MXPA00003801A - Multiply-substituted protease variants - Google Patents

Abstract

Novel proteasevariants derived from the DNA sequences of naturally-occurring or recombinant non-human proteases are disclosed. The variant proteases, in general, are obtained by i(in vitro) modification of a precursor DNA sequence encoding the naturally-occurring or recombinant protease to generate the substitution of a plurality of amino acid residues in the amino acid sequence of a precursor protease. Such variant proteases have properties which are different from those of the precursor protease, such as altered wash performance. The substituted amino acid residue correspond to positions 62, 212, 230, 232, 252 and 257 of i(Bacillus amyloliquefaciens) subtilisin.

Description

MULTIPLELY REPLACED PROTEASE VARIANTS FIELD AND BACKGROUND OF THE INVENTION Serine proteases are a subgroup of carbonylhydrolases. They comprise a diverse class of enzymes that have a high range of specificities and biological functions. Stroud, R. Sci. Amer., 131: 74-88. Despite its functional diversity, the catalytic machinery of serine proteases has been addressed by at least two families of genetically distinct enzymes: 1) subtilisins and 2) bacterial serine proteases, homologous and related to chymotrypsin, of mammals (for example, trypsin and trypsin from S. gre if u s). These two families of serine proteases show remarkably similar catalysis mechanisms. Kraut. J. (1977). Annu. Rev. Bioche. , 46: 331-358. Furthermore, although the primary structure is not related, the tertiary structure of these two enzyme families brings together a catalytic triad, conserved, of amino acids consisting of serine, histidine and aspartate.

REF. : 119513 Subtilisins are serine proteases (with a molecular weight of approximately 27,500) that are secreted in large quantities 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, F.S., et al (1983), Hoppe-Seyler s Z. Physiol. Chem., 364: 1537-1540. The three-dimensional crystallographic structure of the subtilisin of Bacillus aiyloliquefaciens, Bacillus licheniforimis and several natural variants of B. lentus have been reported. These studies indicate that although subtilisin is not genetically related to mammalian serine proteases, it has a similar active site structure. The crystalline X-ray structures of subtilisin contain covalently linked peptide inhibitors (Robertus, J.D., et al. (1972), Biochemistry, 11: 2439-2449) or complex products (Robertus, J.D., et al. (1976), J.

Biol. Chem. , 251: 1097-1103) have also provided information concerning the active site and the putative substrate that binds the subtilisin cleft. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin; Svendsen. B. (1976), Carisberg Res. Commun., 41: 237-291; Markland, F.S. Id. ) as well as at least one report wherein the methionine side chain at residue 222 of subtilisin was converted by hydrogen peroxide to methionine sulfoxide (Stauffer, DC, et al. (1965), J. Biol. Chem., 244: 5333-5338) and extensive site-specific mutagenesis has been carried out (Wells and Estell (1988) (TIBS 13: 291-297).

