EP1124987A2 - Techniques de criblage de variantes de proteases servant dans des compositions detergentes - Google Patents

Techniques de criblage de variantes de proteases servant dans des compositions detergentes

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Publication number
EP1124987A2
EP1124987A2 EP99971044A EP99971044A EP1124987A2 EP 1124987 A2 EP1124987 A2 EP 1124987A2 EP 99971044 A EP99971044 A EP 99971044A EP 99971044 A EP99971044 A EP 99971044A EP 1124987 A2 EP1124987 A2 EP 1124987A2
Authority
EP
European Patent Office
Prior art keywords
amino acid
acid residue
protease
protease variants
detergent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99971044A
Other languages
German (de)
English (en)
Inventor
Chanchal Kumar Ghosh
Phillip Frederick Brode, Iii
Deborah Susan Rauch
Michael Stanford Showell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Procter and Gamble Co
Original Assignee
Procter and Gamble Co
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Filing date
Publication date
Application filed by Procter and Gamble Co filed Critical Procter and Gamble Co
Publication of EP1124987A2 publication Critical patent/EP1124987A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

Definitions

  • 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.
  • Enzymes particularly protease enzymes, are becoming common components of detergent compositions for their ability to clean.
  • 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.
  • detergent systems detergent components present in a wash water
  • a European detergent system typically has about 4500 ppm to about 5000 ppm of detergent components present in the wash water.
  • a Japanese detergent system typically has about 667 ppm of detergent component present in the wash water.
  • detergent systems typically have about 975 ppm of detergent components present in the wash water.
  • 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.
  • 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).
  • 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.
  • 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.
  • a detergent composition comprising a protease variant selected by the screening method discussed above, and a surfactant.
  • 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 adso ⁇ tion 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
  • a detergent composition comprising a protease variant selected from the screening method described above, and a surfactant.
  • 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.
  • 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'.
  • the present invention relates to methods for screening protease variants for use in the various detergent systems that exist throughout the world.
  • 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.
  • 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.
  • 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 adso ⁇ tion activity of the protease variants are effective methods of identifying and/or selecting protease variants that provide effective performance in the various detergent systems.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 adso ⁇ tion, but less mobility, whereas, negatively charged variants provide less adso ⁇ tion, but more mobility. The more mobile negatively charged variants can sample more sites.
  • a detergent composition comprising a protease variant selected by the net charge method described above.
  • 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,
  • 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 adso ⁇ tion 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 adso ⁇ tion step (step c) can be any suitable surface known in the art.
  • the surface is a nylon membrane, more preferably a nylon 66 membrane.
  • 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 Co ⁇ oration of Naperville, IL (formerly Pall Co ⁇ oration of Glen Cove, NY).
  • step c) determining the adso ⁇ tion 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,
  • 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 adso ⁇ tion 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.
  • a detergent composition comprising one or more protease variants selected according to the adso ⁇ tion method described above.
  • 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,
  • Proteases - Proteases are carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides.
  • proteases means a naturally occurring protease or recombinant protease.
  • Naturally-occurring proteases include ⁇ -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.
  • 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.
  • the protease variants are designed to have trypsin-like specificity and preferably also be bleach stable.
  • 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.
  • 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
  • C-X bonds in which X is oxygen or nitrogen 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.
  • 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.
  • the relative order of these amino acids reading from amino to carboxy terminus, is aspartate-histidine-serine.
  • the relative order is histidine-aspartate-serine.
  • 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 pu ⁇ oses 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 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).
  • 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,
  • the preferred protease variant enzymes useful for the present invention comprise the substitution, deletion or insertion of amino acid residues in the following combinations:
  • protease variant including substitutions of the amino acid residues at position 103 and at one or more of the following positions 236 and 245;
  • 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;
  • 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
  • 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:
  • 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.
  • 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.
  • 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
  • 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.
  • 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, 192
  • 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.
  • properties include proteolytic activity, substrate specificity, stability, altered pH profile and/or enhanced performance characteristics.
  • 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,
  • the present invention 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.
  • 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).
  • Fig. 2 herein shows the conserved residues as between B. amyloliquefaciens subtilisin and B. lentus subtilisin.
  • 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.
  • 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.
  • the equivalent amino acid for Vail 65 in Bacillus amyloliquefaciens subtilisin in the other subtilisins is isoleucine for B. lentus and B. licheniformis.
  • the amino acid at position +76 is asparagine (N) in both B. amyloliquefaciens and B. lentus subtilisins.
  • the amino acid equivalent to +76 in Bacillus amyloliquefaciens subtilisin is substituted with aspartate (D).
  • D aspartate
  • 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.
  • 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.
  • residues of the precursor protease for which a tertiary structure has been obtained by x-ray crystallography
  • 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 inco ⁇ orated herein by reference) and can be used as outlined above to determine equivalent residues on the level of tertiary structure.
  • 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.
  • signal sequence 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.
  • 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.
  • "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.
  • Bacillus subtilis 168 also described in US Patent RE 34,606 and US Patent 5,264,366, the disclosure of which are inco ⁇ orated herein by reference
  • 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.
  • 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 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.
  • 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.
  • the gene can be a natural B. lentus gene.
  • a synthetic gene encoding a naturally-occurring or mutant precursor protease may be produced.
  • 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 inco ⁇ orated herein by reference.
  • 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.
  • 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.
  • restriction sites are preferably unique sites within the protease gene so as to facilitate the replacement of the gene segment.
  • 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.
  • 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.
  • 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.
  • 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.
  • variant enzymes having altered thermal stability and/or altered substrate specificity as compared to the precursor.
  • 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.
  • increases or decreases (alteration) of the stability of the variant 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.
  • substitutions are preferably made in Bacillus lentus (recombinant or native-type) subtilisin, although the substitutions may be made in any Bacillus protease.
  • 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.
  • 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 enzymes having trypsin-like specificity hydrolyze the synthetic substrate bVGR-pNA preferentially over the synthetic substrate sucAAPF-pNA.
  • Chymotrypsin-like protease enzymes hydrolyze the latter much faster than the former.
  • 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.
  • 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.
  • the absorbence on the two synthetic substrate proteins may be converted into a specificity ratio.
  • the ratio is determined by the formula specificity of:
  • 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.
  • protease variants having one or more substitutions are screened using the net charge method in accordance with the present invention.
  • 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:
  • a protease variant have one or more substitutions is screened using the adso ⁇ tion 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.
  • the detergent system is filtered.
  • 175 ⁇ l of the filtered detergent system is added to all wells except for those in the first column.
  • a protease variant-containing European detergent system as described above is prepared using calculations for 1000 ⁇ l of 450 rate in individual vials.
  • 450 rate solutions add 16 ⁇ l of detergent/protease variant solution and 209 ⁇ l of detergent system alone in first column of well plate.
  • Starting with the first column take 75 ⁇ l of the above combination, add to the second column. Take 75 ⁇ l from second column and add to the third column. Repeat for successive columns.
  • protease variants that provided effective cleaning are illustrated in rows 1-5 of Table IV below.
  • proteease variants and Subtilisin 309 that did not provide effective cleaning are illustrated in rows 6-9 of Table IV below.

