AU2002325461B2 - Multiply-substituted protease variants with altered net charge for use in detergents - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Genencor International, Inc.

Actual Inventor(s): Ayrookaran J Poulose, Volker Schellenberger, James T Kellis, Christian Paech, Joanne Nadherny, Donald P Naki, Robert M Caldwell, Katherine D Collier Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: MULTIPLY-SUBSTITUTED PROTEASE VARIANTS WITH ALTERED NET CHARGE FOR USE IN

DETERGENTS

Our Ref: 684729 POF Code: 2420/2420 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 1_ i WO 99/20771 PCT/US98/22590 1 A- MULTIPLY-SUBSTITUTED PROTEASE VARIANTS WITH ALTERED NET CHARGE FOR USE IN DETERGENTS Related Applications The present application is a divisional application of Australian application no. 11996/99 Background of the Invention Serine proteases are a subgroup of carbonyl hydrolases. They comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R. Sci. Amer., 131:74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: 1) the subtilisins and 2) the mammalian chymotrypsin-related and homologous bacterial serine proteases trypsin and S. gresius trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut, J. (1977), Annu. Rev. Biochem., 46:331-358, Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.

Subtilisins are serine proteases (approx. MW 27.500) which are secreted in large amounts from a wide variety of Bacillus species and other microorganisms.

The protein sequence of subtilisin has been determined from at least nine different species of Bacillus. Markland, et al. (1983), Hoppe-Seyler's Z. Physiol. Chem., 364:1537-1540. The three-dimensional crystallographic structure of subtilisins from Bacillus amyloliquefaciens, Bacillus licheniformis and several natural variants of B.

lentus have been reported. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, et al. (1972), Biochemistry, 11:2439-2449) or product complexes (Robertus, et al. (1976), J. Biol. Chem., 251:1097-1103) have also provided information regarding the active site and putative substrate binding deft of subtilisin. In addition, a large number of kinetic and chemical 2 modification studies have been reported for subtilisin Svendsen, B. (19765), Carlsberq Res. Commun., 41:237-291; Markland, F.S. Id.) as well as at least one report wherein the side chain of methionine at residue 222 of subtilisin was converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, et al.

(1965), J. Bio. Chem., 244:5333-5338) and extensive site-specific mutagenesis has been carried out (Wells and Estell (1988) TIBS 13:291-297) A common issue in the development of a protease variant for use in a detergent formulation is the variety of wash conditions including varying detergent formulations that a protease variant might be used in. For example, detergent formulations used in different areas have different concentrations of their relevant components present in the wash water. For example, a European detergent system typically has about 4500-5000 ppm of detergent components in the wash water while a Japanese detergent system typically has approximately 667 ppm of detergent components in the wash water. In North America, particularly the United States, a detergent system typically has about 975 ppm of detergent components present in the wash water. Surprisingly, a method for the rational design of a protease variant for use in a low detergent concentration system, a high detergent concentration system, and/or a medium detergent concentration system as well as for use in all three types of detergent concentration systems has been developed.

Summary of the Invention It is an aspect of the invention to provide protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease and thus the protease variant is more effective in a low detergent concentration system than a precursor protease. A low detergent concentration system is a wash system that has less than about 800 ppm of detergent components present in the wash water.

It is another aspect of the invention to provide protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease and thus the protease variant is more effective in a high detergent concentration system than a precursor protease.

A high detergent concentration system is a wash system that has greater than about 2000 ppm of detergent components present in the wash water.

-3- It is another aspect of the invention to provide protease variants.containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease and thus the protease variant is more effective in a medium detergent concentration system than a precursor protease. A medium detergent concentration system is a system that has between about 800 ppm and about 2000 ppm of detergent components present in the wash water.

It is another aspect of the invention to provide protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease and thus the protease variant is more effective in a medium detergent concentration system than a precursor protease. A medium detergent concentration system is a wash system that has between about 800 ppm to about 2000 ppm of detergent components present in the wash water.

It is a further aspect of the invention to provide DNA sequences encoding such protease variants, as well as expression vectors containing such variant DNA sequences.

Still further, another aspect of the invention is to provide host cells transformed with such vectors, as well as host cells which are capable of expressing such DNA to produce protease variants either intracellularly or extracelularly.

