RU2252958C2 - Subtilisin variant (variants), dna encoding the same, expression vector, cleaning composition, animal feed, composition for cloth treatment - Google Patents

Abstract

FIELD: enzymology, protein engineering, in particular enzyme with proteolysis activity.

SUBSTANCE: invention relates to new subtilisin variants obtained by substitution in amino acid sequence of wild-type enzyme following by certain charge alteration in corresponding site, namely negative charge increasing (positive charge decreasing) or vice versa. Subtilisin variants with the first-type substitutions have higher cleaning activities mainly in systems with law detergent concentrations. Subtilisin variants with the second-type substitutions have higher cleaning activities mainly in systems with high detergent concentrations. Variants being effective both in systems with low and high detergent concentration also are disclosed. New subtilisin variants are obtained by expression of DNA mutant sequence in cells of strain Bacillus. Subtilisin muteins of present invention are useful in cleaning compositions and composition for cloth treatment, as well as in animal feed additives.

EFFECT: enzyme of improved effectiveness.

18 cl, 4 dwg, 14 tbl, 2 ex

Description

This application is a partial continuation of US patent application No. 08/956323, filed October 23, 1998, US patent application No. 08/956564, filed October 23, 1998 and US patent application 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 specificities 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 mammalian 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 secrete various species of Bacillus and other microorganisms in large quantities. 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. Chem., 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) 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 residue 222 of subtilisin to methionine sulfoxide under the action 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).

The main problem when creating a variant of a protease intended for use in a detergent is the variety of washing conditions, including detergent compositions, in which this variant of a protease can be used. For example, detergent compositions used in different regions have different concentrations of detergent components in the wash water. For example, the washing system used in Europe usually contains about 4,500-5,000 parts per million washing components in the washing water, while the washing system used in Japan usually contains about 667 parts per million washing components in the washing water. In North America, particularly in the United States, a washing system typically contains approximately 975 parts per million washing components in washing water. Applicants have developed a method for rationally constructing a variant of a protease intended for use in a system with a low concentration of detergent, in a system with a high concentration of detergent and / or in a system with an average concentration of detergent, and also for use in systems with a concentration of detergents of all three types.

Summary of the invention

The object of this invention are protease variants with amino acid substitutions at the position of one or more residues, such that the substitution changes the charge in this position, making it more negative or less positive compared to the precursor protease, as a result of which the protease variant is more effective in a system with a low concentration of detergent than the precursor protease. A low detergent concentration system is a washing system that contains less than 800 parts per million washing components in washing water.

Another object of this invention are protease variants with amino acid substitutions at the position of one or more residues, such that such a substitution changes the charge at this position, making it more positive or less negative compared to the precursor protease, as a result of which the protease variant is more effective in a system with a high concentration of detergent than the precursor protease. A system with a high concentration of detergent is a washing system that contains more than 2000 parts per million washing components in washing water.

Another object of this invention are protease variants with amino acid substitutions at the position of one or more residues, such that such a substitution changes the charge at this position, making it more positive or less negative compared to the precursor protease, as a result of which the protease variant is more effective in a system with an average concentration of detergent than a precursor protease. A system with an average concentration of detergent is a system that contains from about 800 to about 2000 parts per million detergent components in the washing water.

Another object of this invention are protease variants with amino acid substitutions at the position of one or more residues, such that the substitution changes the charge in this position, making it more negative or less positive compared to the precursor protease, as a result of which the protease variant is more effective in a system with an average concentration of detergent than a precursor protease. A system with an average concentration of detergent is a washing system that contains from about 800 to about 2000 parts per million washing components in washing water.

Another object of this invention are DNA sequences encoding such protease variants, and expression vectors containing DNA sequences of such variants.

Another object of this invention are host cells transformed with such vectors and host cells capable of expressing such DNA in order to produce protease variants intracellularly or extracellularly.

Another object of this invention is a cleansing composition containing a variant of the protease of the present invention.

In addition, the object of this invention is animal feed containing the protease variant of the present invention.

The object of this invention is also a composition for treating tissues containing a variant of the protease of the present invention.

