mean the whitening performance on one or more soils, in particular on laundry or dishes. Within the scope of the invention, both the washing or cleaning agent which comprises the protease or the washing or cleaning liquor formed by this agent, as well as the protease itself, have a respective cleaning performance. The cleaning performance of the enzyme thus contributes to the cleaning performance of the agent or the washing or cleaning liquor formed by the agent. The cleaning performance is preferably determined as stated below.

Washing liquor is understood to mean the working solution containing the detergent or cleaning agent which acts on textiles or fabrics or hard surfaces and thus comes into contact with the dirt present on textiles or fabrics or hard surfaces. The washing liquor is usually created when the washing or cleaning process begins and the detergent or cleaning agent is diluted with water, for example in a dishwasher, a washing machine or in another suitable container.

The proteases according to the invention have enzymatic activity, i.e. they are capable of hydrolyzing peptides and proteins, especially in a detergent or cleaning agent. A protease according to the invention is therefore an enzyme which catalyzes the hydrolysis of amide/peptide bonds in protein/peptide substrates and is thereby able to cleave proteins or peptides. Furthermore, a protease according to the invention is preferably a mature protease, i.e. the catalytically active molecule without signal and/or propeptide(s). Unless otherwise stated, the sequences given also refer to mature (processed) enzymes.

In various embodiments of the invention, the protease is a free enzyme. This means that the protease can act directly with all components of an agent and, if the agent is a liquid agent, that the protease is in direct contact with the agent's solvent (e.g. water). In other embodiments, an agent may contain proteases that form an interaction complex with other molecules or that contain a “coating.” Here, a single or several protease molecules can be separated from the other components of the agent by a structure surrounding them. Such a separating structure can arise from, but is not limited to, vesicles such as a micelle or a liposome. The surrounding structure can also be a virus particle, a bacterial cell or a eukaryotic cell. In various embodiments, an agent may contain cells of Bacillus pumilus or Bacillus subtilus expressing the proteases of the invention or cell culture supernatants of such cells.

In the context of the present invention, the feature that a protease has the specified substitutions means that it contains one (of the specified) substitution(s) at the respective position, i.e. at least the specified positions are not otherwise mutated or, for example, by fragmentation of the protease , are deleted. In various preferred embodiments, the proteases described herein have the sequence of SEQ ID NO:1, with the exception of the explicitly mentioned substitutions, i.e. apart from the substituted positions, they are 100% identical to the sequence according to SEQ ID NO:1.

The identity of nucleic acid or amino acid sequences is determined by sequence comparison. This sequence comparison is based on the BLAST algorithm established and commonly used in the prior art (see e.g Altschul et al. (1990) Basic local alignment search tool, J. Mol. Biol., 215:403-410, and Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res., 25:3389-3402 ) and basically happens by associating similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences with one another. A tabular assignment of the relevant positions is called alignment. Another algorithm available in the art is the FASTA algorithm. Sequence comparisons (alignments), especially multiple sequence comparisons, are created using computer programs. The Clustal series, for example, is often used (see e.g Chenna et al. (2003) Multiple sequence alignment with the Clustal series of programs, Nucleic Acid Res., 31:3497-3500), T-Coffee (see e.g. Notredame et al. (2000) T-Coffee: A novel method for multiple sequence alignments , J Mol Biol, 302:205-217 ) or programs based on these programs or algorithms. Sequence comparisons (alignments) are also possible using the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the specified standard parameters, whose AlignX module for sequence comparisons is based on ClustalW, or Clone Manager 10 (Use of the BLOSUM 62 scoring matrix for sequence alignment at the amino acid level). Unless otherwise stated, sequence identity reported herein is determined using the BLAST algorithm.

Such a comparison also allows a statement to be made about the similarity of the compared sequences to one another. It is usually given in percent identity, i.e. the proportion of identical nucleotides or amino acid residues in the same positions or in positions corresponding to one another in an alignment. The broader concept of homology includes conserved amino acid exchanges in amino acid sequences, i.e. amino acids with similar chemical activity, since these usually exert similar chemical activities within the protein. Therefore, the similarity of the compared sequences can also be indicated as percent homology or percent similarity. Identity and/or homology statements can be made about entire polypeptides or genes or just about individual regions. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by similarities in the sequences. Such areas often have identical functions. They can be small and contain only a few nucleotides or amino acids. Such small areas often perform essential functions for the overall activity of the protein. It can therefore make sense to relate sequence matches only to individual, possibly small areas. Unless otherwise stated, identity or homology information in the present application refers to the total length of the nucleic acid or amino acid sequence specified in each case.

In the context of the present invention, the statement that an amino acid position corresponds to a numerically designated position in SEQ ID NO:1 therefore means that the corresponding position is assigned to the numerically designated position in SEQ ID NO:1 in an alignment as defined above. Furthermore, the assignment of the positions depends on the mature protein. This assignment is particularly applicable if the amino acid sequence of a protease according to the invention comprises a higher number of amino acid residues than the protease according to SEQ ID NO:1. Starting from the positions mentioned in the amino acid sequence of the protease according to SEQ ID NO:1, the change positions in a protease according to the invention are those that are assigned to these positions in an alignment.

In addition to the amino acid changes explained above, proteases according to the invention can have further amino acid changes, in particular amino acid substitutions, insertions or deletions. Such proteases are, for example, further developed through targeted genetic modification, i.e. through mutagenesis processes, and optimized for specific purposes or with regard to specific properties (for example with regard to their catalytic activity, stability, etc.). Furthermore, nucleic acids according to the invention can be introduced into recombination approaches and thus used to generate completely new proteases or other polypeptides.

The aim is to introduce targeted mutations such as substitutions, insertions or deletions into the known molecules in order, for example, to improve the cleaning performance of enzymes according to the invention. For this purpose, in particular the surface charges and/or the isoelectric point of the molecules and thereby their interactions with the substrate can be changed. For example, the net charge of the enzymes can be changed in order to influence substrate binding, especially for use in detergents and cleaning agents. Alternatively or additionally, one or more corresponding mutations can increase the stability or catalytic activity of the protease and thereby improve its cleaning performance. Advantageous properties of individual mutations, e.g. individual substitutions, can complement each other. Proteases that have already been optimized with regard to certain properties, for example with regard to their stability during storage, can therefore be further developed further within the scope of the invention.

For the description of substitutions that affect exactly one amino acid position (amino acid exchanges), the following convention is used here: first the naturally present amino acid is designated in the form of the internationally used single-letter code, then follows the associated sequence position and finally the inserted amino acid. Multiple or alternative substitutions within the same polypeptide chain are separated by slashes. “130D/V” means that position 130 has mutated to D or V. In insertions, additional amino acids are named after the sequence position. In the case of deletions, the missing amino acid is represented by a symbol, for example a star or a dash, or a Δ is given before the corresponding position. For example, P9T describes the substitution of proline at position 9 by threonine, P9TH describes the insertion of histidine after the amino acid threonine at position 9, and P9* or ΔP9 describes the deletion of proline at position 9. This nomenclature is known to those skilled in the field of enzyme technology.

A further subject of the invention is therefore a protease which is characterized in that it is obtainable from a protease as described herein as a starting molecule by single or multiple conservative amino acid substitution, the protease in the count according to SEQ ID NO:1 being at least one of the Has amino acid substitutions described above. The term “conservative amino acid substitution” means the exchange (substitution) of an amino acid residue for another amino acid residue, whereby this exchange does not lead to a change in polarity or charge at the position of the exchanged amino acid, e.g. the exchange of a non-polar amino acid residue for another non-polar amino acid residue. Conservative amino acid substitutions within the scope of the invention include, for example: G=A=S, I=V=L=M, D=E, N=Q, K=R, Y=F, S=T, G=A=I=V =L=M=Y=F=W=P=S=T.

