RU2269572C2 - Multisubstituted protease (variants) - Google Patents

Abstract

FIELD: biotechnology, light industry.

SUBSTANCE: invention relates to protease variants obtained by substitution in subtilisin amino acid sequence from Bacillus amyloliquefaciens, Baccilus subtilus, Baccilus licheniformis, and Baccilus lentus of amino acid residues in 103, 236 and/or 245 sites and in one ore more other sites followed by selection of mutants having improved washing action. DNA fragments encoding said protease variants and expression vector also are disclosed.

EFFECT: detergent compositions with improved washing action.

24 cl, 7 dwg, 5 tbl, 3 ex

Description

Related Applications

This application is a partial continuation of application for US patent No. 08/956323, filed October 23, 1998, application for US patent No. 08/956564, filed October 23, 1998, and patent application US No. 08/956324, filed October 23 1998, which are fully incorporated into this description by reference.

BACKGROUND OF THE INVENTION

Serine proteases are a subset of carbonyl hydrolases. They form a class of diverse enzymes with various specific and biological functions. Stroud R., Sci. Amer., 131: 74-88. Despite the functional diversity, the catalytic mechanism of serine proteases is inherent in at least two genetically different families of enzymes: 1) subtilisins and 2) related human chymotrypsin and homologous bacterial serine proteases (such as trypsin and trypsin S.gresius). These two families of serine proteases have extremely similar catalysis mechanisms. Kraut J., (1977), Annu. Rev. Biochem., 46: 331-358. In addition, although the primary structures of the enzymes of these families are not related, their tertiary structures have a conservative catalytic triad of amino acids consisting of serine, histidine, and aspartate.

Subtilisins are serine proteases (approximate molecular weight of 27,500), which are secreted in large quantities by various species of Bacillus and other microorganisms. The subtilisin protein sequence has been determined in at least nine different species of Bacillus. Markland, F.S., et al., (1983), Hoppe-Seyler's Z.Physiol.Chein., 364: 1537-1540. The scientific literature describes the three-dimensional crystallographic structure of subtilisins isolated from Bacillus amyloliquefaciens, Bacillus licheniformis, and several natural variants of B.lentus. These studies show that, although subtilisin does not have a genetic relationship with mammalian serine proteases, it has a similar active site structure. X-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus JD, et al., (1972), Biochemistry, 11: 2439-2449) or product complexes (Robertus JD, et al., (1976), J.Biol.Chem ., 251: 1097-1103), they also provided information on the active site and the putative binding site of subtilisin. In addition, a large number of studies have been conducted on the kinetic and chemical modifications of subtilisin; Svendsen B., (1976), Carlsberg Res. Commun., 41: 237-291; Markland FS, ibid.), While at least one scientific work describes the conversion of the side chain of methionine at the position of the residue 222 of subtilisin to methionine sulfoxide under the influence of hydrogen peroxide (Stauffer DC, et al., (1965), J. Biol. Chem. 244: 5333-5338) and extensive site-specific mutagenesis (Wells and Estell (1988), TIBS 13: 291-297).

Summary of the invention

The object of this invention are protease variants with amino acid substitution at the position of the residue corresponding to position 103 of the subtilisin of Bacillus amyloliquefaciens, and with the substitution of one or more amino acids at the positions of the residues selected from the group comprising the positions of the residues corresponding to positions 1, 3, 4, 8, 10 , 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75 , 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128 , 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188 , 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 21 5, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 subtilisin of Bacillus amyloliquefaciens: moreover, when the substitution is in the position corresponding to the position of residue 103 is combined with substitution at the position corresponding to the position of residue 76, substitution can be made at the positions of one or more residues other than the positions of residues corresponding to positions 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218, 222, 260, 265 or 274 subtilisin of Bacillus amyloliquefaciens.

Although any combination of the above amino acid substitutions may occur, the preferred enzymes of the protease variant of the present invention may be substituted with amino acid residues in the following combinations of positions. All of the indicated positions of the residues correspond to the provisions of the subtilisin of Bacillus amyloliquefaciens:

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

(2) a protease variant with substitutions of amino acid residues at positions 103 and 236 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270, and 275;

(3) a protease variant with substitutions of amino acid residues at positions 103 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131 , 159, 170, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 222, 230, 232, 248, 252, 257, 260, 261, 270 and 275; or

(4) a protease variant with substitutions of amino acid residues at positions 103, 236 and 245 and at one or more of the following positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130 , 131, 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 243, 248, 252, 257, 260, 270, and 275.

More preferred protease variants have a plurality of substitutions selected from the group consisting of the positions of Bacillus amyloliquefaciens subtilisin residues corresponding to the positions indicated in Table 1:

The most preferred protease variants are shown in table 3.

Another object of this invention are DNA sequences encoding such proteae variants, as well as expression vectors containing DNA sequences of such variants.

Another object of this invention are host cells transformed with such vectors, as well as host cells capable of expressing such DNA to produce intracellular or extracellular protease variants.