There is further provided a cleaning composition comprising a protease variant of the present invention.

Additionally, there is provided an animal feed comprising a protease variant of the present invention.

Also provided is a composition for the treatment of a textile comprising a protease variant of the present invention.

There is further provided a method of producing a protease variant that is more effective in a low, medium and high detergent concentration system than a precursor protease including: a) substituting an amino acid at one or more residue positions wherein the substitution alters the charge at that position to make the charge more positive or less negative compared to the precursor protease; b) substituting an amino acid at one or more residue positions wherein the substitution alters the charge at that position to make the charge more negative or less positive compared to the precursor protease; c) testing the variant to determine its effectiveness in a high, medium low detergent concentration system compared to the precursor protease; and d) repeating steps a) c) as necessary to produce a protease variant that is more effective in a low, medium and high detergent concentration system than a precursor protease wherein steps a) and b) can be done in any order.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

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 W089/06276). The symbol denotes the absence of specific amino acid residues as compared to subtilisin BPN'.

Detailed Description of the Invention As noted above, certain geographies have certain wash conditions and, as such, use different types of detergents. For example. Japan uses a low detergent concentration system while Europe uses a high detergent concentration system. As discussed previously, the United States uses a medium detergent concentration system. We have found that different protease variants perform optimally in these different detergent formulations. However, as a result of these observations, one would expect that it would be impossible to find a protease that would work well in all three types of detergents. Surprisingly, this is not the case. A method of rationally designing a protease variant to be used in either a low detergent wyn~aasmlloos.~x

-M

WO 99/20771 PCTr/JUS98/22590 concentration system or a high detergent concentration system or even a medium detergent concentration system as well as one that works in all three detergent concentration systems has been developed, We have found that in order to produce a protease variant that is more efficacious in a low detergent concentration system, it is necessary to replace positively charged residue(s) either with negatively charged residue(s) or neutral residue(s) and/or neutral residue(s) with negatively charged residue(s). In contrast, we note that in order to produce a protease variant that is more efficacious in a high detergent concentration system, it is necessary to replace negatively charged residue(s) either with positively charged residue(s) or neutral residue(s) and/or neutral residue(s) with positively charged residue(s). Further, we have found that many of the protease variants useful in the low detergent concentration system and/or the high detergent concentration system also are effective in a medium detergent concentration system. By balancing these changes, it is possible to produce a protease variant that works well in low detergent concentration systems, low and medium detergent concentration systems, medium and high detergent concentration systems, high detergent concentration systems, or all three detergent concentration systems.

The electrostatic charge of any ionizable amino acid side chain with an acidic or basic function assumes in aqueous solution is a function of the pH. The acidic residues Glu and Asp, in an equilibrium process, lose a proton by dissociation between pH 3 and 6 thereby acquiring a negative charge. In a similar fashion, His, Lys, and Arg gradually deprotonate between pH 5 and 8, pH 8.5 and 11.5, and pH 11 and 14, respectively, thereby losing a positive charge. The proton of Tyr OH increasingly dissociates between pH 8.5 and 11.5, whereby Tyr acquires a negative charge. The dissociation range for the carboxy terminus is pH 1 to 4, yielding a negative charge, and for the amino terminus it is pH 8 to 11, accompanied by the loss of a positive charge. The dissociation range for amino acid side chains given here are average values for many proteins but they are known to be affected by unusual structural configurations in some proteins.

The cumulative effect of all charges determines whether a protein has a net positive or net negative charge at a given pH. The pH at which positive and negative charges are equally effective and convey an electrostatically neutral state to a protein is called the isoelectric point A protein will lose or gain charge when the pH is shifted or when an amino acid with an ionizable side chain residue is A k WO 99/20771 PCT/US98/22590 -6added or removed. An increase in net positive charge can be achieved either by replacing a residue that at a given pH is negatively charged with an uncharged or a positively charged residue, leading to a formal charge change of +1 and +2, respectively. By replacing an uncharged side chain residue with one that is protonated at the given pH the formal charge change would be Similarly, net negative charge can be increased by replacing positively and uncharged side chains with negatively charged side chains at the pH of observation and gain a formal in crease of negative charge by -1 and respectively.