Alternatively or additionally, the protease is characterized in that it is obtainable from a protease according to the invention as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which has a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268 or 269 contiguous amino acids match the parent molecule, with the protease (i) at positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209 , 212 or 256, in particular selected from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W, N212S/T and L256D/E/Q.

For example, it is possible to delete individual amino acids at the termini or in the loops of the enzyme without losing or reducing the proteolytic activity. Furthermore, such fragmentation, deletion, insertion or substitution mutagenesis can also, for example, reduce the allergenicity of the enzymes in question and thus improve their overall usability. Advantageously, the enzymes retain their proteolytic activity even after mutagenesis, i.e. their proteolytic activity corresponds at least to that of the starting enzyme, i.e. in a preferred embodiment the proteolytic activity is at least 80%, preferably at least 90%, of the activity of the starting enzyme. Other substitutions can also have beneficial effects. Both single and several contiguous amino acids can be exchanged for other amino acids.

Advantageous positions for sequence changes, in particular substitutions, of the protease according to SEQ ID NO:1, which are preferably important when transferred to homologous positions of the proteases according to the invention and give the protease advantageous functional properties, are therefore the positions that are in an alignment with the positions described herein correspond, i.e. in the count according to SEQ ID NO:1. The following amino acid residues are located in the wild-type molecule of the protease at the positions mentioned: 3S, 4V, 74N, 99R, 136A, 143R, 154S, 160S, 161Y, 163A, 171V, 181A, 183F, 185Q, 199V, 200Q, 203Y, 209A, 212 N and 256L.

Further confirmation of the correct assignment of the amino acids to be changed, ie in particular their functional correspondence, can be provided by comparison experiments, according to which the two positions assigned to each other on the basis of an alignment are changed in the same way in both proteases being compared and it is observed whether in both the enzymatic activity is changed in the same way. For example, an amino acid exchange in a specific position of the protease from Bacillus lentus according to SEQ ID NO:1 is accompanied by a change in an enzymatic parameter, for example with an increase in the K _M value, and a corresponding change in the enzymatic parameter, for example also a Increase in the K _M value, observed in a protease variant according to the invention, the amino acid exchange of which was achieved by the same introduced amino acid, this can be seen as a confirmation of the correct assignment.

1. (a) Einbringen einer ein- oder mehrfachen konservativen Aminosäuresubstitution in die Protease, wobei die Protease aufweist:
   • i) an den Positionen, die den Positionen 3, 4 und 199 entsprechen, die Aminosäuresubstitutionen S3T, V4I und V199I, und
   • ii) mindestens eine Aminosäuresubstitution an mindestens einer der Positionen, die den Positionen 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 oder 256 entsprechen, insbesondere ausgewählt aus N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W, N212S/T und L256D/E/Q;
2. (b) Veränderung der Aminosäuresequenz durch Fragmentierung, Deletions-, Insertions- oder Substitutionsmutagenese derart, dass die Protease eine Aminosäuresequenz umfasst, die über eine Länge von mindestens 190, 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268 oder 269 zusammenhängenden Aminosäuren mit dem Ausgangsmolekül übereinstimmt, wobei die Protease aufweist:
   • i) an den Positionen, die den Positionen 3, 4 und 199 entsprechen, die Aminosäuresubstitutionen S3T, V4I und V199I, und
   • ii) mindestens eine Aminosäuresubstitution an mindestens einer der Positionen, die den Positionen 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 oder 256 entsprechen, insbesondere ausgewählt aus N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W, N212S/T und L256D/E/Q;
   Sämtliche Ausführungsformen gelten auch für die erfindungsgemäßen Verfahren.

All of the facts mentioned are also applicable to the methods according to the invention for producing a protease. Accordingly, a method according to the invention further comprises one or more of the following method steps:

1. (a) introducing a single or multiple conservative amino acid substitution into the protease, the protease having:
   • i) at positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I, and
   • ii) at least one amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, in particular selected from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W , N212S/T and L256D/E/Q;
2. (b) changing the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease comprises an amino acid sequence which is at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262 in length , 263, 264, 265, 266, 267, 268 or 269 contiguous amino acids match the starting molecule, the protease having:
   • i) at positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I, and
   • ii) at least one amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, in particular selected from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W , N212S/T and L256D/E/Q;
   All embodiments also apply to the methods according to the invention.

In a further embodiment of the invention, a previously described protease is stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer. Increasing the stability during storage and/or use, for example during the washing process, results in the enzymatic activity lasting longer and thus the cleaning performance being improved. In principle, all stabilization options described and/or appropriate in the prior art can be considered. Those stabilizations that are achieved via mutations of the enzyme itself are preferred, since such stabilizations do not require any further work steps after the enzyme has been obtained. Examples of sequence changes suitable for this are mentioned above. Other suitable sequence changes are known from the prior art.

• - Veränderung der Bindung von Metallionen, insbesondere der Calcium-Bindungsstellen, beispielsweise durch Austauschen von einer oder mehreren der an der Calcium-Bindung beteiligten Aminosäure(n) gegen eine oder mehrere negativ geladene Aminosäuren und/oder durch Einführen von Sequenzveränderungen in mindestens einer der Folgen der beiden Aminosäuren Arginin/Glycin;
• - Schutz gegen den Einfluss von denaturierenden Agentien wie Tensiden durch Mutationen, die eine Veränderung der Aminosäuresequenz auf oder an der Oberfläche des Proteins bewirken;
• - Austausch von Aminosäuren, die nahe dem N-Terminus liegen, gegen solche, die vermutlich über nicht-kovalente Wechselwirkungen mit dem Rest des Moleküls in Kontakt treten und somit einen Beitrag zur Aufrechterhaltung der globulären Struktur leisten.

Other stabilization options include:

• - Changing the binding of metal ions, in particular the calcium binding sites, for example by replacing one or more of the amino acids involved in the calcium binding with one or more negatively charged amino acids and / or by introducing sequence changes in at least one of the sequences the two amino acids arginine/glycine;
• - Protection against the influence of denaturing agents such as surfactants through mutations that cause a change in the amino acid sequence on or on the surface of the protein;
• - Exchange of amino acids that are close to the N-terminus with those that presumably come into contact with the rest of the molecule via non-covalent interactions and thus contribute to the maintenance of the globular structure.

Preferred embodiments are those in which the enzyme is stabilized in several ways, since several stabilizing mutations act additively or synergistically.

wobei R jeweils ausgewählt ist aus -COOH, C_1-6-Alkyl-substituierten oder unsubstituierten C_2-6-Dicarbonsäuren, C_1-6-Alkyl-substituierten oder unsubstituierten C_2-6-Carbonsäuren und -OOC-NR² ₂, wobei R² gleich oder verschieden und ausgewählt aus C_1-6-Alkyl oder H sind, sowie Salzen, Estern oder Derivaten davon, vorzugsweise Benzoesäure, Phenylmalonsäure, Benzylmalonsäure, Phenylbernsteinsäure, Benzylbernsteinsäure, Methyl-3-Benzoylpropionat, (S)-3-Phenylbuttersäure und Benzylcarbamat, und Kombinationen davon bestehenden Gruppe ausgewählt ist, kombiniert werden, um die Stabilität der Protease in Wasch- und Reinigungsmitteln weiter zu erhöhen.Alternatively or additionally, the protease according to the invention can be combined with at least one reversible inhibitor compound, which consists of polyols, in particular glycerol and 1,2-propylene glycol, benzamidine hydrochloride, borax, boric acids, boronic acids or their salts or esters or derivatives, in particular phenylboronic acid derivatives or 4-formylphenylboronic acid ( 4-FPBA), compounds of formulas (I) or (II)

where R is each selected from -COOH, C _1-6 alkyl-substituted or unsubstituted C _2-6 dicarboxylic acids, C _1-6 alkyl-substituted or unsubstituted C _2-6 carboxylic acids and -OOC-NR ² ₂ , where R ² are the same or different and selected from C _1-6 alkyl or H, as well as salts, esters or derivatives thereof, preferably benzoic acid, phenylmalonic acid, benzylmalonic acid, phenylsuccinic acid, benzylsuccinic acid, methyl-3-benzoylpropionate, (S)-3- Phenylbutyric acid and benzyl carbamate, and combinations thereof selected from the existing group, can be combined to further increase the stability of the protease in detergents and cleaning agents.

