EP1124987A2

EP1124987A2 - Methods for screening protease variants for use in detergent compositions

Info

Publication number: EP1124987A2
Application number: EP99971044A
Authority: EP
Inventors: Chanchal Kumar Ghosh; Phillip Frederick Brode, Iii; Deborah Susan Rauch; Michael Stanford Showell
Original assignee: Procter and Gamble Co
Current assignee: Procter and Gamble Co
Priority date: 1998-10-23
Filing date: 1999-10-22
Publication date: 2001-08-22
Also published as: AR020951A1; JP2003512012A; AU1223000A; WO2000024924A3; WO2000024924A2

Abstract

Methods for screening protease variants for use in detergent systems are provided. Such methods relate to the net charge of the substitutions of the protease variants and the adsorption properties of the protease variants. Also included are detergent compositions comprising protease variants screened by such methods.

Description

METHODS FOR SCREENING PROTEASE VARIANTS FOR USE IN DETERGENT COMPOSITIONS

FIELD OF THE INVENTION

The present invention relates to methods for screening protease variants, particularly multiply-substituted protease variants, for selecting protease variants that provide effective cleaning performance in detergent compositions.

BACKGROUND OF THE INVENTION

Enzymes, particularly protease enzymes, are becoming common components of detergent compositions for their ability to clean.

However, researchers are continuing to attempt to develop better performing protease enzymes in response to consumers' needs.

A common issue in the screening protease variants for use in detergent compositions and/or detergent systems is the variety of wash conditions including a variety of detergent compositions that protease variants may be used in. For example, detergent systems (detergent components present in a wash water) used in different geographical areas have different concentrations of their respective detergent components present in the wash water. For example, a European detergent system typically has about 4500 ppm to about 5000 ppm of detergent components present in the wash water. While a Japanese detergent system typically has about 667 ppm of detergent component present in the wash water. Whereas in North America, particularly the United States, detergent systems typically have about 975 ppm of detergent components present in the wash water.

In addition to the detergent concentration issues in the various geographies, the various geographical areas also differ regarding the most prevalent types of stains, the typical temperature at which a wash occurs, the water hardness and others.

In light of the complexity of detergent systems throughout the world, a screening method for selecting detergent components, particularly enzymes, more particularly protease enzymes is needed.

ConventionaJ methods for screening and selecting protease enzymes, particularly protease variants that perform effectively in detergent compositions included trial and error. Such methods were time consuming and costly, and offered very little predictability. Accordingly, there is a need for a method for screening protease variants that provided more predictability, and was less time consuming and costly.

SUMMARY OF THE INVENTION

The present invention meets the needs discussed above by providing methods for screening protease variants, especially multiply-substituted protease variants, to determine their chances of performing effectively under various wash conditions (i.e., low, medium and/or high detergent concentration systems).

It has been surprisingly found that screening methods based upon net charge of substitutions made in protease variants and/or adsorption activity of protease variants provide an effective, time-efficient, and relatively inexpensive process for screening and selecting protease variants that perform well in various detergent systems.

In one aspect of the present invention, a method for screening protease variants for use in detergent systems comprising: a) providing one or more protease variants having one or more amino acid residue substitutions; and b) calculating the net charge of said one or more substitutions of said protease variants, and c) optionally, selecting one or more protease variants for use in low, medium and/or high detergent concentration systems based upon the net charge of the one or more substitutions of the protease variant, is provided.

In another aspect of the present invention, a detergent composition comprising a protease variant selected by the screening method discussed above, and a surfactant.

In yet another aspect of the present invention, a method for screening protease variants for use in detergent systems comprising: a) providing one or more protease variants having one or more amino acid residue substitutions; b) adding said one or more protease variants to a detergent system; and c) determining the adsoφtion properties of said one or more protease variants in said detergent system to a surface, preferably a nylon membrane, wherein step c) optionally further comprises: i) measuring the initial quantity of enzyme activity of said one or more protease variants; ii) filtering said one or more protease variants through a nylon membrane; iii) measuring the quantity of enzyme activity of said one or more protease variants filtered through said nylon membrane; iv) determining the quantity of enzyme activity loss of said one or more protease variants adsorbed onto said nylon membrane by determining the difference in quantities between step i) and step iii); and v) converting the quantity of enzyme activity loss to percent of enzyme adsorbed on said nylon membrane; and vi) optionally, selecting said one or more protease variants exhibiting a percent of enzyme adsorbed on said membrane in the range of about 16 %, preferably from about 20% to about 65%, more preferably to about 60% for use in said detergent system, is provided.

In still yet another aspect of the present invention, a detergent composition comprising a protease variant selected from the screening method described above, and a surfactant.

These and other objects, features and advantages will be clear from the following detailed description, examples and appended claims.

Accordingly, the present invention provides effective methods for screening protease variants for use in detergent compositions and/or detergent systems; and efficient methods for screening protease variants for use in detergent compositions and/or detergent systems.

All percentages, ratios and proportions herein are on a weight basis unless otherwise indicated. All documents cited herein are hereby incoφorated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1 A-C depict the DNA and amino acid sequence for Bacillus amyloliquefaciens subtilisin and a partial restriction map of this gene.

Fig. 2 depicts the conserved amino acid residues among subtilisins from Bacillus amyloliquefaciens (BPN)' and Bacillus lentus (wild-type).

Figs. 3A and 3B depict the amino acid sequence of four subtilisins. The top line represents the amino acid sequence of subtilisin from Bacillus amyloliquefaciens subtilisin (also sometimes referred to as subtilisin BPN'). The second line depicts the amino acid sequence of subtilisin from Bacillus subtilis. The third line depicts the amino acid sequence of subtilisin from B. licheniformis. The fourth line depicts the amino acid sequence of subtilisin from Bacillus lentus (also referred to as subtilisin 309 in PCT WO89/06276). The symbol * denotes the absence of specific amino acid residues as compared to subtilisin BPN'.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods for screening protease variants for use in the various detergent systems that exist throughout the world.