A low detergent concentration system includes detergents where less than about 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration system as they have approximately 667 ppm of detergent components present in the wash water.

A medium detergent concentration includes detergents where between about B00 ppm and about 2000ppm of detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems as they have approximately 975 ppm of detergent components present in the wash water. Brazil typically has approximately 1500 ppm of detergent components present in the wash water.

A high detergent concentration system includes detergents where greater than about 2000 ppm of detergent components are present in the wash water.

European detergents are generally considered to be high detergent concentration systems as they have approximately 4500-5000 ppm of detergent components in the wash water.

Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. As mentioned above, Brazil typically has approximately 1500 ppm of detergent components present in the wash water. However, other high suds phosphate builder detergent geographies, not limited to other Latin American countries, may have high detergent concentration systems up to about 6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations of detergent compositions in typical wash solutions throughout the world varies from less than about 800 ppm of detergent composition ("low detergent concentration geographies"), for example about 667 ppm in Japan, to between about 800 ppm to Ir--I~LL- i' rj L. WO 99/20771 PCT/US98/22590 -7 about 2000 ppm ("medium detergent concentration geographies"), for example about 975 ppm in U.S. and about 1500 ppm in Brazil, to greater than about 2000 ppm ("high detergent concentration geographies"), for example about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high suds phosphate builder geographies.

The concentrations of the typical wash solutions are determined empirically.

For example, in the a typical washing machine holds a volume of about 64.4 L 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 L 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.

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

WO 99/20771 PCT/US98/22590 8 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.

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 naturallyoccurring 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 shares 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 the amino to carboxy terminus, is aspartate-histidine-serine. In the chymotrypsin related proteases, the relative order, however, is histidineaspartate-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 purposes 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.

"Recombinant subtilisin" or "recombinant protease" refer to a subtilisin or protease in which the DNA sequence encoding the subtilisin or protease is modified to produce a variant (or mutant) DNA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally-occurring amino acid sequence. Suitable methods to produce such modification, and which may be combined with those disclosed herein, include those disclosed in US Patent RE 34,606, US Patent 5,204,015 and US Patent 5.185,258, U.S. Patent 5.700.676, U.S. Patent 5.801.038. and U.S. Patent 5,763,257.

"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. coil or Pseudomonas and gram positive bacteria such as Micrococcus or Bacillus. Examples of eucaryotic WO 99/20771 PCT/US98/22590 -9organisms from which subtilisin and their genes may be obtained include yeast such as Saccharomyces cerevisiae, fungi such as Aspergillus sp.

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 the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the "precursor ONA 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 known to those skilled in the art (see, for example, EP 0 328299, W089/06279 and the US patents and applications already referenced herein).

These amino acid position numbers refer to those assigned to the mature Bacillus amyloliquefaciens subtilisin sequence presented in Fig. 1. The invention.

however, is not limited to the 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 are made at the equivalent amino acid residue positions in B. lentus corresponding to those listed above.

A residue (amino acid) position of a precursor protease is equivalent to a residue of Bacillus amyloliquefaciens subtilisin if it is either homologous corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in Bacillus amyloliquefaciens subtilisin 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 avoiding the elimination i- ik WO 99/20771 PCT7US98/22590 10 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. Siezen et al. (1991) Protein Eng. 4(7):719-737 shows the alignment of a large number of serine proteases. Siezen et al. refer to the grouping as subtilases or subtilisin-like serine proteases.

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 amyloliquefaciens subtilisin in other subtilisins such as subtilisin from Bacillus lentus (PCT Publication No. W089/06279 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. 3A 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 deletions in the sequence of Bacillus lentus as compared to Bacillus amyloliquefaciens subtilisin. Thus, for example, the equivalent amino acid for Val165 in Bacillus amyloliquefaciens subtilisin in the other subtilisins is isoleucine for B. lentus and B. licheniformis.

"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 0) are within 0.13nm and preferably 0.1nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non- WO 99/20771 PCT/US9822590 11 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.

i.IFFoI-I Fc(h) Rfactor IFoh) TIFo(h)l Equivalent residues which are functionally analogous 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 of 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 incorporated 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 are conserved residues whereas others are not. In the case of residues which are not conserved, the substitution 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 substitutions should not result in a naturally-occurring sequence. The protease variants of the present invention include the mature forms of protease variants, as well as the pro- and prepro-forms of such protease variants. The prepro-forms are the preferred construction since this facilitates the expression, secretion and maturation of the protease variants.