wobei R Wasserstoff, eine Hydroxyl-, eine C_1-6 Alkyl-, eine substituierte C_1-6 Alkyl-, eine C_1-6 Alkenyl oder eine substituierte C_1-6 Alkenyl-Gruppe ist. Bevorzugt ist der Rest R in dem Phenylboronsäurederivat eine C_1-6 Alkyl-Gruppe und hierunter weiter bevorzugt -CH₃, -CH₃CH₂ oder - CH₃CH₂CH₂ sein. Weiter bevorzugt ist der Rest R in dem Phenylboronsäurederivat Wasserstoff. Besonders bevorzugt ist das Phenylboronsäurederivat 4-Formyl-phenylboronsäure (4-FPBA).In the context of the present invention, “phenylboronic acid derivative” is understood to mean a compound with the formula (III). The compound of formula (III) has the following structural formula:

where R is hydrogen, a hydroxyl, a C _1-6 alkyl, a substituted C _1-6 alkyl, a C _1-6 alkenyl or a substituted C _1-6 alkenyl group. The radical R in the phenylboronic acid derivative is preferably a C _1-6 alkyl group and, among these, is more preferably -CH ₃ , -CH ₃ CH ₂ or - CH ₃ CH ₂ CH ₂ . More preferably, the radical R in the phenylboronic acid derivative is hydrogen. The phenylboronic acid derivative 4-formylphenylboronic acid (4-FPBA) is particularly preferred.

The reversible inhibitor compound used can be boric acid.

wobei R jeweils ausgewählt ist aus -COOH, C_1-6-Alkyl-substituierten oder unsubstituierten C_2-6-Dicarbonsäuren, C_1-6-Alkyl-substituierten oder unsubstituierten C_2-6-Carbonsäuren und -OOC-NR² ₂, wobei R² gleich oder verschieden und ausgewählt aus C_1-6-Alkyl oder H sind, sowie Salzen, Estern oder Derivaten davon, und Kombinationen davon bestehenden Gruppe ausgewählt ist, sein, vorzugsweise ausgewählt aus der aus Benzoesäure, Phenylmalonsäure, Benzylmalonsäure, Phenylbernsteinsäure, Benzylbernsteinsäure, Methyl-3-Benzoylpropionat, (S)-3-Phenylbuttersäure und Benzylcarbamat bestehenden Gruppe.The reversible inhibitor compound used can be a compound of the formulas (I) or (II)

where R is each selected from -COOH, C _1-6 alkyl-substituted or unsubstituted C _2-6 dicarboxylic acids, C _1-6 alkyl-substituted or unsubstituted C _2-6 carboxylic acids and -OOC-NR ² ₂ , where R ² is the same or different and selected from C _1-6 alkyl or H, as well as salts, esters or derivatives thereof, and combinations thereof, and combinations thereof, preferably selected from the group consisting of benzoic acid, phenylmalonic acid, benzylmalonic acid, phenylsuccinic acid, Benzyl succinic acid, methyl 3-benzoyl propionate, (S)-3-phenylbutyric acid and benzyl carbamate.

In particularly preferred embodiments, the protease according to the invention is used in agents or compositions that are essentially free of boron-containing compounds. “Substantially free of boron-containing compounds” in this context means that the corresponding agents or compositions contain less than 2% by weight, preferably less than 1% by weight, more preferably less than 0.5% by weight and particularly preferably contain less than 0.1% by weight of boron-containing compounds, based on the total weight of the agent/composition. In very particularly preferred embodiments, these agents/compositions are free of boron-containing compounds, i.e. in particular they do not contain any boric acid and/or phenylboronic acid derivatives.

In a further embodiment of the invention, the protease is characterized in that its cleaning performance is not significantly reduced compared to the wild-type enzyme (SEQ ID NO:1) or a starting variant, i.e. has at least 80% of the reference washing performance, preferably at least 100%, more preferably at least 110% or more.

The cleaning performance can be determined in a washing system that contains a detergent in a dosage of between 2.0 and 8.0 grams per liter of washing liquor and the enzyme. The enzymes to be compared are used in the same concentration (based on active protein). The use of the respective enzymes with the same activity ensures that even if there is a discrepancy in the ratio of active substance to total protein (the values of the specific activity), the respective enzymatic properties, i.e. the cleaning performance on certain types of soiling, are compared become. In general, low specific activity can be compensated for by adding a larger amount of protein. Furthermore, the enzymes to be examined can also be used in the same amount or weight if the enzymes to be examined have a different affinity for the test substrate in an activity test. In this context, the expression “same amount of substance” refers to the same molar use of the enzymes to be examined. The expression “equal weight” refers to an equal weight use of the enzymes to be examined.

The concentration of the protease in the detergent intended for such a washing system is 0.001 to 0.1% by weight, preferably 0.01 to 0.06% by weight, based on active protein.

Washing or cleaning performance is understood to mean the ability of a detergent or cleaning agent to partially or completely remove existing soiling, in particular the whitening performance of one or more soilings on textiles. Examples of such soiling are blood on cotton or chocolate-milk/soot on cotton, cocoa on cotton or porridge on cotton. Within the scope of the invention, both the washing or cleaning agent which comprises the protease, or the washing or cleaning liquor formed by this agent, as well as the protease itself have a respective cleaning performance. The cleaning performance of the protease thus contributes to the cleaning performance of the agent or the washing or cleaning liquor formed by the agent.

Washing or cleaning liquor is understood to mean the working solution containing the detergent or cleaning agent which acts on the textiles or hard surfaces and thus comes into contact with the dirt present on the textiles or hard surfaces. The washing or cleaning liquor is usually created when the washing or cleaning process begins and the washing or cleaning agent is diluted with water, for example in a washing machine or dishwasher or in another suitable container.

A liquid reference detergent for such a washing system can, for example, be composed as follows (all information in percent by weight (wt.%)): 4.4% alkylbenzenesulfonic acid, 5.6% other anionic surfactants, 2.4% C _12-18 Na salts of fatty acids (soaps), 4.4% non-ionic surfactants, 0.2% phosphonates, 1.4% citric acid, 0.95% NaOH, 0.01% defoamer, 2% glycerin, 0.08% Preservatives, 1% ethanol, rest demineralized water. The dosage of the liquid detergent is preferably between 4.5 and 6.0 grams per liter of washing liquor, for example 4.7, 4.9 or 5.9 grams per liter of washing liquor. Washing is preferred in a pH range between pH 7 and pH 10.5, preferably between pH 8 and pH 9.

The cleaning performance against soiling on cotton is determined by measuring the degree of cleaning of the washed textiles. For example, the washing process can take place for 60 minutes at a temperature of 40°C and the water can have a hardness of between 15.5°dH and 16.5°dH (German hardness).

In the context of the invention, the cleaning performance is determined, for example, at 20 ° C or 40 ° C using a liquid detergent such as that specified above, the washing process preferably taking place for 60 minutes at 600 rpm.

The degree of whiteness, i.e. the lightening of the dirt, as a measure of the cleaning performance is determined using optical measuring methods, preferably photometrically. A suitable device for this is, for example, the Minolta CM508d spectrometer. The devices used for the measurement are usually calibrated beforehand with a white standard, preferably a supplied white standard.

Preferred embodiments of proteases according to the invention achieve such advantageous cleaning performance even at low temperatures, in particular in the temperature ranges between 10 ° C and 60 ° C, preferably between 15 ° C and 50 ° C and particularly preferably between 20 ° C and 40 ° C.