As discussed above, Japanese detergent systems are considered low detergent concentration systems since less than 800 ppm of detergent components are present in the wash water. European detergent systems are considered high detergent concentration systems since greater than 2000 ppm of detergent components are present in the wash water. North American, especially the United States, detergent systems are considered medium detergent concentration systems since between about 800 ppm and 2000 ppm of detergent components are present in the wash water.

Latin American detergent systems are typically high suds phosphate builder detergent systems and generally fall within the medium to high detergent concentration systems ranges. For example, Brazil typically has about 1500 ppm of detergent components present in the wash water. Whereas, other Latin American countries may have detergent systems that include as high as 6000 ppm of detergent components present in the wash water.

The concentration of the typical detergent systems discussed above are determined empirically. For example, in the U.S., a typical washing machine holds a volume of about 64.4 1 of wash solution. Accordingly, in order to obtain a concentration of about 975 ppm of detergent within the wash solution, about 62.79 g of detergent composition must be added to the 64.4 1 of wash solution. This amount is the typical amount measured into the wash water by the consumer using the measuring cup provided with the detergent.

Even in light of these complexities that exist in detergent systems throughout the world, it has been found that methods for screening protease enzymes, particularly protease variants, more particularly multiply-substituted protease variants based upon net charge of amino acid residue substitutions present in the protease variants and/or the adsoφtion activity of the protease variants are effective methods of identifying and/or selecting protease variants that provide effective performance in the various detergent systems. Methods for Screening

A. Net Charge Method: A preferred embodiment of the present invention is a method for screening protease variants for use in detergent systems comprising: a) providing one or more protease variants, preferably selected from the protease variants described herein, having one or more amino acid residue substitutions; and b) calculating the net charge of said one or more substitutions of said protease variants.

Preferably, the one or more protease variants is included in a detergent system, more preferably a detergent system described herein.

The calculating step preferably is performed under conditions, preferably detergent system conditions, comprising a pH in the range of from about 7 to about 12, more preferably from about 10 to about 11.

Preferably, the net charge of the one or more substitutions of the protease variant can be calculated relative to the protease variant's precursor protease. More preferably, the net charge of the one or more substitutions of the protease variant is calculated relative to Subtilisin 309 (See PCT WO 89/06276).

The method preferably further comprises the step of: selecting said one or more protease variants having a net positive or neutral charge of said substitutions for use in high detergent concentration systems as described herein.

Alternatively, the method preferably further comprises the step of: selecting said one or more protease variants having a net negative or neutral charge of said substitutions for use in low detergent concentration systems as described herein.

Yet another preferred embodiment includes the method for screening as described above further comprising the step of: selecting said one or more protease variants having a net positive, negative or neutral charge of said substitutions for use in medium detergent concentration systems.

Still yet another preferred embodiment includes the method for screening as described above further comprising the step of: selecting said one or more protease variants having a net neutral charge of said substitutions for use in low, medium and high detergent concentration systems.

In light of the foregoing, it is evident that the detergent concentration, in other words, the amount of detergent components present in a wash water is a factor in determining what protease variants perform effectively in a given detergent system.

Under high detergent component concentration conditions, particularly high surfactant concentration conditions, soil surfaces are covered with a layer of negative charge due to surfactant adsoφtion. Positively charged protease variants are able to effectively compete for soil surfaces that are covered with such a negative charge. Net, under high detergent concentration conditions, positively charged variants adsorb, and are mobile on the surface. As a result of this, positively charged variants provide superior cleaning benefits as compared to negatively charged variants. Negatively charged variants on the other hand are not adsorbed, and move away from the soil surfaces covered with a negatively charged layer of surfactant.

Under low detergent component concentration conditions, particularly low surfactant concentration conditions, the positively charged variants comprise a net positive charge that limits mobility of the protease variant so it cannot sample as many sites as a less positively charged protease variant. As a result, positively charged variants under low detergent concentration conditions provides less effective cleaning than negatively charged variants. Negatively charged variants adsorb to the soil surface less but are more mobile, and therefore, provide superior cleaning benefits as compared to positively charged variants. Net, under low detergent concentration conditions, positively charged variants provide more adsoφtion, but less mobility, whereas, negatively charged variants provide less adsoφtion, but more mobility. The more mobile negatively charged variants can sample more sites.

In light of the foregoing, it is evident that the balancing of adsoφtion and mobility is critical for obtaining superior cleaning performance in a given detergent concentration.

Another preferred embodiment of the present invention is a detergent composition comprising a protease variant selected by the net charge method described above. Preferably such a detergent composition comprises one or more of the following adjunct materials: surfactants, builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilizing systems, chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides, color speckles, silvercare, anti-tarnish and/or anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments and pH control agents as described in U.S. Patent Nos. 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101.

B. Adsorption Method: Another preferred embodiment of the present invention is a method for screening protease variants for use in detergent systems comprising: a) providing one or more protease variants, preferably selected from the protease variants described herein, having one or more amino acid residue substitutions; b) adding said one or more protease variants to a detergent system; and c) determining the adsoφtion properties of said one or more protease variants in said detergent system to a surface.

The detergent system is preferably selected from the group consisting of: low, medium and/or high detergent concentration systems as described herein.

The detergent system preferably has a pH in the range of from about 7 to about 12, more preferably from about 10 to about 11.