Ir^-~i i -u^l~.~uw~rr~3-~3jrc~- -LI WO 99/20771 PCT/US98/22590 12 "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 posttranslational 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 acid 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 herein. 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 (ATCC 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 of 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 -rr-~Z;i;L I- WO 99120771 PCT/US982s590 13 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 subtifis 1168 (also described in US Patent RE 34,606 and US Patent 5.264,366. the disclosure of which are incorporated 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 into 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 presequence 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.

WO 99/120771 PCTIUS9822590 14- This plasmid replicates in hosts in the sense that it contains the well-known elements necessary for plasmid replication: a promoter operably linked to the gene in question (which may be supplied as the gene's own homologous promoter if it is recognized, 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 limitations. 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 encoding the precursor protease. An example of synthetic gene construction is set forth in Example 3 of US Patent 5,204,015, the disclosure of which is incorporated 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 protease 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 C WO 99/20771 PCT/US9822590 15 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 a 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 is 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 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 Km. kI., ratio and/or modified substrate specificity 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.

~f -L WO 99/20771 PCT/US922590 16 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, or are specific to the substrate used to determine these kinetic parameters.

In another aspect of the invention, it has been found that protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease are more effective in a low detergent concentration than a precursor protease.

In a further aspect of the invention, it has been found that protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease are more effective in a high detergent concentration than a precursor protease.

Further, we have found that many of the protease variants useful in the low detergent concentration system and/or the high detergent concentration system also are effective in a medium detergent concentration system.

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

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.

WO 99/20771 PCT/US98/22590 17 Many of the protease variants of the invention are useful in formulating various detergent compositions or personal care formulations such as shampoos or lotions. A number of known compounds are suitable surfactants useful in compositions comprising the protease mutants of the invention. These include nonionic, anionic, cationic or zwitterionic detergents, as disclosed in US 4,404,128 to Barry J. Anderson and US 4,261,868 to Jiri Flora, et at. A suitable detergent formulation is that described in Example 7 of US Patent 5,204.015 (previously incorporated by reference). The art is familiar with the different formulations which can be used as cleaning compositions. In addition to typical cleaning compositions, it is readily understood that the protease variants of the present invention may be used for any purpose that native or wild-type proteases are used. Thus, these variants can be used, for example, in bar or liquid soap applications, dishcare formulations, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile applications, as fusion-cleavage enzymes in protein production, etc. The variants of the present invention may comprise enhanced performance in a detergent composition (as compared to the precursor). As used herein, enhanced performance in a detergent is defined as increasing cleaning of certain enzyme sensitive stains such as grass or blood, as determined by usual evaluation after a standard wash cycle.

Proteases of the invention can be formulated into known powdered and liquid detergents having pH between 6.5 and 12.0 at levels of about .01 to about (preferably to by weight. These detergent cleaning compositions can also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases, as well as builders and stabilizers.

The addition of proteases of the invention to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH is within the above range, and the temperature is below the described protease's denaturing temperature. In addition, proteases of the invention can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.

The present invention also relates to cleaning compositions containing the protease variants of the invention. The cleaning compositions may additionally contain additives which are commonly used in cleaning compositions. These can be selected from, but not limited to, bleaches, surfactants, builders, enzymes and -18bleach catalysts. It would be readily apparent to one of ordinary skill in the art what additives are suitable for inclusion into the compositions. The list provided herein is by no means exhaustive and should be only taken as examples of suitable additives. It will also be readily apparent to one of ordinary skill in the art to only use those additives which are compatible with the enzymes and other components in the composition, for example, surfactant.

When present, the amount of additive present in the cleaning composition is from about 0.01% to about 99.9%, preferably about 1% to about 95%, more preferably about 1% to about The variant proteases of the present invention can be included in animal feed such as part of animal feed additives as described in, for example, US 5,612,055; US 5,314,692; and US 5,147,642.

