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

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

 

This application is a partial continuation of application for U.S. patent No. 08/956323, filed October 23, 1998, application for U.S. patent No. 08/956564, filed October 23, 1998, and applications for U.S. patent 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 the carbonyl hydrolases. They form a diverse class of enzymes with different specificnosti and biological functions. Stroud R., Sci.Amer., 131:74-88. Despite the functional diversity, the catalytic mechanism of serine proteases possesses at least two genetically distinct families of enzymes: 1) subtilisins and 2) related to the disease of a mammal and homologous bacterial serine proteases (such as trypsin and trypsin S.gresius). These two families of serine proteases have very similar mechanisms of catalysis. Kraut J., (1977), Annu. Rev.Biochem., 46:331-358. In addition, although the primary structure of enzymes of these families are not related, their tertiary structures are conservative catalytic triad of amino acids consisting of serine, histidine and aspartate.

Subtilisin are the serine proteases (approximate molecular weight of 27,500), which secrete large quantities of various species of Bacillus and other micro is organisme. Protein sequence of subtilisin are defined at least nine different species of Bacillus. F.S. Markland et al., (1983), Hoppe-Seyler's Z.Physiol.Chem., 364:1537-1540. In the scientific literature describes three-dimensional crystallographic structure of subtilisin isolated from Bacillus amyloliquefaciens, Bacillus licheniformis and several natural variants B.lentus. These studies show that, although subtilisin no genetic relationship with the serine proteases mammals, it has a similar structure of the active site. X-ray crystal structure of subtilisin containing covalently linked peptide inhibitors (Robertus J.D. et al., (1972), Biochemistry, 11:2439-2449) or complex products (Robertus J.D. et al., (1976), J.Biol. Chem., 251:1097-1103), allowed us to obtain information about the active site and the expected substractive site of subtilisin. In addition, there was conducted a large number of kinetic and chemical modification of subtilisin; Svendsen C., (1976), Carlsberg Res.Commun., 41: 237-291; F.S. Markland, ibid), with at least one scientific paper describes the conversion of the side chain of methionine in position residue 222 of subtilisin in methanesulfonic under the action of hydrogen peroxide (Stauffer D.C. 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 protease intended for the use of the program detergent, is the variety of washing conditions, including detergent formulations, which can be used this variant protease. For example, detergent compositions used in different regions have different concentration of detergent components in the wash water. For example, used in Europe washing system usually contains about 4,500 to 5,000 ppm of detergent components in the wash water, while Japan washing system usually has about 667 ppm of detergent components in the wash water. In North America, particularly in the United States, the washing system usually contains approximately 975 ppm of detergent components in the wash water. Applicants have developed a method for the rational design variant protease intended for use in a system with a low concentration of detergent in the system with a high concentration of detergent and/or in the system with an average concentration of detergent, as well as for use in systems with the concentration of the detergents all three types.

A brief statement of the substance of the invention

The object of this invention are protease variants with substitutions of amino acids in the position of one or more residues, are made so that this substitution alters the charge at that position, making it more negative or less positive compared to the protease-before what Estonica, resulting in this variant protease is more effective in a system with a low concentration of detergent than protease-predecessor. System with a low concentration of detergent is a cleaning system that contains less than 800 ppm of detergent components in the wash water.

Another object of this invention are protease variants with substitutions of amino acids in the position of one or more residues, are made so that this substitution alters the charge at that position, making it more positive or less negative compared to the protease-precursor, resulting in the specified variant protease is more effective in a system with a high concentration of detergent than protease-predecessor. System with a high concentration of detergent is a cleaning system that contains more than 2000 ppm of detergent components in the wash water.

Another object of this invention are protease variants with substitutions of amino acids in the position of one or more residues, are made so that this substitution alters the charge at that position, making it more positive or less negative compared to the protease-precursor, resulting in the specified variant protease is a more effective system with cf the days the concentration of detergent, than protease-predecessor. System with an average concentration of detergent is a system which contains from about 800 to about 2000 ppm of detergent components in the wash water.