BRIEF DESCRIPTION OF THE INVENTION One object of the present is to provide a protease variant containing a substitution of an amino acid in one or more residue positions, corresponding to the residue positions selected from the group consisting of 62, 212, 230, 232, 252 and 257 of the subtilisin of Ba ci llus amyl ol i qu ef a ci ens. Although any combination of the amino acid substitutions listed above may be employed, the preferred protease variant enzymes of the present invention comprise the substitution of the amino acid residues in the following combinations. All positions of the residues correspond to the positions of the subtilisin of the Ba ci ll us amyl ol i qu efa ci ens: (1) a protease variant that includes substitutions of the amino acid residues in position 62 and in one or more of the following positions 103, 104, 109, 159, 213, 232, 236, 245, 248 and 252; (2) a protease variant that includes substitutions of the amino acid residues at position 2.12 and at one or more of the following positions 12, 98, 102, 103, 104, 159, 232, 236, 245, 248, and 252; (3) a protease variant that includes substitutions of the amino acid residues at position 230 and at one or more of the following positions 68, 103, 104, 159, 232, 236 and 245; (4) a protease variant that includes substitutions of the amino acid residues at position 232 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 and 275; (5) a protease variant that includes substitutions of the amino acid residues at position 232 and at one or more of the following positions 103, 104, 236 and 245; (6) a protease variant that includes substitutions of the amino acid residues at position 232 and 103 in one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102 , 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 And 275; (7) a protease variant that includes substitutions of the amino acid residues in positions 232 and 104 and in one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 And 275; (8) a protease variant which includes substitutions of amino acid residues at positions 232 and 236 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270, and 275; (9) a protease variant that includes substitutions of the amino acid residues in positions 232 and 245 and in one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, '109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 and 275; (10) a protease variant which includes substitutions of the amino acid residues at position 232, 103, 104, 236 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76 , 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260 , 270 and 275; (11) a protease variant that includes substitutions of amino acid residues at position 252 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248, and 270; (12) a protease variant that includes substitutions of the amino acid residues at position 252 and at one or more of the following positions 103, 104, 236 and 245; (13) a protease variant that includes substitutions of the amino acid residues in position 252 and 103 and in one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248, and 270; (14) a protease variant that includes substitutions of the amino acid residues at position 252 and 104 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248, and 270; (15) a protease variant that includes substitutions of the amino acid residues at position 252 and 236 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248, and 270; (16) a protease variant that includes substitutions of amino acid residues at positions 252 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248, and 270; (17) a protease variant that includes substitutions of the amino acid residues at position 252, 103, 104, 236 and 245, and at one or more of the following positions 1, 9, 12, 61, 62, 68, 97 , 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248, and 270; and (18) a protease variant that includes substitutions of amino acid residues at position 257 and at one or more of the following positions 68, 103, 104, 205, 209, 210, 232, 236, 245, and 275. The most preferred protease variants are the sets of substitutions selected from the group consisting of the residue positions corresponding to the positions found in Table 1 of Bacillus amyl oli quefa ci ens: The most preferred protease variants are the sets of substitutions selected from the group consisting of the residue positions corresponding to the positions found in Table 2 of the subtilisin of Bacillus amyloliquefaciens: An additional goal is to provide DNA sequences that encode those protein variants, as well as expression vectors containing those DNA sequences of the variants. Still further, another object of the invention is to provide host cells transformed with those vectors, as well as host cells that are capable of expressing that DNA to produce protease variants either intracellularly or extracellularly. In addition, a cleaning composition comprising a protease variant of the present invention is provided. Additionally, an animal feed is provided comprising a protease variant of the present invention. Also provided is a composition for the treatment of a textile product, comprising a protease variant of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figures A-C represent the amino acid and DNA sequence for the subtilisin of Bacillus amyloliquefaciens and a partial restriction map of this gene. Figure 2 represents conserved amino acid residues, between subtilisin of Bacillus amyloliquefaciens (BPN) 'and Bacillus lentus (wild type). Figures 3A and 3B depict the amino acid sequence of four subtypes. The line above represents the amino acid sequence of subtilisin of the subtilisin of Bacillus amyl oliquefaciens (sometimes referred to as subtilisin BPN '). The second line represents the amino acid sequence of the subtilisin of Bacillus subtilis. The third line represents the amino acid sequence of the subtilisin of B. licheni formi s. The fourth line represents the amino acid sequence of the subtilisin of Bacillus lentus (also referred to as subtilisin 309 in PCT WO89 / 06276). The symbol * denotes the absence of specific amino acid residues compared to subtilisin BPN '.