Methods for determining protease activity are familiar to those skilled in the field of enzyme technology and are routinely used by them. For example, such processes are disclosed in Tenside, Volume 7 (1970), pp. 125-132. Alternatively, protease activity can be determined via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF). The protease cleaves the substrate and releases pNA. The release of the pNA causes an increase in the absorbance at 410 nm, the time course of which is a measure of the enzymatic activity (see Del Mar et al., 1979). The measurement is carried out at a temperature of 25°C, at pH 8.6, and a wavelength of 410 nm. The measuring time is 5 min and the measuring interval is 20s to 60s. Protease activity is usually reported in protease units (PE). Suitable protease activities are, for example, 2.25, 5 or 10 PE per ml of wash liquor. However, the protease activity is not zero.

An alternative test for determining the proteolytic activity of the proteases according to the invention is an optical measurement method, preferably a photometric method. The test suitable for this involves the protease-dependent cleavage of the substrate protein casein. This is broken down by the protease into a large number of smaller partial products. The entirety of these partial products has an increased absorption at 290 nm compared to non-cleaved casein, whereby this increased absorption can be determined using a photometer and thus a conclusion can be drawn about the enzymatic activity of the protease.

The protein concentration can be determined using known methods, for example the BCA method (bicinchoninic acid; 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method ( Gornall et al., J. Biol. Chem. 177 (1948): 751-766 ) can be determined. In this regard, the active protein concentration can be determined by titrating the active centers using a suitable irreversible inhibitor and determining the residual activity (cf. Bender et al., J. Am. Chem. Soc. 88, 24 (1966): 5890-5913 ) take place.

A further subject of the invention is a protease as described above, which is characterized in that it has at least one chemical modification. A protease with such a change is called a derivative, i.e. the protease is derivatized.

For the purposes of the present application, derivatives are understood to mean proteins whose pure amino acid chain has been chemically modified. Such derivatizations can occur, for example, in vivo by the host cell that expresses the protein. In this regard, couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy. However, derivatizations can also be carried out in vitro, for example by chemically converting a side chain of an amino acid or by covalently binding another compound to the protein. For example, it is possible to couple amines to carboxyl groups of an enzyme to change the isoelectric point. Such another compound can also be another protein, which is bound to a protein according to the invention, for example via bifunctional chemical compounds. Derivatization also means covalent binding to a macromolecular carrier, or non-covalent inclusion in suitable macromolecular cage structures. Derivatizations can, for example, influence the substrate specificity or the binding strength to the substrate or cause a temporary blocking of the enzymatic activity if the coupled substance is an inhibitor. This can be useful, for example, for the storage period. Such modifications may further affect stability or enzymatic activity. They can also serve to reduce the allergenicity and/or immunogenicity of the protein and thus, for example, to increase its skin compatibility. For example, couplings with macromolecular compounds, for example polyethylene glycol, can improve the protein in terms of stability and/or skin compatibility.

Derivatives of a protein according to the invention can also be understood in the broadest sense as preparations of these proteins. Depending on how it is obtained, processed or prepared, a protein can be combined with various other substances, for example from the culture of the producing microorganisms. A protein can also have been specifically mixed with other substances, for example to increase its storage stability. All preparations of a protein according to the invention are therefore also in accordance with the invention. This is also independent of whether it actually develops this enzymatic activity in a particular preparation or not. It may be desired that it has little or no activity during storage and only develops its enzymatic function at the time of use. This can be controlled, for example, using appropriate accompanying substances. In particular, the joint preparation of proteases with specific inhibitors is possible in this regard.

Of all the proteases or protease variants and/or derivatives described above, those whose storage stability and/or whose cleaning performance is improved compared to the initial variant are particularly preferred in the context of the present invention, the cleaning performance being determined in a washing system as described above.

A further subject of the invention is a nucleic acid which codes for a protease according to the invention, and a vector containing such a nucleic acid, in particular a cloning vector or an expression vector.

These can be DNA or RNA molecules. They can be present as a single strand, as a single strand complementary to this single strand, or as a double strand. Especially with DNA molecules, the sequences of both complementary strands must be taken into account in all three possible reading frames. Furthermore, it should be taken into account that different codons, i.e. base triplets, can code for the same amino acids, so that a specific amino acid sequence can be coded by several different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences that can encode one of the proteases described above are included in this subject matter of the invention. The person skilled in the art is able to determine these nucleic acid sequences without any doubt because, despite the degeneracy of the genetic code, defined amino acids can be assigned to individual codons. Therefore, starting from an amino acid sequence, the person skilled in the art can easily determine nucleic acids that code for this amino acid sequence. Furthermore, in nucleic acids according to the invention, one or more codons can be replaced by synonymous codons. This aspect relates in particular to the heterologous expression of the enzymes according to the invention. Each organism, for example a host cell of a production strain, has a specific codon usage. Codon usage refers to the translation of the genetic code into amino acids by the respective organism. Bottlenecks in protein biosynthesis can occur if the codons on the nucleic acid are confronted with a comparatively small number of loaded tRNA molecules in the organism. Although encoding the same amino acid, this results in a codon being translated less efficiently in the organism than a synonymous codon encoding the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, it can be translated more efficiently in the organism.

It is possible for a person skilled in the art to use known DNA and/or amino acid sequences to produce the corresponding nucleic acids up to complete genes using methods that are generally known today, such as chemical synthesis or the polymerase chain reaction (PCR) in conjunction with standard molecular biological and/or protein chemical methods to produce. Such methods are, for example Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press . known.

For the purposes of the present invention, vectors are understood to mean elements consisting of nucleic acids which contain a nucleic acid according to the invention as a characteristic nucleic acid region. They are able to establish this as a stable genetic element in a species or a cell line over several generations or cell divisions. Vectors are special plasmids, i.e. circular genetic elements, especially when used in bacteria. In the context of the present invention, a nucleic acid according to the invention is cloned into a vector. The vectors include, for example, those whose origins are bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements from a wide variety of origins. With the additional genetic elements present, vectors are able to establish themselves as stable units in the relevant host cells over several generations. They can exist extrachromosomally as separate units or integrate into a chromosome or chromosomal DNA.

Expression vectors include nucleic acid sequences which enable them to replicate in the host cells containing them, preferably microorganisms, particularly preferably bacteria, and to express a nucleic acid contained there. Expression is particularly influenced by the promoter(s) that regulate transcription. In principle, expression can take place through the natural promoter originally located in front of the nucleic acid to be expressed, but also through a host cell promoter provided on the expression vector or through a modified or a completely different promoter from another organism or another host cell. In the present case, at least one promoter for the expression of a nucleic acid according to the invention is made available and used for its expression. Expression vectors can also be regulated, for example by changing the cultivation conditions or when the host cells they contain reach a certain cell density or by adding certain substances, in particular activators of gene expression. An example of such a substance is the galactose derivative isopropyl-β-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon). In contrast to expression vectors, the nucleic acid contained in cloning vectors is not expressed.

A further subject of the invention is a non-human host cell which contains a nucleic acid according to the invention or a vector according to the invention, or which contains a protease according to the invention, in particular one which secretes the protease into the medium surrounding the host cell. A nucleic acid according to the invention or a vector according to the invention is preferably transformed into a microorganism, which then represents a host cell according to the invention. Alternatively, individual components, ie nucleic acid parts or fragments of a nucleic acid according to the invention, can also be introduced into a host cell in such a way that the resulting host cell then contains a nucleic acid according to the invention or a vector according to the invention. This procedure is particularly suitable if the host cell already contains one or more components of a nucleic acid according to the invention or a vector according to the invention and the other components are then supplemented accordingly. Methods for transforming cells are established in the prior art and are well known to those skilled in the art. In principle, all cells are suitable as host cells, i.e. prokaryotic or eukaryotic cells. Preferred are those host cells that can be handled in a genetically advantageous manner, for example in terms of transformation with the nucleic acid or vector and its stable establishment, for example single-celled fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological manageability. This applies, for example, to easy cultivability, high growth rates, low requirements for fermentation media and good production and secretion rates for foreign proteins. Preferred host cells according to the invention secrete the (transgenically) expressed protein into the medium surrounding the host cells. Furthermore, the proteases can be modified by the cells that produce them after their production, for example by attaching sugar molecules, formylation, amination, etc. Such post-translational modifications can functionally influence the protease.