The present invention further relates to a cleansing composition comprising a protease variant of the present invention.

In addition, this invention relates to animal feed containing a variant of the protease of the present invention.

The invention also relates to a composition for treating a tissue comprising a protease variant of the present invention.

Brief Description of the Drawings

Figure 1 AC shows the DNA sequence and amino acid sequence for subtilisin Bacillus amyloliquefaciens and a partial restriction map of this gene.

Figure 2 shows the conserved amino acid residues of subtilisins from Bacillus amyloliquefaciens (BPN) 'and Bacillus lentus (wild type).

3A and 3B show the amino acid sequences of four subtilisins. The top row refers to the amino acid sequence of subtilisin from Bacillus amyloliquefaciens (which is sometimes defined as subtilisin BPN '). The second row refers to the amino acid sequence of subtilisin from Bacillus subtilis. The third row relates to the amino acid sequence of subtilisin from B.licheniformis. The fourth row refers to the amino acid sequence of subtilisin from Bacillus lentus (which is called tayuke subtilisin 309 in PCT WO 89/06276). The symbol * means the absence of specific amino acid residues compared to subtilisin BPN '.

DETAILED DESCRIPTION OF THE INVENTION

Proteases are carbonyl hydrolases that typically cleave peptide bonds of proteins or peptides. As used herein, the term "protease" means a natural or recombinant protease. Natural proteases include α-aminoacyl peptide hydrolase, peptidyl amino acid hydrolase, acylaminohydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiolproteinase, carboxylproteinase and metalloproteinase. The scope of this invention includes serine proteases, metalloproteases, thiol proteases and acid proteases, as well as endo and exoproteases.

The present invention relates to protease enzymes that are not natural variants of carbonyl hydrolase (protease variants), since they have different proteolytic activity, resistance, substrate specificity, pH profile and / or efficiency compared to the carbonyl precursor hydrolase from which it is obtained amino acid sequence of the variant. In particular, such protease variants contain a non-naturally occurring amino acid sequence that is obtained by substituting several amino acid residues of the precursor protease with other amino acids. The precursor protease may be a natural or recombinant protease.

In the protease variants of the present invention, any of the nineteen naturally occurring L-amino acids at certain positions of the amino acid residues may be substituted. Such substitutions can be made in the precursor subtilisin (prokaryotic subtilisin, eukaryotic subtilisin, mammalian subtilisin, etc.). In this description of the invention, different amino acids are indicated by conventional one- and three-letter codes. Such codes are provided in Dale, M.W., (1989), Molecular Genetics of Bacteria, John & Sons, Ltd., Appendix B.

The protease variants of this invention are preferably derived from Bacillus subtilisin. More preferably, protease variants are derived from Bacillus lentus subtilisin and / or 309 subtilisin.

Subtilisins are proteases of bacteria or fungi that usually cleave peptide bonds of proteins or peptides. As used herein, the term "subtilisin" means natural or recombinant subtilisin. It is known that different types of microorganisms produce and often secrete a number of natural subtilisins. The amino acid sequences of these subtilisins are not completely homologous. However, the subtilisins included in this group have the same or similar proteolytic activity. This class of serine proteases has a common amino acid sequence defining a catalytic triad that distinguishes them from the class of serine proteases related to chymotrypsin. Both subtilisins and chymotrypsin-related serine proteases have a catalytic triad consisting of aspartate, histidine and serine. In proteases related to subtilisin, these amino acids, when read from the amino terminus to the carboxyl end, are located in the following relative order: aspartate-histidine-serine. However, chymotrypsin-related proteases have the following relative arrangement of these amino acids: histidine-aspartate-serine. Thus, subtilisin refers to a serine protease having a catalytic triad of subtilisin-related proteases. Examples of such subtilisins are, but are not limited to, the subtilisins shown in FIG. 3. The numbering of these amino acids in proteases is usually, and in accordance with the objectives of the present invention, corresponds to the numbers assigned to the sequence of mature subtilisin Bacillus amyloliquefaciens depicted in figure 1.

"Recombinant subtilisin" or "recombinant protease" means a subtilisin or protease in which the DNA sequence encoding a subtilisin or protease is modified to produce a variant (or mutant) of a DNA sequence encoding the substitution, deletion or insertion of one or more amino acids in a natural amino acid sequence . Acceptable methods of making such a modification, which can be used in combination with the methods described in this invention, include the methods described in US patent No. 34606, 5204015, 5185258, 5700676, 5801038 and 5763257.

Non-human subtilisins and their encoding DNA can be obtained from many prokaryotic and eukaryotic microorganisms. Suitable examples of prokaryotic microorganisms are gram-negative microorganisms, such as E. coli or Pseudomonas, and gram-positive bacteria, such as Micrococcus or Bacillus. Examples of eukaryotic microorganisms from which subtilisin and its genes can be obtained are yeast, such as Saccharomyces cerevlsiae, and fungi, such as Aspergillus sp.