DETAILED DESCRIPTION OF THE INVENTION The proteases are carboni lhydroles so they act generally to break the protein bonds of proteins or peptides. As used herein, "protease" means a naturally occurring protease or a recombinant protease. Naturally occurring proteases include the a-aminoacylpeptide hydrolase, the peptidyl hydrolase, the innoacid, the acylamino hydrolase, the serine carboxypeptidase, the allocarboxypeptide me, the thioproprobin, the carboxy i lpro teínas a la me t alopro t eina sa. Also included are the serine proteases, the metalloproteases, the thioproteases and the acid proteases, as well as the endopro tects and the exopro t eases. The present invention includes protease enzymes that are not variants of carbonylhydrolase that occur naturally (protease variants) having a proteolytic activity, stability, specificity for the substrate, pH profile and / or performance characteristics, different, when compared with the precursor carbonylhydrolase from which the amino acid sequence of the variant is derived. Specifically, these protease variants have an amino acid sequence not found in nature, which is derived by the substitution of a plurality of amino acid residues of a precursor protease with different amino acids. The precursor protease can be a protease that occurs naturally or a recombinant protease. The protease variants useful herein encompass the substitution of any of the nineteen amino acids L occurring naturally at the positions of the designated amino acid residues. These substitutions can be made in any precursor subtilisin (prokaryotic, eukaryotic, mammalian, etc.). Throughout this application, several amino acids are referred to by common one- and three-letter codes. These codes are identified in Dale, M.W. (1989), Molecular Genetics of Bacteria, John Wiley & Sons. Ltd .; Ap endi ce B. The protease variants useful herein are preferably derived from a subtilisin of Ba ci l l u s. More preferably, the protease variants are derived from the subtilisin of Bacillus in tus and / or of subtilisin 309. The subtilisins are bacterial or fungal proteases, which generally act to break the peptide bonds of proteins or peptides. As used herein, "subtilisin" means a naturally occurring subtilisin, or a recombinant subtilisin. It is known that a series of subtilisins that occur naturally are produced and often secreted by several microbial species. The amino acid sequences of the members of this series are not fully homologous. However, the subtilisins in this series exhibit the same type or a similar type of proteolytic activity. This class of serine proteases shares a common amino acid sequence, which defines a catalytic triad that distinguishes them from the class of serine proteases related to chymotrypsin. The serine proteases related to subtilisins and chymotrypsin both have a catalytic triad comprising aspartate, histidine and serine. However, in the proteases related to chymotrypsin, the relative order is hi s tine-aspartate-ina. Thus, subtilisin refers herein to a serine protease that has the catalytic triad of proteases related to subtilisin. Examples include, but are not limited to, the subtilisins identified in Figure 3 herein. Generally, and for purposes of the present invention, the numbering of the amino acids in the proteases corresponds to the numbers assigned to the sequence of subtilisin of the Bacillus amyl oli qu efa ci ens madura, presented in Figure 1. " "Recombinant subtilisin" or "recombinant protease" refers to a subtilisin or protease in which the DNA sequence encoding the subtilisin or the protease is modified to produce a variant (or mutant) DNA sequence encoding the substitution, deletion or insertion of one or more amino acids in the sequence of naturally occurring amino acids. Suitable methods for producing such modification, and which may be combined with those described herein, include those described in US Patent No. 34,606, US Patent No. 5,204,015 and US Patent No. 5,185,258, US Patent No. 5,700,676, U.S. Patent No. 5,801,038, and U.S. Patent No. 5,763,257. "non-human subtilisins" and the DNA encoding them can be obtained from any prokaryotic and eukaryotic organisms. Suitable examples of prokaryotic organisms include gram negative organisms such as E. coli or Pseudomonas and gram positive bacteria such as Micrococcus or Bacillus. Examples of eukaryotic organisms from which subtilisin and its genes can be obtained, include yeast such as Saccharomyces cerevisiae, fungi such as Aspergillus sp. A "protease variant" has an amino acid sequence that is derived from the amino acid sequence of a "precursor protease". The precursor proteases include the naturally occurring proteases and the recombinant proteases. The amino acid sequence of the protease variant is "derived" from the amino acid sequence of the parent protease, by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. That modification is of the "precursor DNA sequence" that encodes the amino acid sequence of the precursor protease rather than the manipulation of the enzyme of the precursor protease, per se. Suitable methods for such manipulation of the precursor DNA sequence include the methods described herein, as well as the methods known to those skilled in the art (see, for example, EP 0 328299, WO 89/06279 and US Pat. US Patents and Applications to which reference has already been made herein). Hereby specific amino acid substitutions are identified in one or more residue positions corresponding to the residue positions selected from the group consisting of 62, 212, 230, 232, 252"and 257 subtilisin of Bacillus amyl oli. The preferred variants are those that have combinations of substitutions at residue positions corresponding to the positions of the subtilisin of Bacillus amyloliquefaciens of Table 1. The most preferred variants are those that have combinations of substitutions at the residue positions corresponding to the positions of the subtilisin of Bacillus amyloliquefaciens, of Table 2. The additionally preferred variants are those which have combinations of substitutions at the residue positions corresponding to the positions and to the subtilisin of Bacillus amyl or 1 iquefac iens of the Table 3 XI t.