One aspect of the invention is a composition for the treatment of a textile that includes variant proteases of the present invention. The composition can be used to treat for example silk or wool as described in publications such as RD 216,034; EP 134,267; US 4,533,359; and EP 344,259.

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

All publications and patents referenced herein are hereby incorporated by reference in their entirety.

Example 1 A large number of protease variants were produced and purified using methods well known in the art. All mutations were made in Bacillus lentus GG36 subtilisin.

The protease variants produced were tested for performance in two types of detergent and wash conditions using a microswatch assay described in "Method of assaying for a preferred enzyme and/or detergent", International patent application PCT/US98/027629, published as WO 99/34011.

Tables 1-13 list the variant proteases assayed and the results of testing in two different detergents. All values are given a comparison to the first protease shown in the table a value of 1.32 indicates an ability to release 132% of the stain as opposed to the 100% of the first variant in the table).

Column A shows the charge difference of a variant. For column B, the detergent was 0.67 g/l filtered Ariel Ultra (Procter Gamble, Cincinnati, OH, USA).

Z 4711 WO 99120771 PCT/US98/22590 19 in a solution containing 3 grains per gallon mixed Ca2+/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at 25 6 C (low concentration detergent system).

For column C, the detergent was 3.38 g/l filtered Ariel Futur (Procter Gamble, Cincinnati, OH, USA), in a solution containing 15 grains per gallon mixed Ca2+/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at 40"C (high concentration detergent system).

Table 1 A B C N76D S103A V1041 Q109R 1.00 1.00 N76D S103A V1041 Q109R Q245R +1 0.48 1.41 0 Table 2 A B C V68A N76D S103A V1041 G159D Q236H Q245R 1.00 1.00 V68A N76D S103A V1041 G159D N204D Q236H Q245R -1 1.11 0.03 Table 3 A B C V68A N76D S103A V1041 1.00 1.00 T22K V68A N76D S103A V1041 +1 0.74 1.85

C

Table 4 A B C N76D S103A V1041 M222S 1.00 1.00 N76D S103A V1041 N173R M222S 0 0.66 1.84 012R N76D S103A V1041 M222S 0245R +1 0.41 5.84 Table A B C 012R N76D S103A 1104T S130T M222S 0245R 1.00 1,00 Q12R N76D S103A 1104T S130T M222S Q245R N2610 -1 1.79 0.81 Q12R N76D S103A[1104T S130T R170S N185D M222S N2430 0245R -3 2.87 0.02 Table 6 I A 13 C V68A N76D S103A V1041 G159D 0236H 1.00 1.00 V68A N76D S103A V1041 G159D 0236H 0245R +1 0-94 6801 1 V68A GS13A V1041 159D A232V 0236H Q2458 N252K 13 10,4420.60 1 Table 7 __TA B C V68A N760 S103A V1041 G159D A232V 0236H Q245R 1.00 1.00 V68A N7GD S103A V1041 G159D P21OR A232V Q236H Q245R 1+1 0.44 12.66 Table 8 V6AS103A V1041 {G159D A232V Q236H 0246R, N252K A 0 B .0C V68A S103A V1041 G159D A232V Q236H- Q245R N248D N252K -1 1.961 0.65 Table 9 A B C

I

V6AS103AJ V1041 G21590 A232V 0236H Q245R 100 1.00 V168A S103A V11041 G1590 A232V 0236H K237E Q245R -2 1.27 0.12 Table VSA S10SA V1041 0159D A232V 0236H Q 245R IL257V I 1.001 1.00 V68A IN7BD IS1O3A V104l G159D IA232V d2 36 H IQ245R IL257V -1 11.56 10,48 Table 11 A_ B A C S103A V1041 G159D A232V Q236H Q245R N248D0 22 1.00 1.00 S103A V104l G15SD L217E A232V Q236H 0245R N21 N5K -1 11.90 0.15 Table 12 A B C V04 G159D A232V 0 236H Q245R N248D IN252K 1.00 11.00! N76D S103A IV1041 SlOiG IG159D A232V I0236H Q245R IN2480D 22 -1 11.2810.39 Table 13 A B C N62D S103A V1O4l G1590 T213R A232V Q236H Q245R N248D N252K 1.00 N62D S103A V1041 Q109R IG159D T213R A232V Q236H Q 245R N248D N252K +1 [0,40 1.74a th WO 99/20771 PCT/US98/22590 23 Example 2 The following protease variants were made and tested as noted in Example 1.