Further preferred embodiments represent host cells whose activity can be regulated due to genetic regulatory elements that are provided, for example, on the vector, but can also be present in these cells from the outset. For example, by controlled addition of chemical compounds that serve as activators, by changing the cultivation conditions or when a certain cell density is reached, these can be stimulated to express themselves. This enables economical production of the proteins according to the invention. An example of such a connection is IPTG as described above.

Preferred host cells are prokaryotic or bacterial cells. Bacteria are characterized by short generation times and low demands on cultivation conditions. This allows cost-effective cultivation processes or manufacturing processes to be established. The expert also has a wealth of experience with bacteria in fermentation technology. Gram-negative or gram-positive bacteria may be suitable for a specific production for a variety of reasons that can be determined experimentally in individual cases, such as nutrient sources, product formation rate, time required, etc.

In gram-negative bacteria such as Escherichia coli, a large number of proteins are secreted into the periplasmic space, i.e. into the compartment between the two membranes enclosing the cells. This can be advantageous for special applications. Furthermore, gram-negative bacteria can also be designed in such a way that they release the expressed proteins not only into the periplasmic space, but into the medium surrounding the bacterium. In contrast, Gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of the Actinomycetales do not have an outer membrane, so that secreted proteins are immediately released into the medium surrounding the bacteria, usually the nutrient medium, from which the expressed proteins can be purified. They can be isolated directly from the medium or further processed. In addition, gram-positive bacteria are related or identical to most organisms of origin for technically important enzymes and usually produce comparable enzymes themselves, so that they have similar codon usage and their protein synthesis apparatus is naturally aligned accordingly.

Host cells according to the invention can be modified in terms of their requirements for the culture conditions, have other or additional selection markers or express other or additional proteins. In particular, these can also be host cells that transgenically express several proteins or enzymes.

The present invention is in principle applicable to all microorganisms, in particular to all fermentable microorganisms, particularly preferably to those of the genus Bacillus, and results in Proteins according to the invention can be produced by using such microorganisms. Such microorganisms then represent host cells within the meaning of the invention.

In a further embodiment of the invention, the host cell is characterized in that it is a bacterium, preferably one selected from the group of genera Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferably one selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum , Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas maltophilia.

However, the host cell can also be a eukaryotic cell, which is characterized by the fact that it has a nucleus. A further subject of the invention is therefore a host cell which is characterized in that it has a cell nucleus. In contrast to prokaryotic cells, eukaryotic cells are capable of post-translationally modifying the protein formed. Examples of this are fungi such as actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This can be particularly advantageous, for example, if the proteins are to undergo specific modifications in connection with their synthesis that make such systems possible. The modifications that eukaryotic systems carry out, particularly in connection with protein synthesis, include, for example, the binding of low-molecular compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to reduce the allergenicity of an expressed protein. Co-expression with the enzymes naturally formed by such cells, such as cellulases, can also be advantageous. Furthermore, for example, thermophilic fungal expression systems can be particularly suitable for expressing temperature-stable proteins or variants.

The host cells according to the invention are cultivated and fermented in the usual way, for example in discontinuous or continuous systems. In the first case, a suitable nutrient medium is inoculated with the host cells and the product is harvested from the medium after a period of time to be determined experimentally. Continuous fermentations are characterized by reaching a steady state in which cells partially die over a comparatively long period of time but also grow back and at the same time the protein formed can be removed from the medium.

1. a) Kultivieren einer erfindungsgemäßen Wirtszelle, und
2. b) Isolieren der Protease aus dem Kulturmedium oder aus der Wirtszelle.

Host cells according to the invention are preferably used to produce proteases according to the invention. A further subject of the invention is therefore a process for producing a protease

1. a) cultivating a host cell according to the invention, and
2. b) Isolating the protease from the culture medium or from the host cell.

This subject matter of the invention preferably includes fermentation processes. Fermentation processes are known from the prior art and represent the actual large-scale production step, usually followed by a suitable purification method of the product produced, for example the proteases according to the invention. All fermentation processes that are based on a corresponding process for producing a protease according to the invention represent embodiments of this subject matter of the invention.

Fermentation processes which are characterized in that the fermentation is carried out via a feed strategy are particularly suitable. The media components that are consumed by ongoing cultivation are added. As a result, considerable increases can be achieved both in cell density and in cell mass or dry mass and/or in particular in the activity of the protease of interest. Furthermore, the fermentation can also be designed in such a way that undesirable metabolic products are filtered out or neutralized by adding buffer or appropriate counterions.

The produced protease can be harvested from the fermentation medium. Such a fermentation process is preferred over isolation of the protease from the host cell, ie product preparation from the cell mass (dry mass), but requires the provision of suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems so that the host cells Secrete protease into the fermentation medium. Alternatively, without secretion, the protease can be isolated from the host cell, ie purified from the cell mass, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.

All of the above-mentioned facts can be combined to form processes in order to produce proteases according to the invention.

A further subject of the invention is an agent which is characterized in that it contains a protease according to the invention as described above. The agent is preferably a detergent or cleaning agent. Particularly preferably, the detergent and cleaning agent is essentially free of boron-containing compounds. “Substantially free of boron-containing compounds” in this context means that the agents according to the invention contain less than 2% by weight, preferably less than 1% by weight, more preferably less than 0.5% by weight and particularly preferably less than 0.1% by weight of boron-containing compounds, based on the total weight of the agent. In very particularly preferred embodiments, the detergents and cleaning agents according to the invention are free of boron-containing compounds, i.e. in particular they do not contain any boric acid and/or phenylboronic acid derivatives.

According to the invention, a detergent or cleaning agent is understood to mean all conceivable types of detergent or cleaning agent, both concentrates and undiluted agents, for use on a commercial scale, in the washing machine or for hand washing or cleaning. These include, for example, detergents for textiles, carpets, or natural fibers, for which the term detergent is used. This also includes, for example, dishwashing detergents for dishwashers (automatic dishwashing detergents) or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood or leather, for which the term cleaning agent is used In addition to manual and machine dishwashing detergents, for example also scouring agents, glass cleaners, toilet fresheners, etc. The detergents and cleaning agents in the context of the invention also include washing aids which are added to the actual detergent when washing textiles manually or by machine in order to achieve a further effect . Furthermore, detergents and cleaning agents within the scope of the invention also include textile pre- and post-treatment agents, i.e. those agents with which the item of laundry is brought into contact before the actual laundry, for example to dissolve stubborn dirt, and also those agents that are in one of the actual The step after textile washing gives the laundry other desirable properties such as a pleasant feel, crease resistance or low static charge. The last-mentioned agents include fabric softeners.

The detergents or cleaning agents according to the invention, which can be present as powdery or granular solids, in compressed or post-compacted particle form, as homogeneous solutions or suspensions, can contain, in addition to a protease according to the invention, all known ingredients that are common in such agents, with preferably at least one further ingredient in the means is available. The agents according to the invention can in particular contain surfactants, builders (builders), polymers, glass corrosion inhibitors, corrosion inhibitors, bleaching agents such as peroxygen compounds, bleach activators or bleach catalysts. They can also contain water-miscible organic solvents, other enzymes, enzyme stabilizers, sequestering agents, electrolytes, pH regulators and/or other auxiliary substances such as optical brighteners, graying inhibitors, color transfer inhibitors, foam regulators as well as dyes and fragrances and combinations thereof.