A "protease variant" has an amino acid sequence derived from the amino acid sequence of a "precursor protease" The precursor proteases are natural and recombinant proteases. The amino acid sequence of a protease variant is obtained from the amino acid sequence of a precursor protease by substitution, deletion or insertion of one or more amino acids in the amino acid sequence of the precursor. This is achieved by modifying the “precursor DNA sequence” encoding the amino acid sequence of the precursor protease, rather than manipulating the precursor protease enzyme itself. Suitable methods for such a modification of the DNA precursor sequence include the methods described herein, as well as methods known to those skilled in the art (see, for example, European Patent No. 0328299, WO 89/06279 and the above US patents and patent applications).

In this specification, specific substitutions corresponding to position 103 are identified in combination with one or more of the following substitutions corresponding to positions 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22 , 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98 , 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146 , 147, 158, 159, 160, 166, 167, 170, 173, 174, .177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 subt Isigny Bacillus amyloliquefaciens.

Preferred options are those with a combination of substitutions at the positions of the residues corresponding to the positions of the subtilisin of Bacillus amyloliquefaciens in table 1. More preferred are those with the combinations of substitutions at the positions of the residues corresponding to the positions of the subtilisin of Bacillus amyloliquefaciens in table 3.

Other preferred options are those with combinations of substitutions at the positions of the residues corresponding to the subtilisin residues of Bacillus amyloliquefaciens in Table 2.

The position numbers of these amino acids refer to the numbers assigned to the mature subtilisin sequence of Bacillus amyloliguefaciens shown in FIG. However, this invention is not limited to the mature form of this subtilisin and includes precursor proteases containing amino acid residues at positions that are "equivalent" to the specific identified residues in Bacillus amyloliguefaciens subtilisin. In a preferred embodiment of the present invention, the precursor protease is Bacillus lentus subtilisin and substitutions are made at positions equivalent to the amino acid residues in B.lentus that correspond to the above.

The position of the residue (amino acid) of the precursor protease is equivalent to the position of the Bacillus amyloliquefaciens subtilisin residue if it is homologous (i.e., corresponds to the position in the primary or tertiary structure) or is similar to a specific residue or part of this residue in Bacillus amyloliquefaciens subtilisin (i.e. has the same or similar functional ability to connect, react or chemically interact).

To establish homology with the primary structure, the amino acid sequence of the precursor protease is compared directly with the primary sequence of the subtilisin of Bacillus amyloliquefaciens and, in particular, with a set of residues that are known to be unchanged in subtilisins and determine the nature of the sequence. For example, FIG. 2 shows conservative residues that are common to subtilisins of B. amyloliquefaciens and B.lentus. After performing a comparative analysis of the conserved residues, which allows to determine the insertions and deletions necessary to preserve the primary structure (that is, to avoid the removal of conservative residues as a result of random deletions and insertions), residues equivalent to certain amino acids in the primary sequence of Bacillus amyloliquefaciens subtilisin are determined.

A comparison of conserved residues should preferably reveal the presence of 100% of such residues. However, the presence of more than 75% or at least 50% of the conserved residues is also sufficient to determine equivalent residues. The catalytic triad Asp32 / His64 / Ser221 should be fully preserved. Siezen et al., (1991) Protein Eng., 4 (7): 719-737 performed a comparative analysis of a large number of serine proteases. Siezen et al. define this group as subtilases or subtilisin-like serine proteases.

For example, FIG. 3 shows a comparative analysis of the amino acid sequences of subtilisin from Bacillus amyloliguefaciens, Bacillus subtilis, Bacillus licheniformis (carlsbergensis) and Bacillus lentus, the purpose of which is to identify the maximum homology between these amino acid sequences. A comparison of these sequences shows that in each sequence there are several conserved residues. These conservative residues (for BPN 'and B.lentus) are presented in figure 2.

Thus, these conserved residues can be used to determine the corresponding equivalent amino acid residues of Bacillus amyloliquefaciens subtilisin in other subtilisins, such as subtilisin from Bacillus lentus (PCT publication No. WO 89/06279 of July 13, 1989), the preferred precursor protease enzyme, or subtilisin, defined as PB92 (European Patent No. 0328299), which is homologous to the preferred subtilisin Bacillus lentus. On figa and 3B shows a comparative analysis of the amino acid sequences of some of these subtilisins with the subtilisin sequence of Bacillus amyloliquefaciens, which allows to identify the maximum homology of conserved residues. As can be seen, the sequence of Bacillus lentus has a number of divisions in comparison with subtilisin of Bacillus amyloliquefaciens. For example, the amino acid for Vall65 in the subtilisin of Bacillus amyloliquefaciens is equivalent to isoleucine in the other subtilisins of B.lentus and B.licheniformis.