In These positional numbers of the amino acids refer to those assigned to the subtilisin sequence of the Bacillus amyl olfactory mature, presented in Figure 1. However, the invention is not limited to the mutation of this particular subtilisin but rather is extends to precursor proteases that contain amino acid residues at positions that are "equivalent" to the particular residues identified in the subtilisin of Bacillus amyl oliquefacts. In a preferred embodiment of the present invention, the precursor protease is the subtilisin of Bacillus lentus and the substitutions are made at the equivalent positions of amino acid residues, in B. lentus, which correspond to those listed above. A residue (amino acid) position of a precursor protease is equivalent to a residue of Bacillus amyl olylate subtilisin either homologous (ie corresponding to the position in each primary or tertiary structure) or analogous to a specific residue or portion of that residue in the subtilisin of Bacillus amyl oliquefaciens (that is, having an equal or similar functional ability to combine, react, or interact chemically). To establish homology with the primary structure, the amino acid sequence of a precursor protease is directly compared to the primary sequence of the subtilisin of Bacillus amyloliquefacts and particularly to a set of residues known to be invariant in subtilisin for which the sequence is known For example, Figure 2 of the present shows conserved residues as between the subtilisin of B. amyl oliquefaciens and the subtilisin of B. lentus. After aligning the conserved waste, allowing the insertions and deletions necessary to maintain the alignment (ie, avoiding the elimination of residues conserved through delecióp and arbitrary insertion), the equivalent residues are defined for particular amino acids in the primary sequence of the subtilisin of Bacillus amyloliquefaciens . The alignment of the conserved residues should preferably keep 100. of those residues. However, alignment greater than 75% or as small as 50% of the conserved residues is also adequate to define equivalent residues. The preservation of the catalytic triad Asp32 / Hi s 64 / 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 refers to grouping as subtylases or serine proteases similar to subtilisin. For example, in Figure 3, the amino acid sequence of the subtilisin of Bacillus amyloliquefacts, Bacillus subtilis, Bacillus licheniformis - (cari sbergensi) and Bacillus lentus, are aligned to provide the maximum amount of homology among the amino acid sequences. A comparison of these sequences shows that there is a certain number of residues conserved and contained in each sequence. These conserved residues (such as between BPN 'and B. lentus) are identified in Figure 2. These conserved residues can, therefore, be used to define the corresponding equivalent amino acid residues of the' subtilisin of Bacillus amyloliquefaciens, in other subtilisins such as Bacillus lentus subtilisin (PCT Publication No. WO89 / 06276 published July 13, 1989), the preferred protease precursor enzyme, herein, or the subtilisin referred to as PB92 ( EP 0 328 299), which is largely homologous to the subtilisin of the preferred Bacillus lentus. The amino acid sequences of certain of these subtilisins are aligned in Figures 3A and 3B with the subtilisin sequence of Bacillus amyloliquefaciens, to produce maximum homology of conserved residues. As can be seen, there are a number of deletions in the sequence of Bacillus lentus, when compared with the subtilisin of Bacillus amyloliquefaciens. In this way, for example, the amino acid equivalent for Vall65 in the subtilisin of Bacillus a yloliquefaci ens, in the other subtilisins, is isoleucine for B. lentus and B. licheniformis. The "equivalent residues" can also be defined by determining the homology at the level of the 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 atoms of the main chain, of a particular amino acid residue, of the precursor protease and of the subtilisin of Bacillus amyloliquefacts (N on N, CA on Ca, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. The alignment is achieved after the best model has been oriented and located to give the maximum overlap of the atomic coordinates of the protein atoms, other than hydrogen, of the protease in question for the subtilisin of Bacillus amyloliquefaciens. The best model is the crystallographic model that provides the lowest R factor for the experimental diffraction data, at the highest available resolution.

Factor R = th \ Foíh) \ - \ Fc (h) \? * L Fo (h) \ Equivalent residues _ that are functionally analogous to a specific residue of the subtilisin of Bacillus amyloliquefaciens, are defined as those amino acids of the protease precursor that can adopt a conformation such that, or alter, or modify or contribute to, the structure of the protein, to binding to the substrate or to catalysis, in a defined manner and attributed to a specific residue of the Bacillus subtilisin amyl oliquene ens Furthermore, it is those residues of the parent protease (for which a tertiary structure has been obtained by X-ray crystallography) occupying a position analogous to the extent that, although the atoms of the main chain of the given residue may not meet the criteria of equivalence based on the occupation of a homologous position, the atomic coordinates of at least two of the atoms of the side chain, of the residue, coincide with 0.13 nm of the corresponding side chain atoms of the subtilisin of Bacillus amyl ol ique aciens. The coordinates of the three-dimensional structure of the subtilisin of Bacillus amyloliquefaciens are presented in EPO publication No. 0 251 446 (equivalent to US Pat. No. 5,182,204, the description of which is incorporated herein by reference) and can be used as it was previously sketched, to determine the equivalent residuals at the level of the tertiary structure. Some of the waste identified for substitution is conserved waste, w others are not. In the case of residues that are not conserved, the substitution of one or more amino acids is limited to substitutions that produce a variant having an amino acid sequence that does not correspond to un-a found in nature. In the case of conserved residues, these substitutions will not result in a naturally occurring sequence. The protease variants of the present invention include the mature forms of the protease variants, as well as the proformas and prepro forms of those protease variants. The preproforms are the preferred construction since it facilitates the expression, secretion and maturation of the protease variants.