The surface used in the adsoφtion step (step c) can be any suitable surface known in the art. Preferably the surface is a nylon membrane, more preferably a nylon 66 membrane. Further, it is desirable that the nylon membrane include a hydrophilic surface, preferably comprising about 50% amines and about 50% carboxyl groups. An example of a suitable nylon membrane for use in accordance with the present invention is a BIODYNE®A membrane, which is commercially available from Nalge Nunc International Coφoration of Naperville, IL (formerly Pall Coφoration of Glen Cove, NY).

Preferably, step c) determining the adsoφtion properties further comprises: i) measuring the initial quantity of enzyme activity of said one or more protease variants, preferably by using a pNA assay as described in Brode III, P.F., et al. (1996), Biochemistry, 35:3162-3169; ii) filtering said one or more protease variants through a nylon membrane,

(typically and preferably protease variant samples are contained within one or more wells of a well-plate, such as a 96-well plate, as those of ordinary skill in the art would appreciate); iii) measuring the quantity of enzyme activity of said one or more protease variants filtered through said nylon membrane; iv) determining the quantity of enzyme activity loss of said one or more protease variants adsorbed onto said nylon membrane by determining the difference in quantities between step i) and step iii); and v) converting the quantity of enzyme activity loss to percent of enzyme adsorbed on said nylon membrane.

The adsoφtion method preferably further comprises the step of: selecting said one or more protease variants exhibiting a percent of enzyme adsorbed on said membrane in the range of about 16 %, preferably 20% to about 65%, preferably to about 60% for use in said detergent system. This range of adsorption activity of the protease variant is unexpected based upon the fact that those skilled in the art would expect that enzymes adsorb less provide better cleaning performance. Therefore, one skilled in the art would have expected that the cleaning performance of an enzyme increases as the adsorption activity of the enzyme decreases as described in WO 95/07991, WO 95/30010, WO 95/30011, WO 96/28556, WO 96/28557, WO 96/28558, and WO 96/28566.

Contrary to the teachings of the prior art, based upon the methods of the present invention, it has suφrisingly been found that there is an optimum range of adsoφtion activity of protease variants that provide the best cleaning performance. Therefore, the teachings of the prior art that cleaning performance of an enzyme increases as the adsoφtion activity of the enzyme decreases (especially in the case of protease variants) does not hold true. Because the methods of the present invention show that there is an optimum range of adsoφtion activity of a protease variant that is not too low and is not too high.

Yet another preferred embodiment of the present invention is a detergent composition comprising one or more protease variants selected according to the adsoφtion method described above. Preferably such a detergent composition comprises one or more of the following adjunct materials: surfactants, builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilizing systems, chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioners, hydrolyzable surfactants, perservatives, anti- oxidants, anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides, color speckles, silvercare, anti-tarnish and/or anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments and pH control agents as described in U.S. Patent Nos. 5,705,464, 5,710,1 15, 5,698,504, 5,695,679, 5,686,014 and 5,646,101.

Proteases - Proteases are carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides. As used herein, "protease" means a naturally occurring protease or recombinant protease. Naturally-occurring proteases include α-aminoacylpeptide hydrolase, peptidylamino acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiol proteinase, carboxylproteinase and metalloproteinase. Serine, metallo, thiol and acid protease are included, as well as endo and exo-proteases.

The present invention includes protease enzymes which are non-naturally occurring carbonyl hydrolase variants (protease variants) having a different proteolytic activity, stability, substrate specificity, pH profile and/or performance characteristic as compared to the precursor carbonyl hydrolase from which the amino acid sequence of the variant is derived. Specifically, such protease variants have an amino acid sequence not found in nature, which is derived by replacement of a plurality of amino acid residues of a precursor protease with different amino acids. The precursor protease may be a naturally-occurring protease or recombinant protease. As stated earlier, the protease variants are designed to have trypsin-like specificity and preferably also be bleach stable.

The protease variants useful herein encompass the substitution of any of the nineteen naturally occurring L-amino acids at the designated amino acid residue positions. Such substitutions can be made in any precursor subtilisin (procaryotic, eucaryotic, mammalian, etc.). Throughout this application reference is made to various amino acids by way of common one- and three-letter codes. Such codes are identified in Dale, M.W. (1989), Molecular Genetics of Bacteria, John Wiley & Sons, Ltd., Appendix B.

The protease variants useful herein are preferably derived from a Bacillus subtilisin. More preferably, the protease variants are derived from Bacillus lentus subtilisin and/or subtilisin 309.

Carbonyl Hydrolases - Carbonyl hydrolases are protease enzymes which hydrolyze compounds containing

O

C-X bonds in which X is oxygen or nitrogen. They include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. Naturally-occurring carbonyl hydrolases principally include hydrolases, e.g., peptide hydrolases such as subtilisins or metalloproteases. Peptide hydrolases include -aminoacylpeptide hydrolase, peptidylamino acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiol proteinase, carboxylproteinase and metalloproteinase. Serine, metallo, thiol and acid protease's are included, as well as endo and exo-proteases.

Subtilisins - Subtilisins are bacterial or fungal proteases which generally act to cleave peptide bonds of proteins or peptides. As used herein, "subtilisin" means a naturally-occurring subtilisin or a recombinant subtilisin. A series of naturally-occurring subtilisins is known to be produced and often secreted by various microbial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity. This class of serine proteases share a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. The subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine. In the subtilisin related proteases the relative order of these amino acids, reading from amino to carboxy terminus, is aspartate-histidine-serine. In the chymotrypsin related proteases, the relative order, however, is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases. Examples include, but are not limited to, the subtilisins identified in Fig. 3 herein. Generally, and for puφoses of the present invention, numbering of the amino acids in proteases corresponds to the numbers assigned to the mature Bacillus amyloliquefaciens subtilisin sequence presented in Fig. 1.