This invention further relates to a method for producing a variant protease, which is more effective in a system with a low, medium and high concentration of detergent than a precursor protease, which includes the following stages:

a) substitution of an amino acid in the position of one or more residues so that such a substitution changes the charge in this position, making it more positive or less negative compared to the precursor protease;

b) substitution of an amino acid in the position of one or more residues so that such a substitution changes the charge in this position, making it more negative or less positive compared to the precursor protease;

c) testing the resulting variant to determine its effectiveness in a system with a high, medium and low concentration of detergent compared to the precursor protease;

and

d) re-performing steps a) to c), if necessary, to obtain a variant protease that is more effective in a system with a low, medium and high concentration of detergent than the precursor protease, wherein steps a) and b) can be performed in any okay.

Brief Description of the Drawings

Figure 1 AB 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 line refers to the amino acid sequence of subtilisin from Bacillus amyloliquefaciens (which is sometimes defined as subtilisin BPN '). The second line refers to the amino acid sequence of subtilisin from Bacillus subtilis. The third line refers to the amino acid sequence of subtilisin from B.licheniformis. The fourth line refers to the amino acid sequence of subtilisin from Bacillus lentus (which is called subtilisin 309 in PCT WO 89/06276). The symbol * means the absence of specific amino acid residues in comparison with BPN 'subtiliain.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, different washing modes are used in different regions and, as a result of this, different types of detergents are used. For example, Japan uses a system with a low concentration of detergent, while in Europe uses a system with a high concentration of detergent. As stated above, a system with an average concentration of detergent is used in the United States. Applicants have found that in different detergents, optimal efficiency is achieved using different protease variants. Based on these results, it can be assumed that it is impossible to find a protease that will act effectively in all three types of degenerants. However, it is not. Applicants have developed a method for rationally constructing a variant of a protease suitable for use in a system with a low concentration of detergent, in a system with a high concentration of detergent, or in a system with an average concentration of detergent, that is, it works effectively in systems with all three concentrations of detergent.

Applicants have found that in order to obtain a protease variant that is more effective in a system with a low concentration of detergent, one or more positively charged residues must be replaced with one or more negatively charged residues, neutral residues, and / or one or more neutral residues must be replaced one or more negatively charged residues. It should be noted that in order to obtain a protease variant that is more effective in a system with a high concentration of detergent, one or more negatively charged residues must be replaced by one or more positively charged residues or neutral residues and / or one or more neutral residues replace with one or more positively charged residues. In addition, applicants have found that many protease variants useful in a system with a low concentration of detergent and / or in a system with a high concentration of detergent also work effectively in a system with an average concentration of detergent. By balancing these changes, you can get a variant of the protease that works well in systems with a low concentration of detergent, in systems with a low and medium concentration of detergent, in systems with an average and high concentration of detergent, in a system with a high concentration of detergent, or in systems with all three concentrations detergent.

It is assumed that the electrostatic charge in the side chain of any ionizable amino acid with an acidic or basic function depends on the pH in an aqueous solution. Acidic residues Glu and Asp lose their proton during equilibration due to dissociation at pH 3-6 and acquire a negative charge. Similarly, His, Lys, and Arg gradually deprotonate at pH 5–8, pH 8.5–11.5, and pH 11–14, respectively, thus losing a positive charge. The proton Tyr OH dissociates to a degree at pH 8.5–11.5, as a result of which Tyr takes on a negative charge. The dissociation region for the carboxyl end corresponds to pH 1-4, giving a negative charge, and for the amino terminal corresponds to pH 8-11, accompanied by the loss of a positive charge. The dissociation region for the amino acid side chains indicated here is the average of several proteins, but it is known that the unusual structural configurations of some proteins can influence these values.

The cumulative effect of all charges indicates whether the protein has a total positive or total negative charge at a given pH. The pH at which the positive and negative charges are the same and give the protein an electrostatically neutral state is called the isoelectric point (pI). A protein loses or acquires a charge when a pH shift occurs or when the side chain gains or loses an amino acid with an ionizable residue. The total positive charge can be increased by replacing the negatively charged residue at this pH with an uncharged or positively charged residue, as a result of which the charge formally becomes equal to +1 and +2, respectively. When replacing an uncharged residue in the side chain with a residue protonated at a given pH, the charge formally becomes +1. Similarly, the total negative charge can be increased by replacing positively charged or uncharged side chains with negatively charged side chains at a given pH, resulting in a formal increase in negative charge by -1 and -2, respectively.