"Prosequence" refers to a sequence of amino acids linked 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 proenzyme translation products and, in the absence of a post-translational process, are expressed in this way. A preferred prosequence for producing protease variants is the putative prosequence of the subtilisin of Bacillus amyloid efa cies, although other protease prosequences can be used. . A "signal sequence" or "presequence" refers to any amino acid sequence linked to the N-terminal portion of a protease or to the N-terminal portion of a proprotease that can participate in the secretion of mature or pro-forma forms of the protease. This signal sequence definition is a functional definition that is intended to include all those amino acid sequences encoded by the N-terminal portion of the protease gene that participates to effect the secretion of the protease under native or original conditions. The present invention uses those sequences to effect the secretion of protease variants, as defined herein. A possible signal sequence comprises the first seven amino acid residues of the subtilisin signal sequence of B a ci l l u s s ub t i l i s, fused to the rest of the signal sequence of the subtilisin of Ba ci l l l l l t u s (ATCC 21536). A "prepro" form, of a protease variant, consists of the mature form of the protease, which has a prosequence functionally linked to the amino terminus of the protease and a "pre" sequence or "signal" sequence functionally linked to the amino terminus of the protease. the prosecution. "Expression vector" refers to a DNA construct that contains a DNA sequence that is functionally linked to a suitable control sequence, capable of effecting the expression of that DNA in a suitable host. Those control sequences include a promoter to effect transcription, an optional operator sequence to control that transcription, a sequence encoding the appropriate mRNA ribosome binding sites and sequences, which control the termination of transcription and translation. The vector can be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or it can, in some cases, be integrated into the genome itself. In the present specification "plasmid" and "vector" are sometimes used interchangeably. since the plasmid is the most commonly used form of the vector, currently. However, the invention is intended to include those forms of expression vectors that serve equivalent functions and which are, or become, known in the art. The "host cells" used in the present invention are generally prokaryotic or eukaryotic hosts that have been preferably manipulated through methods described in US Patent RE 34,606 to render them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing the protease is Bacillus strain BG2036 which has deficiency in enzymatically active neutral protease and in alkaline protein (subtilisin). The construction of strain BG2036 is described in detail in U.S. Patent No. 5,264,366. Other host cells for expressing the protease include Bacillus subtilis 1168 (also described in US Patent No. 34,606 and in US Patent No. 5,264,366, the disclosure of which is incorporated herein by reference), as well as any Bacillus strain. adequate such as B. lichenif ormi s r B. lentus, etc. Host cells are transformed or transfected with constructed vectors, using recombinant DNA techniques. These transformed host cells are capable of either replicating vectors encoding the protease variants, or of expressing the desired protease variant. In the case of vectors encoding the preform or prepro form of the protease variant, those variants, when expressed, are typically secreted from the host cell into the host cell's medium. "Functionally linked", when describing the relationship between two DNA regions, simply means that they are functionally related to one another. For example, a presequence is functionally linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most likely involving the breakdown of the signal sequence. A promoter is functionally linked to a coding sequence if it controls the transcription of the sequence; A ribosome binding site is functionally linked to a coding sequence if it is located in a manner that allows translation. Genes encoding the naturally occurring precursor protease can be obtained according to methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes that have putative sequences that encode regions of the protease of interest, prepare genomic libraries of organisms that express the protease, and selectively classify the libraries for the gene of interest by hybridizing the probes. Hybridized clones are altered and sequenced later. 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 is replicated in hosts, in the sense that it contains the well-known elements necessary for plasmid replication: a promoter functionally linked to the gene in question (which can be delivered as the gene's own homologous promoter if recognized, is said, transcribed, by the host), a transcription termination and a polyadenylation region (necessary for the stability of the mRNA transcribed by the host, of the protease gene, in certain eukaryotic host cells that is either exogenous or is supplied by the region) endogenous terminator of the protease gene and, desirably, a selection gene such as a gene with resistance to antibiotics, which allows the continuous maintenance of the culture of the host cells infected by the plasmid, by growth in media containing anti-obesity ico s Plasmids with high copy number also contain an origin of replication for the host, allowing thus, a large number of plasmids are generated in the cytoplasm without chromosomal limitations. However, it is within the scope of the present invention to integrate multiple copies of the protease gene into the host genome. This is facilitated by prokaryotic and eukaryotic organisms that are particularly susceptible to homogenous recombination. The genes can be a B gene. l in natural t. Alternatively, a synthetic gene encoding a mutant precursor protease or occurring naturally can be produced. In this approach, the DNA and / or amino acid sequence of the precursor protease is determined. Subsequently, multiple DNA fragments, single-stranded, synthetic, overlapping, are synthesized, which with hybridization and ligation produce a synthetic DNA that encodes the precursor protease. An example of synthetic gene construction is presented in Example 3 of U.S. Patent No. 5,204,015, the disclosure of which is incorporated herein by reference. Once the precursor, synthetic, or naturally occurring protease gene has been cloned, a number of modifications are carried out to improve the use of the gene beyond the synthesis of the naturally occurring precursor protease. Such modifications include the production of recombinant proteases as described in US Patent No. RE 34,606 and EPO Publication No. 0 251 446 and the production of protease variants described herein. The following example of cassette mutagenesis can be used to facilitate the construction of the protease variants of the present invention, although other methods can be used. First, the gene that occurs naturally and that encodes the protease is obtained, and is subjected to sequencing totally or in part. Then the sequence is scanned for a point at which it is desired to carry out a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded enzyme.