"Equivalent residues" can also be established by determining homology at the level of the tertiary structure for the precursor protease, the tertiary structure of which is investigated by x-ray crystallography. Equivalent residues are those residues for which the atomic coordinates of two or more atoms in the main chain of a certain amino acid residue of the precursor protease and subtilisin Bacillus amyloliquefaciens (N overlaps N, CA overlaps CA, C overlaps C and O overlaps O) after ordering are within 0 13 nm and preferably within 0.1 nm. The primary structure is ordered when the orientation and location of the best model provides maximum overlap of the atomic coordinates of non-hydrogen protein atoms in the protease and subtilisin of Bacillus amyloliquefaciens under consideration. The best model is a crystallographic model that allows you to obtain the smallest R-factor for the experimental diffraction data at the highest resolution.

Equivalent residues that are functionally similar to the specific subtilisin residue of Bacillus amyloliquefaciens are defined as precursor amino acids that can take on such a conformation that they can alter, modify, or stimulate the formation of a protein structure, binding to a substrate, or catalysis similar to a specific and inherent specific residue subtilisin Bacillus amyloliquefaciens. In addition, such residues are precursor protease residues (the tertiary structure of which is established by X-ray crystallography), occupying a similar position in which the atomic coordinates of at least two atoms in the side chain of the residue are 0.13 nm from the corresponding atoms in the side chain of subtilisin Bacillus amyloliquefaciens, although the atoms in the main chain of this residue may not satisfy the equivalence criterion in terms of position homology. The coordinates of the three-dimensional structure of the subtilisin Bacillus amyloliquefaciens are given in EPO publication No. 0251446 (corresponding to US patent No. 5182204, which is incorporated into this description by reference), and, as indicated above, can be used to determine equivalent residues at the level of the tertiary structure.

Some substitutable residues are conserved residues, while other residues are not. In the case of residues that are not conserved, the substitution of one or more amino acids is limited to substitutions, allowing you to get a variant having an amino acid sequence that does not correspond to the natural sequence. In the case of conserved residues, such substitutions should not lead to the formation of a natural sequence. The protease variants of the present invention include mature forms of protease variants, as well as the pro- and preproforms of such protease variants. Preproforms are the preferred construct since they facilitate the expression, secretion and maturation of protease variants.

"Sequence" means the sequence of amino acids associated with the N-terminal portion of the mature form of the protease, the removal of which causes the appearance of a "mature" form of the protease. Many proteolytic enzymes are found in nature in the form of proenzyme translation products and, in the absence of post-translational processing, they are expressed in this form. A preferred sequence for preparing protease variants is the putative sequence of Bacillus amyloliquefaciens subtilisin, although other protease sequences can be used.

"Signal sequence" or "presequence" means any sequence of amino acids associated with the N-terminal portion of the protease or the N-terminal portion of the protease that may participate in the secretion of the mature form or form of the protease. This determination of the signal sequence is functional and includes all amino acid sequences encoded by the N-terminal portion of the protease gene that are involved in the secretion of the protease in vivo. Such sequences are used in the present invention to secrete the protease variants described herein. One acceptable signal sequence contains the first seven amino acid residues of the signal sequence isolated from Bacillus subtilis subtilisin fused to the rest of the Bacillus lentus subtilisin signal sequence (ATCC 21536).

A "prepro" form of a protease variant contains a mature form of a protease having a pro-sequence operably linked to the amino terminus of the protease, and a "pre" or "signal" sequence operably linked to the amino-terminus of the pro-sequence.

“Expression vector” means a DNA-based construct containing a DNA sequence that is operably linked to an acceptable regulatory sequence capable of expressing said DNA in an acceptable host. Such regulatory sequences include a transcriptional promoter, an optional transcriptional control operator sequence, a sequence encoding the ribosome binding sites of acceptable mRNA, and transcriptional and translational termination control sequences. The vector may be a plasmid, phage particle, or simply a potential genomic insertion. After administration to an acceptable host, the vector can replicate and function independently of the host genome, and in some cases, it can integrate into the genome itself. In this specification, the terms “plasmid” and “vector” are sometimes used interchangeably as the plasmid is the most commonly used form of the vector at present. However, other forms of expression vectors that perform equivalent functions and are known in the art are also within the scope of this invention.

The "host cells" used in the present invention are typically prokaryotic or eukaryotic hosts, which are preferably treated according to the methods described in US Pat. No. 3,4606, whereby they lose the ability to secrete an enzymatically active endoprotease. A preferred host cell for protease expression is Bacillus strain BG2036, which lacks an enzymatically active neutral protease and alkaline protease (subtilisin). The construction of strain BG2036 is described in detail in US patent No. 5264366. Other host cells for the expression of the protease are Bacillus subtilis I168 (which are also described in US patent No. 34606 and 5264366 included in this description by reference), as well as any suitable strain of Bacillus, such as B.licheniformis, B. lentus etc.