The variants in Table 14 are protease variants which have both types of substitutions: those which alter the charge at a position to make the charge more negative or less positive and those which alter the charge at a position to make the charge more positive or less negative compared to B. lentus GG36 as well as neutral substitutions that do not affect the charge at a given residue position. This produces protease variants that perform better than a standard in both low detergent concentration systems (column A; 0.67 g/I filtered Ariel Ultra (Procter Gamble, Cincinnati, OH, USA), in a solution containing 3 grains per gallon mixed Ca2+/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at 25°C) and high detergent concentration systems (cotumn B, 3.38 g/I filtered Ariel Futur (Procter Gamble, Cincinnati, OH, USA), in a solution containing 15 grains per gallon mixed Ca2+/Mg 2 hardness, and 0.3 ppm enzyme was used in each well at Table 14 A B N76D S103A V1041 1.00 1.00 V68A S103A V1041 G159D A232V 0236H 0245R N252K 1.41 1.85 V68A N76D S103A V104 G159D T213R A232V Q236H Q245R T260A 1.30 1.73 V68A S103A V1041 G1590 A232V Q236H 0245R N2480 N252K 2,77 1.20 V68A S103A V1041 N140D G159D A232V 0236H Q245R N252K 2,96 1.42 N43K V68A S103A V1041 G159D A232V 0236H Q245R 2.05 1.78 N43D V68A S103A V1041 G159D A232V 0236H Q245R N252K 2.00 1.34 V68A N76D S1O3A V1041 G159D A215R A232V 0236H Q245R 1.67 1.45 Q12R V68A N760 S103A V1041 G1590 A232V Q236H 0245R 2.16 1.72 N760 S103A V1041 V1471 G159D A232V 0236H Q245R N248S K251R 1.35 1.29 V68A N 1D S103A V1041 G159DI A232V 0236H Q245R S256R 2.01 1.72 -V68A N76D S103A V1041 G159D Q206R A232V Q236H 0245R 2.09 1.62 13A V1041 G159D A232V Q236H 245R N248D N252K 1.44 1.41 V68A S1O3A V1041 G159D A232V 0236H 0245R N248D N252K 1.81 1.72 V68A S103A V1041 G159D A232V Q236H Q245R N248D N252K L257R 1.51 1.41 -v 0 'a w tA 'a

C

IV6AA IS103AI V1041 IA232V I2* 25 N248D N2521 1.04 1.501 N76D ISIO3A IV1041 1G159D0 A232V I0236H 0245R L257V 11.92 1.091 tJ

C

Claims (14)