Another object of this invention are protease variants with substitutions of amino acids in the position of one or more residues, are made so that this substitution alters the charge at that position, making it more negative or less positive compared to the protease-precursor, resulting in the specified variant protease is more effective in the system with an average concentration of detergent than protease-predecessor. System with an average concentration of detergent is a cleaning system, which contains from about 800 to about 2000 ppm of detergent components in the wash water.

Another object of this invention are DNA sequences encoding such variant proteases, and expressing the vectors containing the DNA sequence of such options.

Another object of this invention are cell-hosts transformed with such vectors, and cells are the hosts, they are able to Express such DNA, with the aim of producing protease variants intracellular or extracellular.

Another object of this invention is a cleansing compositions is Oia, containing variant protease of the present invention.

In addition, an object of this invention is a pet food containing a variant protease of the present invention.

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

This invention further relates to a method for obtaining a variant of a protease, which is a more efficient system with low, medium and high concentration of detergent than protease-predecessor, which includes the following stages:

a) substitution of the amino acid in position of one or more residues so, what is the substitution alters the charge at that position, making it more positive or less negative compared to the protease-predecessor;

b) substitution of amino acids in the position of one or more residues so, what is the substitution alters the charge at that position, making it more negative or less positive compared to the protease-predecessor;

c) test received options to determine its effectiveness in the system of high, medium and low concentration of detergent compared to protease predecessor;

and

d) re-execution stages a)to C) if necessary to obtain the variant protease, which is more effective in a system with low medium and high concentration of detergent than protease-predecessor, and stages a) and b) can be performed in any order.

Brief description of drawings

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

Figure 2 shows a conservative amino acid residues of subtilisin from Bacillus amyloliquefaciens (BPN' and Bacillus lentus (wild type).

On figa and 3B shows the amino acid sequences of the four subtilisin. The top row 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 .licheniformis. The fourth line refers to the amino acid sequence of subtilisin from Bacillus lentus (which is referred to as 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

As mentioned above, different regions use different modes of washing and, as a consequence, use detergents of different types. For example, in Japan, the system is used with desconocerlos detergent, while in Europe, the system has a high concentration of detergent. As mentioned above, the United States has used the system with an average concentration of detergent. Applicants have discovered that different detergents optimum efficiency is achieved by using different variants of the protease. Based on these results it can be assumed that it is impossible to find a protease, which will effectively act divergent all three types. However, this is not the case. Applicants have developed a method for the rational design variant protease suitable for use in the system with a low concentration of detergent in the system with a high concentration of detergent or in the system with an average concentration of detergent, that is, it is effective in systems with all three concentrations of detergent.

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

It is assumed that the electrostatic charge in the side chain of any of an ionisable amino acids with acidic or basic function depends in aqueous solution from pH. Acidic residues Glu and Asp in the process of balancing lose due to proton dissociation at pH 3-6 and acquire a negative charge. Similarly, His, Lys and Arg gradually deprotonated respectively at pH 5-8, pH 8.5-11.5 and pH 11-14, thus losing the positive is the long. Proton Tight HE dissociates in extent at pH 8,5-11,5, resulting in Tight acquires a negative charge. The field of dissociation for carboxyl end corresponds pH 1-4, giving a negative charge, and for aminobenzo corresponds to pH 8-11, accompanied by loss of positive charge. This area of dissociation for the side chains of amino acids represents average values for several proteins, but it is known that these values can influence the unusual structural configuration of some proteins.

The cumulative effect of all charges indicates whether the total protein or positive total negative charge at this pH. The pH at which the positive and negative charges are the same and report the squirrel electrostatically neutral state, is called the isoelectric point (PI). Protein loses or gains a charge when there is a shift in pH or when a side chain acquires or loses an amino acid with an ionisable residue. The total positive charge can be increased by replacing the negatively charged residue at this pH uncharged or positively charged residue, resulting in the charge formally becomes equal to +1 and +2. When replacing uncharged residue in the side chain residue, protourban the m at this pH, the charge formally becomes equal to +1. Similarly, the total negative charge can be increased by replacing positively charged or uncharged side chains negatively charged side chains at this pH, resulting in a formal increase in negative charge respectively, -1 and-2.