Host cells are transformed or transfected with vectors constructed by recombinant DNA methods. Such transformed host cells are capable of replicating vectors encoding protease variants or expressing the desired protease variant. In the case of vectors encoding the pre- or preproform of a protease variant, such variants, when expressed, are usually secreted from the host cell into the medium containing the host cells.

The term “operably linked”, used to describe the relationship between two regions of DNA, means that they are functionally related to each other. For example, a presequence is operably linked to a peptide if it acts as a signal sequence involved in the secretion of the mature form of the protein, causing cleavage of the specified signal sequence. A promoter is operably linked to a coding sequence if it controls sequence transcription; the ribosome binding site is operably linked to the coding sequence, if its position makes translation possible.

Genes encoding a natural precursor protease can be obtained by conventional methods known in the art. These methods typically include the synthesis of labeled probes having imaginary sequences encoding regions of the protease of interest, creating genomic libraries of microorganisms expressing the protease, and screening these libraries for the desired gene by hybridization with the probes. Then, positively hybridizing clones are mapped and sequenced.

The cloned protease is used to transform the host cell to express the protease. The protease gene is then ligated into a large copy number plasmid. This plasmid is replicated in the hosts, because it contains well-known elements necessary for the replication of the plasmid: a promoter operably linked to the gene in question (which can be introduced as a homologous promoter of a gene, if it is recognized, that is, transcribed, by the host), the termination region transcription and the polyadenylation region (which is necessary to ensure the stability of the mRNA transcribed by the host from the protease gene in certain eukaryotic host cells), which is exogenous or is supported by the endogenous region of the protease gene terminator, and a selectable gene, in particular an antibiotic resistance gene, is desirable, which makes it possible to continuously cultivate plasmid-infected host cells by growing in media containing antibiotics. Plasmids with a large number of copies also have a source of replication for the host, which allows to obtain large quantities of plasmids in the cytoplasm without chromosomal restrictions. However, the present invention also relates to the integration of several copies of the protease gene into the host genome. This is facilitated by prokaryotic and eukaryotic microorganisms, which are particularly susceptible to homologous recombination.

The gene may be a natural B.lentus gene. Alternatively, a synthetic gene encoding a natural or mutant precursor protease can be obtained. In this case, the DNA sequence and / or amino acid sequence of the precursor protease is determined. Then several overlapping fragments of synthetic single-stranded DNA are synthesized, which, after hybridization and ligation, form synthetic DNA encoding a precursor protease. An example of constructing a synthetic gene is given in Example 3 of US Pat. No. 5,204,015, which is incorporated herein by reference.

After cloning a natural or synthetic precursor protease gene, a number of modifications are performed to enhance gene synthesis compared to a natural precursor protease. Such modifications include the production of recombinant proteases, as described in US patent No. RE 34606 and in publication EPO No. 0251446, as well as the production of protease variants described here.

You can use the following method of cassette mutagenesis, which facilitates the construction of variants of the protease of the present invention, although it is possible to use other methods. First, a natural gene encoding a protease is obtained, which is sequenced in whole or in part. The sequence is then scanned to find the point at which it is desirable to mutate (deletion, insertion or substitution) of one or more amino acids in the encoded enzyme. The sequences flanking this point are examined for the presence of restriction sites to replace a short gene segment with a pool of oligonucleotides, which in the expressed state must encode different mutants. Such restriction sites are preferably unique saigas in the protease gene, facilitating the replacement of a gene segment. However, any convenient restriction site that is not redundant in the protease gene can be used, provided that the fragments of the gene resulting from the restriction can be reassembled into the desired sequence. If there are no restriction sites in places located at a convenient distance from the selected point (from 10 to 15 nucleotides), such sites can be created by replacing the nucleotides in the gene so that neither the reading frame nor the encoded amino acids are changed in the final construct. Mutation of a gene in order to change its sequence so that it matches the desired sequence is carried out using primer M13 in accordance with known methods. The task of localizing acceptable flanking regions and determining the necessary changes to obtain two convenient sequences at the restriction site is greatly facilitated by the presence of an excessive genetic code, a restriction map of the gene enzyme and a large number of different restriction enzymes. It should be noted that in the presence of a convenient flanking restriction site, the above method should be used only in the case of a flanking region that does not have the desired site.

After cloning of natural or synthetic DNA, the restriction sites flanking the positions intended for mutation are cleaved by related restriction enzymes and several oligonucleotide cassettes complementary to the terminal regions are ligated into the gene. This method allows to simplify mutagenesis, since all oligonucleotides can be synthesized so that they have the same restriction sites, and synthetic linkers are not required to create restriction sites.

As used herein, the term "proteolytic activity" means the rate of hydrolysis of peptide bonds per milligram of active enzyme. Many methods are known for measuring proteolytic activity (K.M. Kalisz, "Microbial Proteinases", Advances in Biochemical Engineering / Biotechnology, A.Fiechter ed., 1988). In addition to or as an alternative to the modified proteolytic activity, other properties such as K _m , k _cat , k _cat / K _m ratio, substrate specificity and / or activity profile depending on pH can be changed in the enzyme variants of the present invention. These enzymes can be designed for a specific substrate, which is expected to take place, for example, in the preparation of peptides or in hydrolytic processes used in washing.