1. 2. A method of producing a subtilisin variant that is more effective than a precursor subtilisin in a high detergent concentration system more than about 2000 ppm detergent components present in the wash water, comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the overall charge of the precursor subtilisin to make a subtilisin variant having a more positive charge or less negative charge compared to the precursor; b) testing the variant in a high detergent system having more than about 2000 ppm detergent components present in the wash water by comparing the ability of the precursor and the variant to remove a stain, wherein the effectiveness of the precursor is given a value of 1.0 and the variant with improved effectiveness achieves a value greater than 1.0; and I c) producing said variant having improved effectiveness. oo
2. 3. A method of producing a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system having Sbetween about 800 ppm and about 2000 ppm detergent components present in the wash water, comprising: 0 a) substituting an amino acid residue at one or more positions in a precursor c 10 subtilisin wherein the substitution alters the overall charge of the precursor subtilisin to make a subtilisin variant having a more positive charge or less negative charge compared to the precursor; b) testing the variant in a medium detergent system having between about 800 ppm and about 2000 ppm detergent components present in the wash water by comparing the ability of the precursor and the variant to remove a stain, wherein the effectiveness of the precursor is given a value of 1.0 and the variant with improved effectiveness achieves a value greater than 1.0; and c) producing said variant having improved effectiveness.
3. 4. A method of producing a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system having between about 800 ppm and about 2000 ppm detergent components present in the wash water, comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the overall charge of the precursor to make a subtilisin variant having a more negative charge or less positive charge compared to the precursor; b) testing the variant in a medium detergent system having between about 800 Sppm and about 2000 ppm detergent components present in the wash water by comparing the ability of the precursor and the variant to remove a stain, wherein O the effectiveness of the precursor is given a value of 1.0 and the variant with 00 improved effectiveness achieves a value greater than 1.0; and Sc) producing said variant having improved effectiveness. IND A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in both a low detergent concentration system and a high c 10 detergent concentration system comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more positive or less negative compared to the precursor subtilisin; b) substituting an amino acid residue at one or more positions in the precursor subtilisin wherein the substitution alters the charge of the position to make the charge more negative or less positive compared to the precursor subtilisin; c) obtaining a subtilisin variant of step a) and step b) wherein the overall change in charge compared to the precursor is neutral or positive; d) testing the variant obtained in step c) in both a low detergent concentration system having less than about 800 ppm detergent components present in the wash water and a high detergent concentration system having greater than about 2000 ppm detergent components present in the wash water; and e) selecting the variant having a higher stain release effectiveness than said precursor subtilisin in both the low concentration system and the high concentration system, wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than
4. 6. A method according to claim 5, wherein the overall change in charge of O the variant compared to the precursor is neutral. 00
5. 7. A method according to claim 5, wherein the overall change in charge of Sthe variant compared to the precursor is positive 1. IND
6. 8. A method according to any one of claims 5 to 7, wherein step b) is performed before step a). N
7. 9. A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a low detergent concentration system having less than about 800 ppm detergent components present in the wash water, comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more negative or less positive compared to the precursor; b) obtaining a subtilisin variant of step a) wherein the overall change in charge compared to the precursor is negative; c) testing the variant obtained in step b) in a low detergent concentration system having less than about 800 ppm detergent components present in the wash water; and d) selecting the variant having a higher stain release effectiveness in the low detergent concentration system compared to said precursor, wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a high detergent concentration system, comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more positive or less negative compared to the precursor; b) obtaining a subtilisin variant of step a) wherein the overall change in charge compared to the precursor is positive; c) testing the variant obtained in step b) in a high detergent concentration system having more than about 2000 ppm detergent components present in the wash water; and d) selecting the variant having a higher stain release effectiveness in the high detergent concentration system compared to said precursor, wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than
8. 11. A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more negative or less positive compared to the precursor; b) obtaining a subtilisin variant of step a) wherein the overall change in charge compared to the precursor is negative; c) testing the variant obtained in step b) in a medium detergent concentration system having between about 800 ppm and about 2000 ppm detergent components present in the wash water; and d) selecting the variant having a higher stain release effectiveness in the medium detergent concentration system than said precursor, wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than
9. 12. A method for selecting a subtilisin variant that is more effective than a precursor subtilisin in a medium detergent concentration system, comprising: a) substituting an amino acid residue at one or more positions in a precursor subtilisin wherein the substitution alters the charge of the position to make the charge more positive or less negative compared to the precursor; b) obtaining a subtilisin protease variant of step a) wherein the overall change in charge compared to the precursor is positive; c) testing the variant obtained in step b) in a medium detergent concentration system having between about 800 ppm to about 2000 ppm detergent components present in the wash water; and d) selecting the variant having a stain release effectiveness in the medium detergent concentration system than said precursor, wherein the effectiveness of the precursor is given a value of 1.0 and the variant achieves a value of greater than
10. 13. A method according to any one of the preceding claims, wherein the variant is derived from a Bacillus subtilisin.
11. 14. A method according to claim 13, wherein the variant is derived from Bacillus lentus subtilisin.
12. 15. A method according to any one of the preceding claims, wherein the detergent composition is formulated into a powdered or liquid detergent having a ph between 6.5 and 12.0.
13. 16. A method according to any one of the preceding claims substantially as hereinbefore described.
14. 17. A protease variant when produced by a method according to any one of claims 1 to 16. DATED: 18 October 2005 PHILLIPS ORMONDE FITZPATRICK Attorneys for: GENENCOR INTERNATIONAL, INC KM W:\documents'A\2OO5\O9-20 5\Notes\IRN684729 Clean claims 30.9.05.doc