System with a low concentration of detergent provides content in the washing water of less than 800 ppm of detergent components. Japanese detergents are generally considered systems with a low concentration of detergent as they contain approximately 667 ppm of detergent components in the wash water.

System with an average concentration of detergent includes detergents, characterized by the presence of about 800-2000 ppm of detergent components in the wash water. The detergents used in North America, are generally regarded as systems with an average concentration of detergent, as they are characterized by the presence of approximately 975 ppm of detergent components in the wash water. In Brazil commonly used detergents, providing approximately 1500 ppm of detergent components in the wash water.

System with a high concentration of detergent includes detergents, which provide a presence in the wash water more than 2000 parts per million of detergents componentlibrary detergents are generally considered systems with a high concentration of detergent, as they provide approximately 4,500 to 5,000 ppm of detergent components in the wash water.

The detergents used in Latin America, are usually vysokoparnymi, phosphatidylcholine detergents, so they can be classified as systems with medium and high concentrations of detergent, as characterized by the presence of from 1500 to 6000 ppm of detergent components in the wash water. As mentioned above, the Brazilian detergents are usually characterized by the presence of about 1500 ppm of detergent components in the wash water. However, in other regions, which are characterized by the use of vysokoparnym modified phosphate detergents and are not limited to Latin America, can be used in systems with a high concentration of detergent, ensuring that the washing water up to 6000 ppm of detergent components.

In light of the above it becomes evident that the concentration of detergent in a typical washing solutions used in different regions of the world vary from less than 800 parts per million ("regions of application systems with a low concentration of detergent"), for example, about 667 parts per million in Japan, up to about 800-2000 ("regions of application systems with an average concentration of detergent"), for example, about 975 parts per million in the United States and okolo parts per million in Brazil, up to more than 2000 parts per million ("regions of application systems with a high concentration of detergent"), for example, about 4,500 to 5,000 parts per million in Europe and about 6000 parts per million in the regions of application vysokoparnym modified phosphate detergents.

The normal concentration of the wash solutions are determined empirically. For example, in U.S. conventional washing machine holds approximately 64.4 l of wash solution. Thus, to get in the wash solution concentration equal to approximately 975 parts per million of detergent, 64,4 l washing solution you want to add about 62,79 g detergent composition. This quantity measures the consumer using a measuring Cup that came with the detergent.

Proteases are carbonyl hydrolases that break down the peptide bonds of proteins or peptides. Used here is the term "protease" means a natural or recombinant protease. The natural proteases include hydrolase α-aminoacylated, hydrolase peptidyl-amino acids, allamandola, the serine carboxypeptidase, metallocarboxypeptidase, tolerations, carboxypeptidase and metalloproteinase. In the scope of this invention include serine proteases, metalloproteases, terproteksi and acid protease, as well as endo - and ectoprocta.

The present invention relates to Fe the cops protease, which are not natural options carbonyl hydrolases (variant protease), because they have a different proteolytic activity, stability, specificity to the substrate, pH profile and/or efficiency compared with carbonyl hydrolases predecessor, from which was derived amino acid sequence. In particular, such protease variants contain amino acid sequence not found in nature, which is obtained by substitution of several amino acid residues of the protease-precursor of other amino acids. Protease-precursor may be natural or recombinant protease.

In embodiments, the protease of the present invention can be substituted for any of the nineteen natural L-amino acids in certain positions of amino acid residues. Such substitution can be made in the subtilisin-predecessor (prokaryotic subtilisin, eukaryotic subtilisin, subtilisin mammals etc). In this description, different amino acids are denoted by the conventional one - and three-letter codes. These codes are listed in the directory Dale M.W., (1989), Molecular Genetics of Bacteria, John Wiley & Sons, Ltd., Appendix B.