Protease Variants - A "protease variant" has an amino acid sequence which is derived from the amino acid sequence of a "precursor protease." The precursor proteases include naturally-occurring proteases and recombinant proteases. The amino acid sequence of the protease variant is "derived" from the precursor protease amino acid sequence by substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the "precursor DNA sequence" which encodes the amino acid sequence of the precursor protease rather than manipulation of the precursor protease enzyme per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein, as well as methods know to those skilled in the art (see, for example, EP 0 328 299, WO 89/06279 and the U.S. patents and applications already referenced herein).

In a preferred embodiment, the protease variants which are protease enzymes useful in the methods of the present invention comprise protease variants including a substitution of an amino acid residue with another naturally occurring amino acid residue at an amino acid residue position corresponding to position 103 of Bacillus amyloliquefaciens subtilisin in combination with a substitution of an amino acid residue with another naturally occurring amino acid residue at one or more amino acid residue positions corresponding to positions 1, 3, 4, 8, 9, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 1 11, 1 14, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin; wherein when said protease variant includes a substitution of amino acid residues at positions corresponding to positions 103 and 76, there is also a subtitution of an amino acid residue at one or more amino acid residue positions other than amino acid residue positions corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 or 274 of Bacillus amyloliquefaciens subtilisin; and one or more cleaning adjunct materials.

While any combination of the above listed amino acid substitutions may be employed, the preferred protease variant enzymes useful for the present invention comprise the substitution, deletion or insertion of amino acid residues in the following combinations:

(1) a protease variant including substitutions of the amino acid residues at position 103 and at one or more of the following positions 236 and 245;

(2) a protease variant including substitutions of the amino acid residues at positions 103 and 236 and at one or more of the following positions: 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 21 1, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270 and 275;

(3) a protease variant including substitutions of the amino acid residues at positions 103 and 245 and at one or more of the following positions: 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 170, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 222, 230, 232, 248, 252, 257, 260, 261, 270 and 275; and

(4) a protease variant including substitutions of the amino acid residues at positions 103, 236 and 245 and at one or more of the following positions: 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 243, 248, 252, 257, 260, 270 and 275.

A more preferred protease variant useful in the cleaning compositions of the present invention include a substitution set (one substitution set per row in the following Table I) selected from the group consisting of: Table I

An even more preferred protease variant useful in the cleaning compositions of the present invention include a substitution set (one substitution set per row in the following Table II) selected from the group consisting of:

Table II

Recombinant Proteases/Recombinant Subtilisins - A "recombinant protease" or "recombinant subtilisin" refers to a protease or subtilisin in which the DNA sequence encoding the naturally-occurring protease or subtilisin, respectively, is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the protease or subtilisin amino acid sequence. Suitable modification methods are disclosed herein, and in U.S. Patent Nos. RE 34,606, 5,204,015 and 5,185,258.

Non-Human Proteases/Non-Human Subtilisins - "Non-human proteases" or "non-human subtilisins" and the DNA encoding them may be obtained from many procaryotic and eucaryotic organisms. Suitable examples of procaryotic organisms include gram negative organisms such as E. coli or Pseudomonas and gram positive bacteria such as Micrococcus or Bacillus. Examples of eucaryotic organisms from which carbonyl hydrolase and their genes may be obtained include yeast such as Saccharomyces cerevisiae, fungi such as Aspergillus sp. and non- human mammalian sources such as, for example, bovine sp. from which the gene encoding the protease chymosin or subtilisin chymosin can be obtained. A series of proteases and/or subtilisins can be obtained from various related species which have amino acid sequences which are not entirely homologous between the members of that series but which nevertheless exhibit the same or similar type of biological activity. Thus, non-human protease or non-human subtilisin as used herein have a functional definition which refers to proteases or subtilisins, respectively, which are associated, directly or indirectly, with procaryotic and eucaryotic sources.

Variant DNA Sequences - Variant DNA sequences encoding such protease or subtilisin variants are derived from a precursor DNA sequence which encodes a naturally-occurring or recombinant precursor enzyme. The variant DNA sequences are derived by modifying the precursor DNA sequence to encode the substitution of one or more specific amino acid residues encoded by the precursor DNA sequence corresponding to positions 103 in combination with one or more of the following positions 1, 3, 4, 8, 9, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin; wherein when said protease variant includes a substitution of amino acid residues at positions corresponding to positions 103 and 76, there is also a subtitution of an amino acid residue at one or more amino acid residue positions other than amino acid residue positions corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 or 274 of Bacillus amyloliquefaciens subtilisin. Although the amino acid residues identified for modification herein are identified according to the numbering applicable to B. amyloliquefaciens (which has become the conventional method for identifying residue positions in all subtilisins), the preferred precursor DNA sequence useful for the present invention is the DNA sequence of Bacillus lentus as shown in Fig. 3.

In a preferred embodiment, these variant DNA sequences encode the substitution, insertion or deletion of the amino acid residue corresponding to position 103 of Bacillus amyloliquefaciens subtilisin in combination with one or more additional amino acid residues corresponding to positions 1, 3, 4, 8, 9, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101 , 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin; wherein when said protease variant includes a substitution of amino acid residues at positions corresponding to positions 103 and 76, there is also a subtitution of an amino acid residue at one or more amino acid residue positions other than amino acid residue positions corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 or 274 of Bacillus amyloliquefaciens subtilisin. More preferably, these variant DNA sequences encode the protease variants described herein.

Although the amino acid residues identified for modification herein are identified according to the numbering applicable to B. amyloliquefaciens (which has become the conventional method for identifying residue positions in all subtilisins), the preferred precursor DNA sequences useful for the present invention is the DNA sequence of Bacillus lentus as shown in Fig. 3.