In one aspect of this invention, the aim is to provide a protease variant with altered, preferably improved detergent characteristics, as compared to the precursor protease in at least one detergent composition and / or at least one wash schedule.

The washing conditions to which the protease variant may be exposed are very diverse, including different detergents, different volumes of washing water, different temperatures of washing water and duration of washing. For example, detergent compositions used in different regions are characterized by different concentrations of detergent components in the washing water. For example, detergents used in Europe usually contain about 4,500-5,000 parts per million detergent components in washing water, while detergents used in Japan usually contain about 667 parts per million detergent components in washing water. In North America, in particular in the USA, detergents usually contain about 975 parts per million detergent components in washing water.

A low detergent concentration system includes detergents characterized by less than 800 parts per million detergent components in the washing water. Japanese detergents are generally considered low detergent systems because they contain approximately 667 parts per million detergent components in washing water.

A system with an average concentration of detergent includes detergents characterized by the presence of from about 800 to about 2000 parts per million detergent components in the washing water. Detergents used in North America are generally considered systems with an average concentration of detergent, as they contain approximately 975 parts per million detergent components in the washing water. In Brazil, detergents containing approximately 1,500 parts per million detergent components in washing water are commonly used.

A system with a high concentration of detergent includes detergents characterized by the presence of more than 2000 parts per million detergent components in the washing water. European detergents are usually considered systems with a high concentration of detergent, as they contain approximately 4,500-5,000 parts per million detergent components in the washing water.

The detergents used in Latin America are usually high-foam, phosphate-modified detergents, so they can be classified as systems with medium and high concentrations of detergent, as they are characterized by the presence of from 1500 to 6000 parts per million detergent components in washing water. As indicated above, Brazilian detergents typically contain approximately 1,500 parts per million detergent components in the wash water. However, in other regions that are characterized by the use of high-foam phosphate-modified detergents, which are not limited to Latin America, systems with a high concentration of detergent containing up to 6,000 parts per million detergent components in washing water can be used.

In light of the foregoing, it becomes apparent that detergent concentrations in typical washing solutions used in various regions of the world vary from less than 800 ppm (“regions where low detergent concentrations are used”), for example, about 667 ppm in Japan , up to about 800-2000 ppm ("regions of application of systems with an average concentration of detergent"), for example, about 975 ppm in the USA and about 1,500 parts per million in Brazil, and more than 2,000 ppm ("regions of application of systems you Oka detergent concentration "), such as about 4500-5000 ppm in Europe and about 6000 ppm in the application regions vysokopennyh modified phosphate detergents.

Concentrations of conventional washing solutions are determined empirically. For example, in the United States, a conventional washing machine holds approximately 64.4 liters of washing solution. Thus, in order to obtain a concentration of approximately 975 parts per million detergent in the washing solution, approximately 62.79 g of the washing composition must be added to 64.4 L of the washing solution. It is this quantity that the consumer measures with the help of a measuring cup attached to the detergent.

Another example is the use of different washing temperatures in different regions. The temperature of washing water in Japan is usually lower than in Europe.

According to one aspect, the present invention includes a variant of the protease, which has a higher detergent effect in at least one washing mode.

Another objective of this invention is the substitution in the position corresponding to position 103, in combination with one or more substitutions selected from the group including positions corresponding to positions 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 26 8, 269, 270, 271, 272, 274 and 275 subtilisin of Bacillus amyloliquefaciens, which is important for improving the washing action of this enzyme.

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

Based on the results obtained in the study of protease variants, it can be noted that these mutations in the subtilisin of Bacillus amyloliquefaciens are important for the proteolytic activity, effectiveness and / or stability of these enzymes, as well as for the cleansing or washing action of such enzyme variants.

Many protease variants of this invention are useful for preparing various detergent compositions or personal care products, such as shampoos or lotions. In compositions containing the protease mutants of this invention, a number of known compounds that are acceptable surfactants can be used. These substances are nonionic, anionic, cationic or zwitterionic detergents described in US Pat. No. 4,404,128 to Barry J. Anderson and US Pat. No. 4,262,868 to Jiri Flora et al. A suitable detergent composition is described in Example 7 of US Pat. No. 5,204,015 (previously incorporated herein by reference). Various formulations are known in the art that can be used as cleansing compositions. It is understood that, in addition to conventional cleansing compositions, the protease variants of this invention can be used in any field where native proteases or wild-type proteases are used. So, these options can be used, for example, in applications of solid or liquid soap, dishwashing preparations, solutions or products for cleaning contact lenses, compositions for hydrolysis of peptides, waste disposal, tissue processing, as well as cleaving enzymes for the production of proteins and etc. Variants of the present invention may impart higher efficacy to the detergent composition (compared to the precursor). Higher detergent efficacy is defined as better removal of stains susceptible to a particular enzyme, such as plant stains or blood stains, according to a routine assessment after a standard wash cycle.