The low detergent concentration system provides less than 800 parts per million detergent components in the wash 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 approximately 800-2000 parts per million detergent components in the washing water. Detergents used in North America are usually considered systems with an average concentration of detergent, as they are characterized by the presence of approximately 975 parts per million detergent components in the washing water. In Brazil, detergents are commonly used to provide approximately 1,500 parts per million detergent components in washing water.

A system with a high concentration of detergent includes detergents that ensure the presence in the washing water of more than 2000 parts per million detergent components. European detergents are generally considered systems with a high concentration of detergent, as they provide 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 detergent concentrations, since they are characterized by the presence of 1,500 to 6,000 parts per million detergent components in washing water. As mentioned above, Brazilian detergents are usually characterized by the presence of approximately 1,500 parts per million detergent components in the washing water. However, in other regions that are characterized by the use of high-foam phosphate-modified detergents and which are not limited to Latin America, systems with a high concentration of detergent can be used to ensure that up to 6,000 parts per million detergent components are in the wash water.

In light of the foregoing, it becomes apparent that the concentrations of detergents in typical washing solutions used in different regions of the world vary from less than 800 parts per million (“regions of application of systems with low concentration of detergent”), for example, about 667 parts per million in Japan, up to about 800-2000 ("regions of application of systems with an average concentration of detergent"), for example, about 975 parts per million in the USA and about 1500 parts per million in Brazil, up to more than 2000 parts per million ("regions of application of systems with high concentration detergent "), for example, about 4,500-5,000 ppm in Europe and about 6,000 ppm in the regions where high-foam phosphate-modified detergents are used.

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.

Proteases are carbonyl hydrolases that 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, which 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 amino acid sequence obtained. In particular, such protease variants contain an amino acid sequence not found in nature, which is obtained by replacing 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 Wiley & 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 shown 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, division 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. RE 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 cerevisiae, 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 per se. Suitable methods for modifying the DNA sequence of the precursor 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).

The position numbers of these amino acids refer to the numbers assigned to the mature subtilisin sequence of Bacillus amyloliquefaciens 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 amyloliquefaciens 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 precursor protease residue (amino acid) 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 the 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 conserved residues that are common to B. amyloliquefaciens and B.lentus subtilisins. After performing a comparative analysis of the conserved residues, which allows to determine the necessary insertions and division while maintaining the primary structure (that is, to avoid the removal of conservative residues in the case 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, figure 3 shows a comparative analysis of the amino acid sequences of subtilisin from Bacillus amyloliquefaciens, Bacillus subtilis. Bacillus licheniformis (carlshergensis) 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 subtilisin Bacillus amyloliquefaciens in other subtilisins, such as subtilisin from Bacillus lentus (PCT publication No. WO 89/06279 of July 13, 1989), the preferred enzyme protease precursor, 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, in the sequence of Bacillus lentus there are a number of deletions compared to subtilisin of Bacillus amyloliquefaciens. For example, the amino acid for Val165 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. The preferred sequence for obtaining protease variants is the putative Bacillus amyloliquefaciens subtilisin sequence, 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 subtills 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 in accordance with the methods described in US Pat. No. 3,4606, which results in the loss of their 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 protease expression are Bacillus subtills I168 (also described in US Pat. Nos. RE 34606 and 5264366, which are incorporated herein by reference), as well as any suitable Bacillus strain such as B.licheniformis, B. lentus etc.