These recombinant DNA sequences encode protease variants having a novel amino acid sequence and, in general, at least one property which is substantially different from the same property of the enzyme encoded by the precursor protease DNA sequence. Such properties include proteolytic activity, substrate specificity, stability, altered pH profile and/or enhanced performance characteristics.

Specific substitutions corresponding to positions 103 in combination with one or more of the following positions 1, 3, 4, 8, 9, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin; wherein when said protease variant includes a substitution of amino acid residues at positions corresponding to positions 103 and 76, there is also a subtitution of an amino acid residue at one or more amino acid residue positions other than amino acid residue positions corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 or 274 wherein the numbered positions correspond to the naturally-occurring subtilisin from Bacillus amyloliquefaciens or to equivalent amino acid residues in other carbonyl hydrolases or subtilisins (such as Bacillus lentus subtilisin) are described herein. Further, specific substitutions corresponding to one or more of the following positions 62, 212, 230, 232, 252 and 257 wherein the numbered positions correspond to the naturally-occurring subtilisin from Bacillus amyloliquefaciens or to equivalent amino acid residues in other carbonyl hydrolases or subtilisins (such as Bacillus lentus subtilisin) are described herein. These amino acid position numbers refer to those assigned to the mature Bacillus amyloliquefaciens subtilisin sequence presented in Fig. 1. The present invention, however, is not limited to the use of mutation of this particular subtilisin but extends to precursor proteases containing amino acid residues at positions which are "equivalent" to the particular identified residues in Bacillus amyloliquefaciens subtilisin. In a preferred embodiment of the present invention, the precursor protease is Bacillus lentus subtilisin and the substitutions, deletions or insertions are made at the equivalent amino acid residue in B. lentus corresponding to those listed above.

A residue (amino acid) of a precursor protease is equivalent to a residue of Bacillus amyloliquefaciens subtilisin if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in Bacillus amyloliquefaciens subtilisin (i.e., having the same or similar functional capacity to combine, react or interact chemically).

In order to establish homology to primary structure, the amino acid sequence of a precursor protease is directly compared to the Bacillus amyloliquefaciens subtilisin primary sequence and particularly to a set of residues known to be invariant in subtilisins for which sequence is known. For example, Fig. 2 herein shows the conserved residues as between B. amyloliquefaciens subtilisin and B. lentus subtilisin. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of Bacillus amyloliquefaciens subtilisin are defined. Alignment of conserved residues preferably should conserve 100%) of such residues. However, alignment of greater than 75% or as little as 50%) of conserved residues is also adequate to define equivalent residues. Conservation of the catalytic triad, Asp32/His64/Ser221 should be maintained. For example, in Fig. 3 the amino acid sequence of subtilisin from Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis (carlsbergensis) and Bacillus lentus are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved residues contained in each sequence. These conserved residues (as between BPN' and B. lentus) are identified in Fig. 2.

These conserved residues, thus, may be used to define the corresponding equivalent amino acid residues of Bacillus lentus (PCT Publication No. WO89/06279 published July 13, 1989), the preferred protease precursor enzyme herein, or the subtilisin referred to as PB92 (EP 0 328 299), which is highly homologous to the preferred Bacillus lentus subtilisin. The amino acid sequences of certain of these subtilisins are aligned in Figs. 3 A and 3B with the sequence of Bacillus amyloliquefaciens subtilisin to produce the maximum homology of conserved residues. As can be seen, there are a number of deletion in the sequence of Bacillus lentus as compared to Bacillus amyloliquefaciens subtilisin. Thus, for example, the equivalent amino acid for Vail 65 in Bacillus amyloliquefaciens subtilisin in the other subtilisins is isoleucine for B. lentus and B. licheniformis. Thus, for example, the amino acid at position +76 is asparagine (N) in both B. amyloliquefaciens and B. lentus subtilisins. In the protease variants of the invention, however, the amino acid equivalent to +76 in Bacillus amyloliquefaciens subtilisin is substituted with aspartate (D). The abbreviations and one letter codes for all amino acids in the present invention conform to the Patentin User Manual (GenBank, Mountain View, CA) 1990, p. 101.

"Equivalent residues" may also be defined by determining homology at the level of tertiary structure for a precursor protease whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the precursor protease and Bacillus amyloliquefaciens subtilisin (N on N, CA on CA, C on C and O on O) are within 0.13nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non- hydrogen protein atoms of the protease in question to the Bacillus amyloliquefaciens subtilisin. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.

Σ_h\ Fo(h)\-\ Fc(h)\

Rfactor =

Σ_h\ Fo(h)\

Equivalent residues which are functionally analogues to a specific residue of Bacillus amyloliquefaciens subtilisin are defined as those amino acids of the precursor protease which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the Bacillus amyloliquefaciens subtilisin. Further, they are those residues of the precursor protease (for which a tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the extent that, although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two fo the side chain atoms of the residue lie with 0.13nm of the corresponding side chain atoms of Bacillus amyloliquefaciens subtilisin. The coordinates of the three dimensional structure of Bacillus amyloliquefaciens subtilisin are set forth in EPO Publication No. 0 251 446 (equivalent to US Patent 5,182,204, the disclosure of which is incoφorated herein by reference) and can be used as outlined above to determine equivalent residues on the level of tertiary structure.

Some of the residues identified for substitution, insertion or deletion are conserved residues whereas others are not. In the case of residues which are not conserved, the replacement of one or more amino acids is limited to substitutions which produce a variant which has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such replacements should not result in natural-occurring sequence. The protease variants of the present invention include the mature forms of protease variants, as well as the pro- and pre-pro-forms of such protease variants. The prepro-forms are the preferred construction since this facilitates the expression, secretion and maturation of the protease variants.