The proteases of this invention may be included in known powder and liquid detergents with a pH of from 6.5 to 12.0 and a concentration of from about 0.01% by weight. up to about 5% weight. (preferably from 0.1% to 0.5%). These detergent cleansing compositions may contain other enzymes, in particular known proteases, amylases, cellulases, lipases or endoglycosidases, as well as additives and stabilizers.

The incorporation of the proteases of this invention into conventional cleansing compositions is not subject to any particular limitations. In other words, any temperature and pH values acceptable for a given detergent are also acceptable for the compositions of the present invention if the pH is within the above range and this temperature is below the denaturation temperature of the described protease. In addition, the proteases of this invention can be used in a cleaning composition without detergents, alone or in combination with additives and stabilizers.

The present invention also relates to cleansing compositions comprising the protease variants of this invention. Cleansing compositions may additionally contain additives that are commonly used in such compositions. These additives include, but are not limited to, bleaches, surfactants, modifiers, enzymes, and bleach catalysts. One skilled in the art will appreciate that additives are selected for their suitability for a given composition. The list here is not exhaustive and should only be considered as examples of acceptable additives. The person skilled in the art should also understand that it is necessary to use only those additives that are compatible with enzymes and other components of the composition, for example with a surfactant.

The amount of additive in the cleaning composition, if used, 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 protease variants of the present invention can be introduced into animal feed as part of the additives, as described, for example, in US patent No. 5612055, 5314692 and 5147642.

One aspect of the invention is a tissue treatment composition comprising the protease variants of the present invention. This composition can be used for processing, for example, silk or wool, as described in publications such as RD 216034, European patent No. 134267, US patent No. 4533359 and European patent No. 344259.

Further, the present invention is illustrated by examples, which do not limit the scope of the claims.

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

EXAMPLE 1

A large number of protease variants were obtained and purified using methods well known in the art. All mutations were made in the subtilisin GG36 of Bacillus lentus. These options are shown in table 3.

EXAMPLE 2

A large number of protease variants obtained in Example 1 were tested for effectiveness in two types of detergents and washing conditions using the microscopic analysis described in US patent No. 60/068796 "An improved method of assaying for a preferred enzyme and / or preferred detergent composition" .

Table 4 presents the protease variants analyzed and the test results in two different detergents. Column A shows the data for a filtered detergent with a concentration of 0.67 g / l Ariel Ultra (Procter & Gamble, Cincinnati, Ohio, USA) in a solution containing mixed hardness of 42.78 g / m ³ (3 grains / gallon) Ca ²⁺ / Mg ²⁺ and 0.3 ppm of enzyme in each container at 20 ° C. Column B shows data for a filtered detergent with a concentration of 3.38 g / l Ariel Futur (Procter & Gamble, Cincinnati, Ohio, USA) in a solution containing 213.9 g / m ³ (15 grains / gallon) of mixed hardness Ca ²⁺ / Mg ²⁺ and 0.3 ppm of enzyme in each container at 40 ° C.

EXAMPLE 3

Table 5 presents the analyzed protease variants of Example 1 and the test results in four different detergents. These protease variants were subjected to the same efficacy tests as in Example 2 using the following detergents. Column A shows the data for a filtered detergent with a concentration of 0.67 g / l Ariel Ultra (Procter & Gamble, Cincinnati, Ohio, USA) in a solution containing mixed hardness of 42.78 g / m ³ (3 grains / gallon) Ca ²⁺ / Mg ²⁺ and 0.3 ppm of enzyme in each container at 20 ° C. Column B shows data for a filtered detergent with a concentration of 3.38 g / l Ariel Futur (Procter & Gamble, Cincinnati, Ohio, USA) in a solution containing 213.9 g / m ³ (15 grains / gallon) of mixed hardness Ca ²⁺ / Mg ²⁺ and 0.3 ppm of enzyme in each container at 40 ° C. Column C shows data for a detergent with a concentration of 3.5 g / L HSP1 (Procter & Gamble, Cincinnati, Ohio, USA) in a solution containing 114.08 g / m ³ (8 grains / gallon) of mixed hardness Ca ^{2 +} / Mg ²⁺ and 0.3 ppm of enzyme in each container at 20 ° C. Column D shows the data for a detergent with a concentration of 1.5 g / L Tide CT (Procter & Gamble, Cincinnati, Ohio, USA) in a solution containing 42.78 g / m ³ (3 grains / gallon) of mixed hardness Ca ²⁺ / Mg ²⁺ and 0.3 ppm of enzyme in each container at 20 ° C.