Host cells are transformed or transfected with vectors constructed 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 a pro- 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, simply 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 gene promoter, 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 and and supported by the endogenous terminator region of the protease gene and the selectable gene is desired, in particular antibiotic resistance gene that enables continuous preservation plasmid in the culture infected host cells by growth 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 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 desired to mutate (divide, insert, or replace) one or more amino acids in the encoded enzyme. Sequences flanking this point are examined for the presence of restriction sites to replace a short gene segment with a pool of oligonucleotides, which must be encoded by different mutants during expression. Such restriction sites are preferably unique sites in the protease gene that facilitate 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 restriction sites are absent in places located at a convenient distance from the selected point (from 10 to 15 nucleotides), such sites can be created by replacing nucleotides in the gene so that the reading frame and encoded amino acids are not 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 achieve 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 for measuring proteolytic activity are known (KMKalisz, "Microbial Proteinases", Advances in Biochenical 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 , ratio k _cat / K _m , 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 the invention, the goal is to obtain a variant of the protease with altered proteolytic activity compared with the precursor protease, since an increase in such activity (in numerical terms) allows more efficient use of this enzyme on the target substrate. In addition, variant enzymes with altered heat resistance and / or altered substrate specificity compared to the precursor are of particular interest. In some cases, lower proteolytic activity may be desirable, for example, a decrease in proteolytic activity is necessary when using the synthesizing activity of proteases (for example, for the synthesis of peptides). The need for a decrease in proteolytic activity may arise in those cases when it can destroy the product of this synthesis. Conversely, in some cases, it is desirable to increase the proteolytic activity of the variant enzyme compared to its predecessor. In addition, it is sometimes desirable to increase or decrease (change) the stability of a variant, for example, alkali resistance or heat resistance. An increase or decrease in kinetic parameters such as k _cat , K _m or k _cat / K _m is made depending on the subtrate.

Another aspect of the invention is the discovery that protease variants with amino acid substitutions at the position of one or more residues, such that such a substitution changes the charge at this position, making it more negative or less positive compared to the precursor protease, are more effective at lower detergent concentration than the precursor protease.

Another distinguishing feature of this invention is the discovery that protease variants with amino acid substitutions at the position of one or more residues, such that such a substitution changes the charge at this position, making it more positive or less negative compared to the precursor protease, more effective at high detergent concentrations than the precursor protease.

In addition, applicants have found that many protease variants that are effective in a system with a low concentration of detergent and / or in a system with a high concentration of detergent also work effectively in a system with an average concentration of detergent.

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

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 cleaning or washing action of such variant enzymes.

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. Andersen 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, such as 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 alone or in combination with additives and stabilizers in a detergent-free cleansing composition.

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 illustrative and should be considered only 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.

The present invention is further illustrated by examples which do not limit the scope of the following 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 by methods well known in the art. All mutations were made in the subtilisin of Bacillus lentus GG36.

The obtained protease variants 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".

Tables 1-13 show the protease variants analyzed and the test results in two different detergents. All values are given in comparison with the first protease shown in the table (i.e., 1.32 corresponds to 132% spot removal compared to 100% obtained for the first option in the table).

Column A shows the charge difference of the variant. Column B 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 25 ° C (system with a low concentration of detergent). Column C 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 (system with a high concentration of detergent).

EXAMPLE 2

The following protease variants were obtained and tested analogously to example 1.

The options shown in table 14 are protease variants having both types of substitutions: substitutions that change the charge in the specified position, making it more negative or less positive, and substitutions that change the charge in the specified position, making it more positive or less negative compared to with B.lentus GG36, as well as neutral substitutions that do not change the charge at the position of this residue. This allows you to get protease variants that are more effective than standard protease, as in systems with a low concentration of detergent (column A; 0.67 g / l filtered Ariel Ultra (Procter & Gamble, Cincinnati, Ohio, USA) in 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 tank at 25 ° C), as well as in systems with a high concentration of detergent ( column B; 3.38 g / l filtered Ariel Futur (Procter & Gamble, Cincinnati, Ohio, USA.) in a solution containing 213.9 g / m ³ (15 grains / gallon) mixed w stkosti Ca ²⁺ / Mg ^2+, and 0.3 ppm enzyme into each bottle at 40 ° C).

Table 14 A IN N76D S103A V104I 1.00 1.00 V68a S103A V104I G159d A232V Q236H Q245R N252K 1.41 1.85 V68a N76D S103A V104I G159d T213r A232V Q236H Q245R T260A 1.30 1.73 V68a S103A V104I G159d A232V Q236H Q245R N248D N252K 2.77 1.20 V68a S103A V104I N140D G159d A232V Q236H Q245R N252K 2.96 1.42 N43K V68a S103A V104I G159d A232V Q236H Q245R 2.05 1.78 N43d V68a S103A V104I G159d A232V Q236H Q245R N252K 2.00 1.34 V68a N76D S103A V104I G159d A215R A232V Q236H Q245R 1.67 1.45 Q12R V68a N76D S103A V104I G159d A232V Q236H Q245R 2.16 1.72 N76D S103A V104I V147I G159d A232V Q236H Q245R N248S K251R 1.35 1.29 V68a N76D S103A V104I G159d A232V Q236H Q245R S256R 2.01 1.72 V68a N76D S103A V104I G159d Q206R A232V Q236H Q245R 2.09 1.62 S103A V104I G159d A232V Q236H Q245R N248D N252K 1.44 1.41 G20r V68a S103A V104I G159d A232V Q236H Q245R N248D N252K 1.81 1.72 V68a S103A V104I G159d A232V Q236H Q245R N248D N252K L257R 1.51 1.41 V68a S103A V104I A232V Q236H Q245R N248D N252K 1.04 1.50 N76D S103A V104I G159d A232V Q236H Q245R L257V 1.92 1.09