Advantageous ingredients of agents according to the invention are disclosed in the international patent application WO 2009/121725 , starting there on page 5, penultimate paragraph, and ending on page 13 after the second paragraph. Reference is expressly made to this disclosure and the content of the disclosure therein is incorporated into the present patent application.

An agent according to the invention advantageously contains the protease according to the invention in an amount of 2 µg to 20 mg, preferably from 5 µg to 17.5 mg, particularly preferably from 20 µg to 15 mg and very particularly preferably from 50 µg to 10 mg per g of the agent . In various embodiments, the concentration of the protease (active enzyme) described herein in the agent is >0 to 1% by weight, preferably 0.0001 or 0.001 to 0.1% by weight based on the total weight of the agent or composition.

An agent according to the invention increasingly preferably contains the protease in an amount of 1 × 10 ^-8 to 5% by weight, from 0.0001 to 1% by weight, from 0.0005 to 0.5% by weight, from 0.001 up to 0.1% by weight, based on active protein and based on the total weight of the detergent.

The embodiments of the present invention include all solid, powdery, liquid, gel or pasty dosage forms of agents according to the invention, which may also consist of several phases and may be in compressed or non-compressed form. The agent can be in the form of a free-flowing powder, in particular with a bulk density of 300 g/l to 1200 g/l, in particular 500 g/l to 900 g/l or 600 g/l to 850 g/l. The solid dosage forms of the agent also include extrudates, granules, tablets or pouches. Alternatively, the agent can also be liquid, gel or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste. Liquid assets are generally preferred. Furthermore, the agent can be present as a one-component system. Such means consist of one phase. Alternatively, a means can also consist of several phases. Such a product is therefore divided into several components.

Detergents or cleaning agents according to the invention can only contain a protease. Alternatively, they can also contain further hydrolytic enzymes or other enzymes in a concentration appropriate for the effectiveness of the agent. A further embodiment of the invention therefore represents agents which further comprise one or more further enzymes. Further enzymes which can be preferably used are all enzymes which can develop a catalytic activity in the agent according to the invention, in particular a lipase, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, β-glucosidase, pectinase, carrageenase, perhydrolase, Oxidase, oxidoreductase or other proteases - which can be distinguished from the proteases according to the invention - and mixtures thereof. Further enzymes are advantageously contained in the agent in an amount of 1 × 10 ^-8 to 5 percent by weight based on active protein. Increasingly preferred is each further enzyme in an amount of 1 × 10 ^-7 to 3% by weight, from 0.00001 to 1% by weight, from 0.00005 to 0.5% by weight, from 0.0001 up to 0.1% by weight and particularly preferably from 0.0001 to 0.05% by weight in agents according to the invention, based on active protein. The enzymes particularly preferably show synergistic cleaning performance against certain dirt or stains, ie the enzymes contained in the agent composition support each other in their cleaning performance. Very particularly preferably, such a synergism exists between the protease contained according to the invention and a further enzyme of an agent according to the invention, including in particular between the protease mentioned and an amylase and/or a lipase and/or a mannanase and/or a cellulase and/or a pectinase . Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients of the agent according to the invention.

Textile detergents preferred according to the invention have at least one protease and at least one amylase. In a further preferred embodiment of the invention, textile detergents have at least one protease and at least one cellulase. In a further preferred embodiment, textile detergents have at least one protease and at least one lipase. In a further preferred embodiment, textile detergents have at least one protease, at least one amylase and at least one lipase. In a further preferred embodiment, textile detergents have at least one protease, at least one amylase and at least one cellulase. In a further preferred embodiment, textile detergents have at least one protease, at least one amylase, at least one cellulase and at least one lipase. Textile detergents which have 3 to 10 different enzymes are particularly preferred, with textile detergents which have 3 to 10 different types of enzymes being particularly preferred in terms of their cleaning performance against a very wide range of stains.

Examples of proteases are the subtilisins BPN' from Bacillus amyloliquefaciens and Carlsberg from Bacillus licheniformis, the protease PB92, the subtilisins 147 and 309, the protease from Bacillus lentus, subtilisin DY and the enzymes that can be assigned to the subtilases, but no longer to the subtilisins in the narrower sense thermitase, proteinase K and the proteases TW3 and TW7. Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase® from the company Novozymes. The subtilisins 147 and 309 are sold by the company Novozymes under the trade names Esperase® and Savinase®, respectively. Protease variants are derived from the protease from Bacillus lentus DSM 5483. Other usable proteases are, for example, those under the trade names Durazym®, Relase(D, Everlase®, Nafizym®, Natalase®, Kannase®, Progress Uno 101L® and Ovozyme® from the company Novozymes, which under the trade names Purafect®, Purafect® OxP, Purafect® Prime, Excellase®, Properase®, Preference P100® and Preference P300® from the company Danisco/DuPont, sold under the trade name Lavergy pro 104 LS® from the company BASF, sold under the trade name Protosol® from the company Advanced Biochemicals Ltd., sold under the trade name Wuxi® from the company Wuxi Snyder Bioproducts Ltd., which is sold under the trade names Proleather® and Protease P® by Amano Pharmaceuticals Ltd., and under the trade name Proteinase K-16 by Kao Corp. available enzymes. The proteases from Bacillus gibsonii and Bacillus pumilus, which are disclosed in, are also particularly preferably used WO 2008/086916 , WO 2007/131656 , WO 2017/215925 , WO 2021/175696 and WO 2021/175697 . Further proteases that can be used advantageously are disclosed in, for example WO 91/02792 , WO 2008/007319 , WO 93/18140 , WO 01/44452 , GB 1243784 A , WO 96/34946 , WO 02/029024 and WO 03/057246 . Other proteases that can be used are those that are naturally present in the microorganisms Stenotrophomonas maltophilia, in particular Stenotrophomonas maltophilia K279a, Bacillus intermedius and Bacillus sphaericus.

Examples of amylases are the α-amylases from Bacillus licheniformis, Bacillus amyloliquefaciens or Bacillus stearothermophilus and, in particular, their further developments, which have been improved for use in detergents or cleaning agents. The enzyme from Bacillus licheniformis is available from the company Novozymes under the name Termamyl® and from the company Danisco/DuPont under the name Purastar® ST. Further development products of this α-amylase are available under the trade names Duramyl® and Termamyl® ultra (both from Novozymes), Purastar® OxAm (Danisco/DuPont) and Keistase® (Daiwa Seiko Inc.). The α-amylase from Bacillus amyloliquefaciens is sold by the company Novozymes under the name BAN®, and derived variants of the α-amylase from Bacillus stearothermophilus under the names BSG® and Novamyl®, also by the company Novozymes. Furthermore, the α-amylase from Bacillus sp. A 7-7 (DSM 12368) and the cyclodextrin glucanotransferase (CGTase) from Bacillus agaradherens (DSM 9948) should be highlighted. The amylolytic enzymes can also be used, which are in WO 95/26397 , WO 96/23873 , WO 99/23211 , WO 00/60060 , WO 2003/002711 , WO 2003/054177 , WO 2006/002643 , WO 2007/079938 , WO 2011/100410 and WO 2013/003659 are revealed. Fusion products of all the molecules mentioned can also be used. In addition, the further developments of α-amylase from Aspergillus niger and A. oryzae available under the trade name Fungamyl® from the company Novozymes are suitable. Other commercial products that can be used advantageously include Amylase-LT® and Stainzyme® or Stainzyme® ultra or Stainzyme® plus as well as Amplify™ 12L or Amplify Prime™ 100L, the latter also from the company Novozymes, as well as the PREFERENCE S® series from the company Danisco /DuPont, including, for example, PREFERENCE S100®, PREFERENCE S1000® or PREFERENCE S210®. Variants of these enzymes obtainable through point mutations can also be used according to the invention.

Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases are cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. B. the fungal cellulases from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum, which are in US 4435307 , US 5648263 , US 5691178 , US 5776757 and WO 89/09259 are revealed. Particularly suitable cellulases are the alkaline or neutral cellulases with color-care properties. Examples of such cellulases are cellulases which are in EP 0495257 , EP 0531372 , WO 96/11262 , WO 96/29397 and WO 98/08940 are described. Other examples are cellulase variants, such as those in WO 94/07998 , EP 0531315 , EP 3212777 , EP 3502243 , EP 3653705 , EP 3653706 , US 5457046 , US 5686593 , US 5763254 , WO 95/24471 , WO 98/12307 and WO 99/01544 and WO 2019/122520 are described. Examples of cellulases with endo-1,4-glucanase activity (EC 3.2.1.4) are in WO 2002/099091 described, for example those with a sequence of at least 97% identity to the amino acid sequence of positions 1 to 773 of SEQ ID NO:2 of WO 2002/099091 . Another example may include a GH44 xyloglucanase, e.g. a xyloglucanase enzyme with a sequence of at least 60% identity to positions 40 to 559 of SEQ ID NO:2 of WO 2001/062903 . Commercially available cellulases include Celluzyme™, Carezyme™, Carezyme Premium™, Celluclean™ (e.g. Celluclean™ 5000L and Celluclean™ 4000T), Celluclean Classic™, Cellusoft™, Endolase®, Renozyme® and Whitezyme™ (Novozymes A/S) , Clazinase™ and Puradax HA™ (Genencor International Inc.), KAC-500(B)™ (Kao Corporation), Revitalenz™ 1000, Revitalenz™ 2000 and Revitalenz™ 3000 (DuPont), and Ecostone® and Biotouch® (AB Enzymes ).

Lipases or cutinases, for example, can be used as further enzymes, in particular because of their triglyceride-cleaving activities, but also in order to generate peracids in situ from suitable precursors. Suitable lipases and cutinases are those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples are lipase from Thermomyces, e.g. from T. lanuginosus (formerly called Humicola lanuginosa), as in EP 0258068 and EP 0305216 described, cutinase from Humicola, e.g. H. insolens ( WO 96/13580 ), lipase from strains of Pseudomonas (some of them now renamed Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes ( EP 0218272 ), P. cepacia ( EP 0331376 ), P. sp. Strain SD705 ( WO 95/06720 & WO 96/27002 ), P. wisconsinensis ( WO 96/12012 ), Streptomyces lipases of the GDSL type ( WO 2010/065455 ), cutinase from Magnaporthe grisea ( WO 2010/107560 ), cutinase from Pseudomonas mendocina ( US 5389536 ), lipase from Thermobifida fusca ( WO 2011/084412 ), lipase from Geobacillus stearothermophilus ( WO 2011/084417 ), lipase from Bacillus subtilis (WO 2011/084599), and lipase from Streptomyces griseus ( WO 2011/150157 ) and S. pristinaespiralis ( WO 2012/137147 ). Preferred lipases include, for example, the lipases originally available from Humicola lanuginosa (Thermomyces lanuginosus) or further developed therefrom, in particular those with one or more of the following amino acid exchanges starting from the lipase mentioned in the positions D96L, T213R and / or N233R, particularly preferably T213R and N233R. Preferred commercial lipase products include Lipolase™, Lipex™, Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (Genencor / DuPont) and Lipomax (Gist-Brocades).

To increase the bleaching effect, oxidoreductases, for example oxidases, oxygenases, catalases, peroxidases such as halo-, chloro-, bromo-, lignin-, glucose or manganese peroxidases, dioxygenases or laccases (phenol oxidases, polyphenol oxidases) can be used according to the invention. Advantageously, preferably organic, particularly preferably aromatic, compounds that interact with the enzymes are added in order to increase the activity of the relevant oxidoreductases (enhancers) or to ensure the flow of electrons in the case of very different redox potentials between the oxidizing enzymes and the soils (mediators).

In the cleaning agents described here, the enzymes to be used can also be formulated together with accompanying substances, for example from fermentation. In liquid formulations, the enzymes are preferably used as enzyme liquid formulation(s).

The enzymes are generally not provided in the form of pure protein, but rather in the form of stabilized, storable and transportable preparations. These prefabricated preparations include, for example, the solid preparations obtained by granulation, extrusion or lyophilization or, in particular in the case of liquid or gel-like agents, solutions of the enzymes, advantageously as concentrated as possible, with little water and/or mixed with stabilizers or other auxiliaries.

Alternatively, the enzymes can be encapsulated for both the solid and liquid dosage forms, for example by spray drying or extrusion of the enzyme solution together with a preferably natural polymer or in the form of capsules, for example those in which the enzymes are enclosed as in a solidified gel or in those of the core-shell type, in which an enzyme-containing core is covered with a water-, air- and/or chemical-impermeable protective layer. Additional active ingredients, such as stabilizers, emulsifiers, pigments, bleaches or dyes, can also be applied in superimposed layers. Such capsules are applied using methods known per se, for example by shaking or rolling granulation or in fluid bed processes. Such granules are advantageously low-dust, for example by applying polymeric film formers, and are storage-stable due to the coating.

Furthermore, it is possible to assemble two or more enzymes together so that a single granulate has several enzyme activities.

The enzymes can also be incorporated into water-soluble films, such as those used, for example, in the packaging of detergents and cleaning agents in unit dosage form. Such a film allows the enzymes to be released after contact with water. As used herein, "water soluble" refers to a film structure that is preferably completely water soluble. Such a film preferably consists of (completely or partially hydrolyzed) polyvinyl alcohol (PVA).

A further subject of the invention is a process for cleaning textiles or hard surfaces, which is characterized in that an agent according to the invention is used in at least one process step. In various embodiments, the method described above is characterized in that the protease is at a temperature of 0°C to 100°C, preferably 20°C to 60°C, more preferably 20°C to 40°C and most preferably at 30°C is used.

This includes both manual and mechanical processes, with mechanical processes being preferred due to their more precise controllability, for example in terms of the quantities used and exposure times are. Methods for cleaning textiles are generally characterized by the fact that various cleaning-active substances are applied to the items to be cleaned in several process steps and washed off after the exposure time, or that the items to be cleaned are treated in some other way with a detergent or a solution or dilution of this agent.

Since proteases according to the invention naturally already have a hydrolytic activity and also develop this in media that otherwise have no cleaning power, such as in mere buffer, a single and / or the only step of such a method can consist of a protease according to the invention as the only cleaning-active component is brought into contact with the soiling, preferably in a buffer solution or in water. This represents a further embodiment of this subject matter of the invention.

Alternative embodiments of this subject matter of the invention also represent processes for the treatment of textile raw materials or for textile care, in which a protease according to the invention becomes active in at least one process step. Among these, processes for textile raw materials, fibers or textiles with natural components are preferred, and especially for those with wool or silk.

Finally, the invention also covers the use of the proteases described herein in washing or cleaning agents, for example as described above, for (improved) removal of protein-containing soiling, for example from textiles or hard surfaces.

All facts, objects and embodiments that are described for proteases according to the invention and agents containing them are also applicable to this subject matter of the invention. Therefore, at this point, express reference is made to the disclosure in the appropriate place with the note that this disclosure also applies to the above methods and uses according to the invention.