"Prosequence" refers to a sequence of amino acids bound to the N-terminal portion of the mature form of a protease which when removed results in the appearance of the "mature" form of the protease. Many proteolytic enzymes are found in nature as translational proenzyme products and, in the absence of post-translational processing, are expressed in this fashion. A preferred prosequence for producing protease variants is the putative prosequence of Bacillus amyloliquefaciens subtilisin, although other protease prosequences may be used.

A "signal sequence" or "presequence" refers to any sequence of amino acids bound to the N'terminal portion of a protease or to the N-terminal portion of a proprotease which may participate in the secretion of the mature or pro forms of the protease. This definition of signal sequence is a functional one, meant to include all those amino sequences encoded by the N- terminal portion of the protease gene which participate in the effectuation of the secretion of protease under native conditions. The present invention utilizes such sequences to effect the secretion of the protease variants as defined here. One possible signal sequence comprises the first seven amino acid residues of the signal sequence from Bacillus subtilis subtilisin fused to the remainder of the signal sequence of the subtilisin from Bacillus lentus JATCC 21536). A "prepro" form of a protease variant consists of the mature form of the protease having a prosequence operably linked to the amino terminus of the protease and a "pre" or "signal" sequence operably linked to the amino terminus of the prosequence.

"Expression vector" refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently or the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid" and "vector" are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.

The "host cells" used in the present invention generally are procaryotic or eucaryotic hosts which preferably have been manipulated by the methods disclosed in US Patent RE 34,606 to render them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing protease is the Bacillus strain BG2036 which is deficient in enzymatically active neutral protease and alkaline protease (subtilisin). The construction of strain BG2036 is described in detail in US Patent 5,264,366. Other host cells for expressing protease include Bacillus subtilis 168 (also described in US Patent RE 34,606 and US Patent 5,264,366, the disclosure of which are incoφorated herein by reference), as well as any suitable Bacillus strain such as B. licheniformis, B. lentus,etc).

Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the protease variants or expressing the desired protease variant. In the case of vectors which encode the pre- or prepro-form of the protease variant, such variants, when expressed, are typically secreted from the host cell in to the host cell medium.

"Operably linked, "when describing the relationship between two DNA regions, simply means that they are functionally related to each other. For example, a prosequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. The genes encoding the naturally-occurring precursor protease may be obtained in accord with the general methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes having putative sequences encoding regions of the protease of interest, preparing genomic libraries from organisms expressing the protease, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced.

The cloned protease is then used to transform a host cell in order to express the protease. The protease gene is then ligated into a high copy number plasmid. This plasmid replicates in hosts in the sense that it contains the well-known elements necessary for plasmid replication: a promote operably linked to the gene in question (which may be supplied as the gene's own homologous promoter if it is recognized, i.e. transcribed by the host), a transcription termination and polyadenylation region (necessary for stability of the mRNA transcribed by the host from the protease gene in certain eucaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the protease gene and, desirably, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid-infected host cells by growth in antibiotic-containing media. High copy number plasmids also contain an origin of replication for the host, thereby enabling large numbers of plasmids to be generated in the cytoplasm without chromosomal limitation. However, it is within the scope herein to integrate multiple copies of the protease gene into host genome. This is facilitated by procaryotic and eucaryotic organisms which are particularly susceptible to homologous recombination. The gene can be a natural B. lentus gene. Alternatively, a synthetic gene encoding a naturally-occurring or mutant precursor protease may be produced. In such an approach, the DNA and/or amino acid sequence of the precursor protease is determined. Multiple, overlapping synthetic single-stranded DNA fragments are thereafter synthesized, which upon hybridization and ligation produce a synthetic DNA enclding the precursor protease. An example of synthetic gene construction is set forth in Example 3 of US Patent 5,204,105, the disclosure of which is incoφorated herein by reference.

Once the naturally-occurring or synthetic precursor protease gene has been cloned, a number of modifications are undertaken to enhance the use of the gene beyond synthesis of the naturally-occurring precursor protease. Such modifications include the production of recombinant proteases as disclosed in US Patent RE 34,606 and EPO Publication No. 0 251 446 and the production of protease variants described herein.

The following cassette mutagenesis method may be used to facilitate the construction of the proteases variants of the present invention, although other methods may be used. First, the naturally-occurring gene encoding the protease is obtained and sequenced in whole or in part. Then the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded enzyme. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which, when expressed will encode various mutants. Such restriction sites are preferably unique sites within the protease gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the protease gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by M13 primer extension in accord with generally known methods. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site if available, the above method need be used only in connection with the flanking region which does not contain a site.

Once the naturally-occurring DNA or synthetic DNA is cloned, the restriction sites flanking the positions to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites. As used herein, proteolytic activity is defined as the rate of hydrolysis of peptide bonds per milligram of active enzyme. Many well known procedures exist for measuring proteolytic activity (K. M. Kalisz, "Microbial Proteinases," Advances in Biochemical Engineering/Biotechnology, A. Fiechter ed., 1988). In addition to or as an alternative to modified proteolytic activity, the variant enzymes of the present invention may have other modified properties such as K_m, k_cat, k_cat/K_m ratio and/or modified substrate specifically and/or modified pH activity profile. These enzymes can be tailored for the particular substrate which is anticipated to be present, for example, in the preparation of peptides or for hydrolytic processes such as laundry uses.