Claims (18)

1. A variant of subtilisin with the substitution of an amino acid residue in one or more positions, where the specified substitution changes the charge in this position, making it more negative or less positive compared to the subtilisin precursor, and where the variant of subtilisin has a higher stain removal efficiency than the specified precursor in a system with a low concentration of detergent having less than approximately 800 ppm (parts per million) of detergent components in the washing water, and is selected from the group consisting of options presented in tables 2, 5, 8, 9, 10, 11 and 12. 2. The subtilisin variant according to claim 1, obtained from Bacillus subtilisin. 3. Variant of subtilisin according to claim 2, obtained from subtilisin of Bacillus lentus. 4. DNA encoding a variant of subtilisin according to claim 1. 5. An expression vector comprising the DNA sequence of claim 4, operably linked to an acceptable regulatory sequence capable of expressing said DNA in a Bacillus strain cell. 6. A cleansing composition comprising a protease, wherein said protease is a variant of subtilisin according to claim 1. 7. Animal feed containing as an additive a protease, wherein said protease is a variant of subtilisin according to claim 1. 8. A composition for treating tissue containing a protease, wherein said protease is a variant of subtilisin according to claim 1. 9. A variant of subtilisin with substitution of the amino acid residue in one or more positions, where the substitution changes the charge in this position, making it more positive or less negative than the subtilisin precursor, and where the variant of subtilisin has a higher stain removal efficiency than the specified precursor in a system with a high concentration of detergent having more than approximately 2000 ppm (parts per million) of detergent components in the washing water, and is selected from the group consisting of of the options presented in Tables 1, 3, 4, 6, 7 and 13. 10. The subtilisin variant of claim 9, obtained from Bacillus subtilisin. 11. The subtilisin variant of claim 10, obtained from subtilisin of Bacillus lentus. 12. DNA encoding a variant of subtilisin according to claim 9. 13. An expression vector comprising the DNA sequence of claim 12, operably linked to an acceptable regulatory sequence capable of expressing said DNA in a Bacillus strain cell. 14. A cleansing composition comprising a protease, wherein said protease is a variant of subtilisin according to claim 9. 15. Animal feed containing as an additive a protease, where the specified protease is a variant of subtilisin according to claim 9. 16. A composition for treating tissue containing a protease, wherein said protease is a variant of subtilisin according to claim 9. 17. A variant of subtilisin with substitution of the amino acid residue in one or more positions, where the substitution changes the charge in this position, making it more positive or less negative than the subtilisin precursor, and where the variant of subtilisin has a higher stain removal efficiency than said precursor both in a system with a low concentration of detergent having less than about 800 ppm (parts per million) of detergent components in washing water, and in a system with a high concentration tracer detergent having more than approximately 2000 ppm (parts per million) of detergent components in the washing water, and is selected from the group consisting of the options presented in table 14. 18. A variant of subtilisin with substitution of an amino acid residue in one or more positions, where the substitution changes the charge in this position, making it more negative or less positive compared to the subtilisin precursor, and where the variant of subtilisin has a higher stain removal efficiency than said precursor both in a system with a low concentration of detergent having less than about 800 ppm (parts per million) of detergent components in washing water, and in a system with a high concentration tracer detergent having more than approximately 2000 ppm (parts per million) of detergent components in the washing water, and is selected from the group consisting of the options presented in table 14. Conventional priority is set from 10.23.1997 in accordance with the filing date of applications 08 / 956,323, 08 / 956,324 and 08 / 956,564 to the US Patent Office.

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