EXAMPLES

Detergent matrix used Chemical name % by weight of active substance in the raw material % by weight of active substance in the formulation water demin. 100 rest READ 96 12-18 FAEOS 70 4-8 Palm kernel oleic acid 30 2-4 FAEO 100 8-14 HEDP 60 0.5-2 citric acid 100 3-5 NaOH 50 0.5-2 defoamer 100 <1% Glycerin 99.5 1-3 1,2-propanediol 100 8-12 Monoethanolamine 100 4-8 Soil repellent polymer 30 0.5-1 Protease stabilizer 100 0.5-1.5 Without opt. Brighteners, perfume, dye and enzymes. Dosage 3.17 g/L pH 8.2 to 8.4

Determination of cleaning performance

Mini wash test

1. 1. CFT CS038 (Eigelb/Pigment (eingetrocknet))
2. 2. CFT C-05 (Blut/Milch/Tinte)
3. 3. H-MR-B (Milch/Ruß)

Washing test with Bacillus subtilis culture supernatants containing the expressed protease variants. The supernatants are used with the same activity as the benchmark (1.2 mg/L active enzyme protein in the wash liquor). The washing performance of the protease variants according to the invention is based on the washing performance of the benchmark, which is set equal to 100% (sum of the soils tested, corrected by the performance of the washing liquor alone).
Conditions: 40°C, 16° dH water, 1h
Protease 1 (P1): SEQ ID NO:1-S3T-V4I-V199I-S154D-Q200L-Y203W-L256E
Soiling:

1. 1. CFT CS038 (egg yolk/pigment (dried))
2. 2. CFT C-05 (Blood/Milk/Ink)
3. 3. H-MR-B (milk/soot)

Punched out tissues (diameter = 10 mm) were placed in microtiter plates, washing solution was preheated to 40°C, final concentration 3.17 g/L, solution and enzyme were added to the soil for 1 hour at 20°C or 40°C and 600 rpm incubated, then the soiling was rinsed several times with clear water, allowed to dry and the brightness was determined using a colorimeter. The lighter the fabric, the better the cleaning performance. What is measured here is the L value = brightness, the higher the brighter. The cleaning performance is given in % based on the initial variant, corrected by the performance of the detergent without protease. Performance in the washing test at 20°C Performance in the washing test at 40°C Benchmark P1 Benchmark P1 CS-038 100% 87% 100% 120% C-05 100% 134% 100% 164% H-MR-B 100% 110% 100% 98%

A protease was used as a benchmark, which differs from the protease P1 according to the invention only by a change at position 99, such that the benchmark protease has the amino acid E (Glu) at position 99, while protease P1 has the amino acid R (Arg; as in wild type). Surprisingly, the back mutation at position 99 leads to improved cleaning performance.

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (14)

Protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence specified in SEQ ID NO:1 over its total length to at least 70% and increasingly preferably to at least 71%, 72%, 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91 %, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5% and 98% identical, the protease, based on the numbering according to SEQ ID NO:1, having the amino acid substitutions S3T, V4I and V199I at (i) the positions corresponding to positions 3, 4 and 199 and (ii) at least one, and increasingly preferably two, three, four, five or six, of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256 correspond to at least one and increasingly preferably two, three, four, five or six further amino acid substitution (s) selected from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G , Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181D, F183R , Q185R, Q200L, Y203W, A209K and L256E, existing group are selected. Protease after Claim 1 , wherein the protease (iii) does not have the amino acid Glu (E) or Asp (D) at the position corresponding to position 99. Protease after Claim 1 or 2 , wherein the protease (iii) has the amino acid Arg (R) at the position corresponding to position 99. Protease according to one of the preceding claims, wherein the protease has one of the following amino acid substitution variants, each based on the numbering according to SEQ ID NO:1: (a) S3T-V4I-V199I-Q200L-Y203W; (b) S3T-V4I-V199I-Q200L-Y203W-S154D-L256E; (c) S3T-V41-V199I-Q200L-Y203W-A209K-S154D-L256E; (d) S3T-V4I-V199I-Q200I-Y203Y-A209W-S154D-L256E; (e) S3T-V4I-V199I-Q200I-Y203W-A209W-S154D-L256E; (f) S3T-V4I-V199I-Q200L-Y203W-A209W-S154D-L256E; (g) S3T-V4I-V199I-Q200I-Y203W-A209K-S154D-L256E; (h) S3T-V4I-V199I-Q200I-Y203Y-A209K-S154D-L256E; and (i) S3T-V4I-V199I-Q200L-Y203W-A209K-S1S4D-L2S6E. Protease, characterized in that (a) it consists of a protease according to one of the Claims 1 until 4 as a starting molecule is available through single or multiple conservative amino acid substitution, whereby the protease, based on the numbering according to SEQ ID NO:1, has the amino acid substitutions S3T, V4I at (i) the positions that correspond to positions 3, 4 and 199 and V199I and (ii) at least one of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, at least one other Amino acid substitution selected from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W , A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181 D, F183R, Q185R, Q200L, Y203W, A209K and L256E. (b) them from a protease according to one of the Claims 1 until 4 as a starting molecule is obtainable by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265 in length , 266, 267, 268 or 269 contiguous amino acids correspond to the starting molecule, the protease, in each case based on the numbering according to SEQ ID NO:1, making the amino acid substitutions at (i) the positions that correspond to positions 3, 4 and 199 S3T, V4I and V199I and (ii) at least one of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, at least one further amino acid substitution selected from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/ V/W, A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E. A method for producing a protease, comprising introducing (a) the amino acid substitutions S3T, V4I and V199I at the positions corresponding to positions 3, 4 and 199 based on the numbering according to SEQ ID NO:1, and (b) at least one amino acid substitution in at least one of the positions related to the number merization according to SEQ ID NO:1 correspond to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256, selected from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/S/T, Y203K/V/W, A209K/W, N212S/T and L256D /E/Q, preferably S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E, into a starting molecule that has an amino acid sequence that has at least 70% sequence identity with the amino acid sequence specified in SEQ ID NO:1 above Total length. Procedure according to Claim 6 , further comprising one or more of the following process steps: (a) introducing a single or multiple conservative amino acid substitution, the protease, in each case based on the numbering according to SEQ ID NO:1, at (i) the positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I and (ii) at least one of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209, 212 or 256 correspond to at least one further amino acid substitution selected from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L /S/T, Y203K/V/W, A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E; (b) changing the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease comprises an amino acid sequence which is at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262 in length , 263, 264, 265, 266, 267, 268 or 269 contiguous amino acids correspond to the starting molecule, the protease, in each case based on the numbering according to SEQ ID NO:1, at (i) the positions corresponding to positions 3, 4 and 199, the amino acid substitutions S3T, V4I and V199I and (ii) at least one of the positions corresponding to positions 74, 136, 143, 154, 160, 161, 163, 171, 181, 183, 185, 200, 203, 209 , 212 or 256, at least one further amino acid substitution selected from N74D/E/Q, A136Q, R143L/W/Y, S154D/Q, S160G, Y161T, A163G, V171 L, A181D, F183R, Q185R, Q200A/L/ S/T, Y203K/V/W, A209K/W, N212S/T and L256D/E/Q, preferably S154D, S160G, A181D, F183R, Q185R, Q200L, Y203W, A209K and L256E. Nucleic acid encoding a protease according to one of the Claims 1 until 5 or coding for one according to a method of Claims 6 until 7 obtained protease. Vector containing a nucleic acid Claim 8 , in particular a cloning vector or an expression vector. Non-human host cell that secretes a nucleic acid Claim 8 or a vector Claim 9 contains, or which contains a protease according to one of the Claims 1 until 5 includes, or one according to a method of Claims 6 until 7 obtained protease includes, in particular one that secretes the protease into the medium surrounding the host cell. Method for producing a protease comprising a) cultivating a host cell according to Claim 10 and b) isolating the protease from the culture medium or from the host cell. Agent, in particular a detergent or cleaning agent, characterized in that it contains at least one protease according to one of the Claims 1 until 5 or one according to a procedure of Claims 6 until 7 contains the protease obtained, the agent being essentially free of boron-containing compounds. Process for cleaning textiles or hard surfaces, characterized in that in at least one process step an agent Claim 12 is applied. Use of a protease according to one of the Claims 1 until 5 or one according to a procedure of Claims 6 until 7 obtained protease in a detergent or cleaning agent for removing peptide or protein-containing stains.