Claims (24)

1. A protease variant with an amino acid sequence selected from the group of sequences shown in FIG. 3, having an amino acid substitution at the residue position corresponding to positions 103, 236 and / or 245, and one or more amino acids at the positions of residues selected from the group, consisting of the provisions of residues corresponding to the provisions of 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48 , 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111 , 114, 116, 117, 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170, 173 , 174, 177, 181, 182, 183, 1 84, 185, 188, 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228, 230, 232, 237, 238, 240, 242, 243, 244, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275, where the specified variant of the protease has a higher washing effect than the natural protease. 2. The protease variant of claim 1, obtained from Bacillus subtilisin. 3. The protease variant according to claim 2, obtained from subtilisin of Bacillus lentus. 4. The protease variant of claim 1, wherein the amino acid residues corresponding to positions 103 and 245 are replaced. 5. The protease variant according to claim 4, where the replacement further includes replacing an amino acid residue selected from the group consisting of the positions of the residues corresponding to positions 68, 76, 104, 159, 212, 222, 230, 232, 236, 248 and 252. 6. The protease variant according to claim 4, further comprising replacing the amino acid residue corresponding to position 159. 7. The protease variant of claim 1, wherein the amino acid residues corresponding to positions 103 and 236 are replaced. 8. The protease variant according to claim 7, where the replacement further includes replacing an amino acid residue selected from the group consisting of the positions of the residues corresponding to positions 68, 76, 104, 159, 212, 230, 232, 245, 248 and 252. 9. The protease variant of claim 7, further comprising replacing the amino acid residue corresponding to position 159. 10. The protease variant according to claim 9, further comprising replacing the amino acid residue corresponding to position 104, 232 and 245. 11. The protease variant according to claim 1, containing a set of substitutions selected from the group consisting of a set of positions of the residues indicated in each row of table 1, where the position number of the residue corresponds to the position in the amino acid sequences of subtilisin: Bacillus amyloliquefaciens (Figure 3). 12. The protease variant according to claim 11, containing a set of substitutions selected from the group consisting of sets of positions of the residues indicated in each row of Table 2, where the position number of the residue corresponds to the position in the amino acid sequences of Bacillus amyloliquefaciens subtilisin (Figure 3). 13. The protease variant according to claim 11, containing a set of substitutions selected from the group consisting of the positions of the residues indicated in each row of Table 3, where the position number of the residue corresponds to the position in the amino acid sequences of the subtilisin Bacillus amyloliquefaciens (Figure 3). 14. A variant of a protease with an amino acid sequence selected from the group of sequences shown in FIG. 3, having an amino acid substitution at a residue position corresponding to positions 103 and 236, and one or more amino acid substitutions at a residue position selected from the group consisting of residue positions corresponding to the provisions of 1, 8, 9, 10, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 211 , 212, 213, 215, 217, 230, 232, 248, 252, 257, 260, 270, and 275, wherein said protease variant has a higher detergent action than natural protease. 15. The protease variant of claim 14, further comprising replacing the amino acid residue at position 159. 16. A protease variant with an amino acid sequence selected from the group of sequences shown in FIG. 3, having an amino acid substitution at a residue position corresponding to positions 103 and 245, and one or more amino acid substitutions at a residue position selected from the group consisting of residue positions corresponding to the provisions of 1, 8, 9, 10, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 170, 183, 185, 205, 209, 210 , 211, 212, 213, 215, 217, 222, 230, 232, 248, 252, 257, 260, 261, 270 and 275, where the indicated variant of the protease has a higher washing effect than the natural prot ease. 17. The protease variant according to clause 16, further comprising replacing the amino acid residue at position 159. 18. A variant of a protease with an amino acid sequence selected from the group of sequences shown in FIG. 3, comprising replacing an amino acid at a residue position corresponding to positions 103, 236 and 245, and replacing one or more amino acids at a position of residues selected from the group consisting of positions of residues corresponding to positions 1, 8, 9, 10, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210 , 211, 212, 213, 215, 217, 230, 232, 243, 248, 252, 257, 260, 270 and 275 of the subtilisin Bacillus amyloliquefaciens (Figure 3), where the specified version of the prosthesis has a higher washing action than natural protease. 19. The protease variant of claim 18, further comprising replacing the amino acid residue at position 159. 20. A DNA fragment encoding a variant of the protease according to claim 1 and having a nucleotide sequence in accordance with the genetic code that determines the amino acid sequence of the variant of the protease according to claim 1. 21. A DNA fragment encoding a variant of the protease according to 14 and having a nucleotide sequence that, in accordance with the genetic code, determines the amino acid sequence of the variant of the protease according to 14. 22. The DNA fragment encoding the variant of the protease according to clause 16 and having a nucleotide sequence, in accordance with the genetic code, which determines the amino acid sequence of the variant of the protease according to clause 16. 23. A DNA fragment encoding the variant of the protease according to claim 18 and having a nucleotide sequence, in accordance with the genetic code, determining the amino acid sequence of the variant of the protease according to claim 18. 24. The expression vector comprising the sequence of the DNA fragment according to claim 20, functionally associated with regulatory sequences and providing expression of a variant of the protease according to claim 1.

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