The sequences that flank this point are evaluated with respect to the presence of the restriction sites to re-place a short segment of the gene with a stagnation of oligonucleotides that when expressed will encode several mutants. These restriction sites are preferably unique sites within the protease gene, in order to facilitate the replacement of the segment of the gene. However, any restriction site that is not too redundant in the protease gene can be used, with the proviso that the fragments of the gene, generated by the restriction digestion, can be reassembled in the proper sequence. If the restriction sites are not present at sites within a convenient distance from the selected point (10 to 15 nucleotides), those sites are generated by substituting nucleotides in the gene, in such a way that neither the reading frame nor the encoded amino acids, are changed in the final construction. The mutation of the gene, to change its sequence and adapt to the desired sequence, is achieved by the extension of the M13 primer, in accordance with generally known methods. The task of locating suitable flank regions and evaluating the changes necessary to arrive at two sequences of convenient restriction sites 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 flank restriction site is available, the above method needs to be used only in relation to the flank region that does not contain a site. Once 1"synthetic DNA or naturally occurring DNA is cloned, the restriction sites flanking the positions to be mutated, are digested with the innate restriction enzymes, and a plurality of complementary oligonucleotide cassettes, of the extreme terms, they are linked in the gene.The mutagenesis is simplified by this method, because all the oligonucleotides can be synthesized so that they have the same restriction sites, and synthetic binders are not necessary, to create the sites of restriction.