Abstract

L'invention concerne des techniques de criblage de variantes de protéases servant dans des systèmes détergents. Ces techniques se rapportent à la charge nette des substitutions des variantes de protéases et aux propriétés d'adsorption desdites variantes. L'invention traite également de compositions détergentes comprenant des variantes de protéases criblées par de telles techniques.
EP99971044A 1998-10-23 1999-10-22 Techniques de criblage de variantes de proteases servant dans des compositions detergentes Withdrawn EP1124987A2 (fr)

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PT1523553E (pt) * 2002-01-16 2010-03-03 Genencor Int Variantes de protease de substituições múltiplas
TWI319007B (en) * 2002-11-06 2010-01-01 Novozymes As Subtilase variants
US7888093B2 (en) 2002-11-06 2011-02-15 Novozymes A/S Subtilase variants
CN101778940B (zh) * 2007-06-06 2017-04-05 丹尼斯科美国公司 改进多种蛋白质性质的方法
CA2743123A1 (fr) * 2008-11-11 2010-05-20 Danisco Us Inc. Compositions et procedes comprenant un variant de subtilisine
CN105925555B (zh) 2010-05-06 2020-12-22 丹尼斯科美国公司 包含枯草杆菌蛋白酶变体的组合物和方法
EP2705146B1 (fr) * 2011-05-05 2018-11-07 Danisco US Inc. Procédés et compositions comprenant des variants de la sérine protéase
US20130005048A1 (en) * 2011-06-29 2013-01-03 Monika Felten Indicator device
CN105349569B (zh) * 2015-11-19 2020-05-12 湖北大学 一组不同启动子介导的蛋白酶高通量筛选载体及筛选方法
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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