In one aspect of the invention, the objective is to secure a variant protease having altered proteolytic activity as compared to the precursor protease, since increasing such activity (numerically larger) enables the use of the enzyme to more efficiently act on a target substrate. Also of interest are variant enzymes having altered thermal stability and/or altered substrate specificity as compared to the precursor. In some instances, lower proteolytic activity may be desirable, for example a decrease in proteolytic activity would be useful where the synthetic activity of the proteases is desired (as for synthesizing peptides). One may wish to decrease this proteolytic activity, which is capable of destroying the product of such synthesis. Conversely, in some instances it may be desirable to increase the proteolytic activity of the variant enzyme versus its precursor. Additionally, increases or decreases (alteration) of the stability of the variant, whether alkaline or thermal stability, may be desirable. Increases or decreases in k_cat, K_m or K_ca/K_m are specific to the substrate used to determine these kinetic parameters.

In another aspect of the invention, it has been determined that substitutions at positions corresponding to 103 in combination with one or more of the following positions 1, 3, 4, 8, 9, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 1 19, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin are important in modulating overall stability and/or proteolytic activity of the enzyme.

These substitutions are preferably made in Bacillus lentus (recombinant or native-type) subtilisin, although the substitutions may be made in any Bacillus protease.

Based on the screening results obtained with the variant proteases, the noted mutations in Bacillus amyloliquefaciens subtilisin are important to the proteolytic activity, performance and/or stability of these enzymes and the cleaning or wash performance of such variant enzymes.

Methods and procedures for making the enzymes used in the detergent and cleaning compositions of the present invention are known and are disclosed in PCT Publication No. WO 95/10615.

The enzymes of the present invention have trypsin-like specificity. That is, the enzymes of the present invention hydrolyze proteins by preferentially cleaving the peptide bonds of charged amino acid residues, more specifically residues such as arginine and lysine, rather than preferentially cleaving the peptide bonds of hydrophobic amino acid residues, more specifically phenylalanine, tryptophan and tyrosine. Enzymes having the latter profile have a chymotrypsin- like specificity. Substrate specificity as discussed above is illustrated by the action of the enzyme on two synthetic substrates. Protease's having trypsin-like specificity hydrolyze the synthetic substrate bVGR-pNA preferentially over the synthetic substrate sucAAPF-pNA. Chymotrypsin-like protease enzymes, in contrast, hydrolyze the latter much faster than the former. For the puφoses of the present invention the following procedure was employed to define the trypsin-like specificity of the protease enzymes of the present invention:

A fixed amount of a glycine buffer at a pH of 10 and a temperature of 25 °C is added to a standard 10 ml test tube. 0.5 ppm of the active enzyme to be tested is added to the test tube. Approximately, 1.25 mg of the synthetic substrate per mL of buffer solution is added to the test tube. The mixture is allowed to incubate for 15 minutes at 25 °C. Upon completion of the incubation period, an enzyme inhibitor, PMSF, is added to the mixture at a level of 0.5 mg per mL of buffer solution. The absorbency or OD value of the mixture is read at a 410 nm wavelength. The absorbence then indicates the activity of the enzyme on the synthetic substrate. The greater the absorbence, the higher the level of activity against that substrate.

To then determine the specificity of an individual enzyme, the absorbence on the two synthetic substrate proteins may be converted into a specificity ratio. For the puφoses of the present invention, the ratio is determined by the formula specificity of:

[activity on sAAPF-pNA]/[activity on bVGR-pNA] An enzyme having a ratio of less than about 10, more preferably less than about 5 and most preferably less than about 2.5 may then be considered to demonstrate trypsin-like activity.

Such variants generally have at least one property which is different from the same property of the protease precursor from which the amino acid sequence of the variant is derived.

The following examples are meant to exemplify the methods for screening protease variants of the present invention, but are not necessarily meant to limit or otherwise define the scope of the invention.

EXAMPLE I

Several different protease variants having one or more substitutions are screened using the net charge method in accordance with the present invention. The protease variants are added to a detergent system (A = North American detergent system having a detergent concentration of between about 975 ppm and 1050 ppm; B = European detergent system having a detergent concentration of between about 4500 ppm and 5100 ppm; C = Latin American detergent system having a detergent concentration of between about 1500 ppm and 2000 ppm; and D = Japanese detergent system having a detergent concentration of between 650 ppm to about 700 ppm) having a pH in the range of about 7 to about 12. Net charges of the one or more substitutions of the protease variants are determined relative to Subtilisin 309. The protease variants that provide effective cleaning of stains, such as BMI stain, under certain detergent systems are set forth in Table III:

Table III

EXAMPLE II A protease variant have one or more substitutions is screened using the adsoφtion method in accordance with the present invention. Although this example is detailed regarding a single variant, the method can be repeated for many protease variants. The initial enzyme activity of the protease variant is measured using a pNA assay. A European detergent system having a detergent concentration of between about 5000 ppm (2.5 g of detergent Ariel Futur (Procter & Gamble, Cincinnati, OH, USA) in 500 ml with a mixed Ca²⁺/Mg²⁺ harness of 15 gpg (750 μl addition of 10,000 gpg artificial hardness = 15 gpg/500 ml)) is prepared and allowed to settle. After settling, the detergent system is filtered. Next, in a 96-well plate 175 μl of the filtered detergent system is added to all wells except for those in the first column. Then, a protease variant-containing European detergent system as described above is prepared using calculations for 1000 μl of 450 rate in individual vials. Using 450 rate solutions, add 16 μl of detergent/protease variant solution and 209 μl of detergent system alone in first column of well plate. Starting with the first column, take 75 μl of the above combination, add to the second column. Take 75 μl from second column and add to the third column. Repeat for successive columns.

Next, transfer 100 μl of dilutions into a 96-well silent screen plate (nylon 66 Biodyne® A membrane Btm, 0.45 um pore) into respective columns, i.e. column 1 into column 1 on silent screen plate, etc. After last transfer, let equilibrate for 5 minutes.

Then the solutions are vacuumed from membrane plate into another empty 96-well plate.