As used herein, proteolytic activity is defined as the rate of hydrolysis of peptide bonds per milligram of active enzyme. There are many well-known procedures for measuring proteolytic activity (K.M. Kalisz. "Microbial Proteinases "Advances in Biochemi cal Engineering / Bio technology, A. Fiechter ed., 1988). In addition to, or as an alternative to, the modified proteolytic activity, the variant enzymes of the present invention may have other modified properties such as Km, Koat, Kcat / Km ratio and / or the specificity for the modified substrate and / or the activity profile in the pH, modified. These enzymes can be produced for the particular substrate that is anticipated to be present, for example, in the preparation of peptides or for hydrolytic processes such as laundry uses. In one aspect of the invention, the aim is to ensure a protease variant having an altered, preferably improved wash performance compared to a precursor protease, at least in a detergent formulation and under at least one set of washing conditions. There are a variety of washing conditions, including variable detergent formulations, the volume of the wash water, the temperature of the wash water and the wash time, to which a protease variant may be exposed. For example, formulations of detergents used in different areas have different concentrations of their relevant components that are present in the wash water. For example, a European detergent typically has from about 4,500 to 5,000 ppm of detergent components, in the wash water, while a. Japanese detergent typically has approximately 667 ppm of detergent components in the wash water. In North America, particularly in the United States, a detergent typically has approximately 975 pppi of detergent components present in the wash water. A low concentration system includes detergents where less than about 800 ppm of the detergent components are present in the wash water. Japanese detergents are typically considered as a low detergent concentration system since they have approximately 667 ppm of detergent components present in the wash water. An average detergent concentration system includes detergents wherein between about 800 ppm and 2,000 ppm of detergent components are present in the wash water. American detergents are generally considered to be medium concentration detergent systems since they have approximately 975 ppm of detergent components present in the wash water. The systems in Brazil have. typically about 1,500 ppm of detergent components present in the wash water. A high detergent concentration system includes detergents where more than about 2,000 ppm of the detergent components are present in the wash water. European detergents are generally considered as high concentration detergent systems since they have approximately 4,500 to 5,000 ppm of detergent components in the wash water. Latin American detergents are generally detergents with phosphate additives, which produce a lot of foam, and the range of detergents used in Latin America can fall in both average and high detergent concentrations, since they vary from 1,500 to 6,000 ppm of detergent components in the wash water. As mentioned above, Brazil typically has approximately 1,500 ppm of detergent components present in the wash water. However, other geographies with detergents that have high-foam phosphate additives, not limited to other Latin American countries, can have systems of high concentration of detergents of up to approximately 6,000 ppm of detergent components present in the wash water. In view of the foregoing, it is evident that the concentrations of the detergent compositions in the typical washing solutions, throughout the world, vary from less than about 800 ppm of detergent composition ("geographies with low detergent concentration"), for example from approximately 667 ppm in Japan, to approximately between 800 ppm and approximately 2,000 ppm ("geographies with average detergent concentrations"), for example from approximately 975 ppm in the United States and approximately 1,500 ppm in Brazil, up to more than approximately 2,000 ppm ("geographies with high concentration of detergents"), for example from approximately 4,500 ppm to approximately 5,000 ppm in Europe and approximately 6,000 ppm in geographies with high foam phosphate additives. The concentrations of the typical washing solutions are determined empirically. For example, in the United States, a typical washing machine contains a volume of approximately 64.4 liters of washing solution. Accordingly, to obtain a concentration of about 975 ppm of detergent, within the wash solution, approximately 62.79 g of detergent composition should be added to the 64.4 liters of wash solution. This amount is the typical amount measured in the wash water by the consumer using the measuring cup provided with the detergent. As an additional example, different geographies use different wash temperatures. The temperature of the wash water in Japan is typically lower than that used in Europe Accordingly, one aspect of the present invention includes a protease variant that exhibits improved wash characteristics in at least one set of wash conditions. In another aspect of the invention, it has been determined that the substitution of an amino acid in one or more residue positions corresponding to the residue positions selected from the group consisting of 62, 212, 230, 232, 252 and 257 of the subtilisin of Ba ci llus amyl ol i qu efa ci on s, are important to improve the washing characteristics of the enzyme. Based on the results of selective classification obtained with the variant proteases, the mutations observed in the subtilisin of Ba ci lyl amyl ol i qu efa ci in s, are important for the proteolytic activity, for the characteristics and / or stability of these enzymes and the cleaning or washing characteristics of these enzyme variants. Many of these protease variants of the invention are useful in the formulation of "various detergent compositions or personal care formulations, such as shampoos or lotions." A number of known compounds are useful and suitable surfactants in compositions comprising the mutants of protease of the invention These include nonionic, anionic, cationic or amphoteric ionic detergents as described in U.S. Patent No. 4,404,128-issued to Barry J. Anderson and U.S. Patent No. 4,261,868 issued to Jiri Flora, et al. A suitable detergent formulation is that described in Example 7 of US Patent No. 5,204,015 (previously incorporated by reference.) The technique is familiar to the different formulations that can be used as cleaning compositions. typical cleaning agents it is easily understood that the protease variants of the present and invention can be used for any purpose for which native or wild-type proteases are used. In this way, these variants can be used, for example, in bar or liquid soap applications, formulations for cleaning dishes, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, applications in products textiles, as fusion break enzymes in the production of proteins, etc. The variants of the present invention may comprise improved characteristics, in some detergent composition (if compared to the precursors). As used herein, improved performance in a detergent is defined as the increase in cleaning, of certain enzyme sensitive inks such as grass or blood, as determined by the usual evaluation after the standard wash cycle. The proteases of the invention can be formulated in known liquid and powdered detergents, having a pH between 6.5 and 12.0 at levels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight. These cleansing detergent compositions may also include other enzymes such as known proteases, amylases, celluloses, lipases or endoglycosidases, as well as additives and stabilizers. The addition of proteases of the invention to conventional cleaning compositions does not create any special limitations on use. In other words, any suitable temperature and pH for the detergent are also suitable for the compositions herein, as long as the pH remains within the above range, and as long as the temperature is below the denaturing temperature of the proteases, described. In addition, the proteases of the invention can be used in a cleaning composition without detergents, again, either alone or in combination with additives and stabilizers. The present invention also relates to cleaning compositions containing the protease variants of the invention. The cleaning compositions may also contain additives that are commonly used in cleaning compositions. These can be selected from, although not limited to, bleaches, surfactants, additives, enzymes and bleaching catalysts. It will be apparent to a person of ordinary skill in the art which additives will be suitable for inclusion in the compositions. The list provided herein is not intended to be exhaustive and should be considered as examples of suitable additives. It will also be apparent to a person of ordinary skill in the art to use only those additives that are compatible with the enzymes and other components that are in the composition, for example, the surfactant. When present, the amount of additive present in the cleaning compositions is from about 0.01% to about 99.9%, preferably from about 1% to about 95, more preferably from about 1% to about 80%. The variant proteases of the present invention can be included in animal feeds, as part of the feed additives, as described, for example, in U.S. Patent No. 5,612,055; U.S. Patent No. 5,314,692; and U.S. Patent No. 5,147,642. One aspect of the invention is a composition for the treatment of a textile product, which includes the variants 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 intended to limit the scope of the indications. All the publications and patents to which reference has been made, 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 the subtilisin of Bacillus lentus GG36. The variants are presented in Table 4.

XI Ex empí o 2 A large number of protease variants, produced in Example 1, were analyzed with respect to their performance characteristics in two types of detergents and washing conditions, using a test of my fabric chromosome, described in "an improved method for assay a preferred enzyme and / or preferred detergent composition ", US Patent Serial No. 60 / 068,796. Table 5 lists the protease variants tested and the results of the analysis in two different detergents. For column A, the detergent was Ariel Ultra, filtered, 0.67 g / 1 (Procter &Gamble, Cincinnati, OH, United States of America), and a solution containing 3 grains 3.785 liters (one gallon) of hardness. Ca2 + / Mg2 + mixed, and 0.3 ppm of enzymes were used in each well, at 20 ° C. For column B, the detergent was Ariel Futur, filtered, of 3.38 g / 1 (Procter &Gamble, Cincinnati, OH, United States of America), in a solution containing 15 grains per 3,785 liters (one gallon) of mixed Ca2 + / Mg2 + hardness, and 0.3 ppm of enzymes were used in each well at 40 ° C.

E j us 3 Table 6 lists the protease variants tested, of Example 1, and the results of the analysis in four different detergents. The same analysis of performance characteristics, which for Example 2, were carried out in the protease variants mentioned, with the following detergents. For column A, the detergent was Ariel Ultra (Procter &Gamble, Cincinnati, OH, United States of America) filtered, at 0.67 g / 1, in a solution containing a 3 grain hardness 3,785 liters (one gallon) of Ca2 + / Mg2 + mixed, and 0.3 ppm of enzymes were used in each well, at 20 ° C. For column B, the detergent was Ariel Futur (Procter &Gamble, Cincinnati, OH, United States of America) filtered, at 3.38 g / 1, in a solution containing 15 grains 3.785 liters (one gallon) of Ca2 + / Mg2 + mixed, and 0.3 ppm of enzymes were used in each well, at 40 ° C. For column C, the detergent was HSP1 (Procter &Gamble, Cincinnati, OH, United States of America) at 3.5 g / 1, in a solution containing a hardness of 8 grains 3.785 liters (one gallon) of Ca2 + / Mg2 + mixed, and 0.3 ppm of enzymes were used in each well at 20 ° C. For column D, the detergent was Tide KT (Procter &Gamble, Cincinnati, OH, United States of America) at 1.5 g / 1, in a solution containing a 3 grain hardness 3.785 liters (one gallon) of Ca2 + / Mg2 + mixed, and 0.3 ppm of enzymes were used in each well, at 20 ° C.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates. Having described the invention as above, the content of the following is claimed as property:

Claims (13)

1. A protease variant, characterized in that it comprises replacing an amino acid in one or more residue positions corresponding to the residue positions selected from the group consisting of 212, 232, 252 and 257 of the subtilisin of Bacillus amyl olicaecic ens.

2. The protease variant according to claim 1, characterized in that it is derived from the subtilisin of a Bacillus.

3. The protease variant according to claim 2, characterized in that it is derived from the subtilisin of Bacillus 1 entu s.

4. A DNA, characterized in that it encodes a protease variant according to claim 1.

An express ion vector, characterized in that it encodes the DNA according to claim 4.

6. A host cell, characterized in that it is transformed with the expression vector according to claim 5.

7. A cleaning composition, characterized in that it comprises the protease variant according to claim 1.

8. An animal feed, characterized in that it comprises the protease variant according to claim 1.

9. A composition for the treatment of a textile product, characterized in that it comprises the protease variant according to claim 1.

10. The protease variant according to claim. -1, characterized in that it comprises a set of substitutions, selected from the group consisting of the residue positions corresponding to the positions found in Table 1 of the subtilisin of Bacillus amyl ol i quefaci ens.

11. The protease variant according to claim 10, characterized in that it comprises a set of substitutions selected from the group consisting of residue positions corresponding to the positions found in Table 2, of the subtilisin of Bacillus amyl ol and quefa ci ens

12. The protease variant according to claim 10, characterized in that it comprises a set of substitutions selected from the group consisting of the residue positions corresponding to the positions found in Table 3, of the subtilisin of Bacillus amyl oliquefaciens.

13. The protease variant according to claim 1, characterized in that it also includes substitutions of amino acid residues that are in one or both of positions 62 and 230.