In another 96-well plate, add 200 μl pNA in tris buffer (20 to 220) to wells. Quickly transfer 20 μl of just vacuumed solutions (after filter) to pNA plate. (Time is important here!). Read plate immediately at A410nm at 25°C. Repeat same process, replacing vacuumed solutions with the initial prepared detergent/protease variant solutions. The activity of initial (before filter) and final (after filter) detergent/protease variant solutions is measured and the difference in activity will give percent enzyme adsorbed on to filter paper (membrane).

The protease variants that provided effective cleaning are illustrated in rows 1-5 of Table IV below. Protease variants and Subtilisin 309 that did not provide effective cleaning are illustrated in rows 6-9 of Table IV below.

Table IV

Having described the invention in detail with reference to preferred embodiments and the examples, it will be clear to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention and the invention is not to be considered limited to what is described in the specification.

Claims

What is claimed is:

1. A method for screening protease variants for use in detergent systems comprising: a) providing one or more protease variants having one or more amino acid residue substitutions; and b) calculating the net charge of said one or more amino acid residue substitutions of said one or more protease variants, preferably comprising i) adding said one or more protease variants to a detergent system having a pH in the range of from 7 to 12, preferably in the range of 10 to 11, such that the net charge of said one or more amino acid residue substitutions in said one or more protease variants can be calculated; and c) optionally, selecting said one or more protease variants having a net positive or neutral charge of said one or more amino acid residue substitutions for use in high detergent concentration systems, preferably having greater than 2000 ppm detergent components present in the wash water; and d) optionally, selecting said one or more protease variants having a net negative or neutral charge of said one or more amino acid residue substitutions for use in low detergent concentration systems; preferably having less than 800 ppm detergent components present in the wash water; and e) optionally, selecting said one or more protease variants having a net positive, negative or neutral charge of said one or more amino acid residue substitutions for use in medium detergent concentration systems; preferably having between 800 ppm to 2000 ppm detergent components present in the wash water; and f) optionally, selecting said one or more protease variants having a net neutral charge of said one or more amino acid residue substitutions for use in low, medium and high detergent concentration systems.

2. The method according to Claim 1 wherein said one or more protease variants are selected from multiply-substituted protease variants including a substitution of an amino acid residue with another naturally occurring amino acid residue at an amino acid residue position corresponding to position 103 of Bacillus amyloliquefaciens subtilisin in combination with a substitution of an amino acid residue with another naturally occurring amino acid residue at one or more amino acid residue positions corresponding to positions 1, 3, 4, 8, 9, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 1 1 1, 114, 1 16, 1 17, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin; wherein when said protease variant includes a substitution of amino acid residues at positions corresponding to positions 103 and 76, there is also a subtitution of an amino acid residue at one or more amino acid residue positions other than amino acid residue positions corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 or 274 of Bacillus amyloliquefaciens subtilisin.

3. A detergent composition comprising a protease variant selected according to the method of Claim 1.

4. A method for screening protease variants for use in detergent systems comprising: a) providing one or more protease variants having one or more amino acid residue substitutions; b) adding said one or more protease variants to a detergent system, preferably having a pH in the range of 7 to 12, more preferably 10 to 11; and c) determining the adsoφtion properties of said one or more protease variants in said detergent system to a surface; and d) optionally, selecting said one or more protease variants exhibiting a percent of enzyme adsorbed on said nylon membrane in the range of 16% to 65%), preferably 20%) to 60%, for use in said detergent system.

5. The method according to Claim 4 wherein said detergent system comprises greater than 2000 ppm of detergent components in the wash water.

6. The method according to Claim 5 wherein said detergent system comprises from about 4500 ppm to about 5500 ppm of detergent components in the wash water.

7. The method according to Claim 4 wherein said surface comprises a nylon membrane.

8. The method according to Claim 7 wherein the determining step c) comprises the following steps: i) measuring the initial quantity of enzyme activity, preferably using a pNA assay, of said one or more protease variants; ii) filtering said one or more protease variants through said nylon membrane, preferably a nylon 66 membrane, wherein said nylon membrane preferably comprises a hydrophilic surface; iii) calculating the quantity of enzyme activity of said one or more protease variants filtered through said nylon membrane; iv) determining the quantity of enzyme activity loss of said one or more protease variants adsorbed onto said nylon membrane by determining the difference in quantities between step i) and step iii); and v) converting the quantity of enzyme activity loss to percent of enzyme adsorbed on said nylon membrane.

9. The method according to Claim 8 wherein said hydrophilic surface comprises 50% amines and 50%) carboxyl groups.

10. The method according to Claim 4 wherein said one or more protease variants are selected from multiply-substituted protease variants including a substitution of an amino acid residue with another naturally occurring amino acid residue at an amino acid residue position corresponding to position 103 of Bacillus amyloliquefaciens subtilisin in combination with a substitution of an amino acid residue with another naturally occurring amino acid residue at one or more amino acid residue positions corresponding to positions 1, 3, 4, 8, 9, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin; wherein when said protease variant includes a substitution of amino acid residues at positions corresponding to positions 103 and 76, there is also a subtitution of an amino acid residue at one or more amino acid residue positions other than amino acid residue positions corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 or 274 of Bacillus amyloliquefaciens subtilisin; preferably said multiply-substituted protease variants comprise a substitution set selected from the group consisting of:

68A/76D/103 A 1041/159D/236H/245R

101 G/l 03 A/1041/159D/232V/236H/245R/248D/252K

103 A/1041/159D/232V/236H/245R^/248D/252K

103 A 1041/159D/230V/236H/245R

68A/76D/103A/104I/159D/213R/232V/236H/245R 260A..

11. A detergent composition comprising one or more protease variants selected according to the method of Claim 4.