Variants of the protease according to this invention is preferably derived from subtilisin Bacillus. More preferably, the variants of the FR is SHL get out of subtilisin Bacillus lentus and/or subtilisin 309.

Subtilisin are proteases of bacteria or fungi, which usually break down peptide bonds of proteins or peptides. Used here is the term "subtilisin" means a natural or recombinant subtilisin. It is known that different types of microorganisms produce and often secrete a number of natural subtilisin. Amino acid sequences of these subtilisins are not completely homologous. However, subtilisin included in this group have the same or similar proteolytic activity. This class of serine proteases has an overall amino acid sequence that determines the catalytic triad, which distinguishes them from the class of serine proteases related to the disease. As subtilisin and related chymotrypsin serine proteae have a catalytic triad consisting of aspartate, histidine and serine. In proteases related to subtilisin, these amino acids when reading from aminobenzo to the carboxyl end are located in the following relative order: aspartate-histidine-serine. However, related chymotrypsin protease have the following relative order of these amino acids: histidine-aspartate-serine. Thus, subtilisin belongs to the serine protease with a catalytic triad related subtilisin proteases. PR is measures such subtilisins are but not limited to, subtilisin shown in figure 3. The numbering of these amino acids in the protease normally and in accordance with the purposes of the present invention corresponds to the numbers assigned to the sequence of the Mature subtilisin Bacillus amyloliquefaciens shown in figure 1.

"Recombinant subtilisin" or "recombinant protease" means subtilisin or protease, in which the DNA sequence encoding subtilisin or protease, modified with the possibility of producing variant or mutant) DNA sequence that encodes the substitution, division or insertion of one or several amino acids in the natural amino acid sequence. Acceptable methods of performing this modification, which can be used in combination with the techniques described in this invention include the methods described in U.S. patent No.'RE 34606, 5204015, 5185258, 5700676, 5801038 and 5763257.

"Subtilisin not belonging to the person" and encoding their DNA, can be obtained from many prokaryotic and eukaryotic microorganisms. Acceptable 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, which can be obtained subtilisin and his genes are yeasts such as Saccharomyces cerevisiae, and fungi, such as Aspergillus sp.

"Variant protease has the amino acid sequence derived from the amino acid sequence of the protease-predecessor". The protease-predecessors are natural and recombinant protease. Amino acid sequence variants of the protease derived from the amino acid sequence of the protease-predecessor by substitution, deletion or insertion of one or several amino acids in the amino acid sequence of the precursor. This is achieved by modification of the DNA sequence of the predecessor, the coding amino acid sequence of the protease-predecessor, and not the manipulation of enzyme protease predecessor per se. Acceptable methods of modification of the DNA sequence of the precursor include methods described here, as well as methods known to experts in this field (see, for example, European patent No. 0328299, WO 89/06279 the above-cited patents and patent applications U.S.).

Non of the provisions of these amino acids refer to the numbers assigned to the sequence of the Mature subtilisin Bacillus amyloliquefaciens shown in figure 1. However, this invention is not limited to the Mature form of subtilisin and includes protease precursor containing amino acid residues in positions that are "equivalent" to skretny identified residues in subtilisin from Bacillus amyloliquefaciens. In a preferred embodiment of the present invention, the protease-predecessor is a subtilisin Bacillus lentus and replacements made at positions equivalent to amino acid residues in .lentus that meet specified above.

The position of the residue (amino acid) protease-predecessor is equivalent to the position of rest of the subtilisin from Bacillus amyloliquefaciens, if it is homologous (i.e. corresponds to the position in the primary or tertiary structure) or analogous to a specific residue or portion of that residue in the subtilisin of Bacillus amyloliquefaciens (i.e. has the same or similar functional capacity to unite, react, or chemically interact).

To establish homology to primary structure, the amino acid sequence of the protease-predecessor compare directly with the primary sequence of subtilisin from Bacillus amyloliquefaciens and, in particular, with a set of residues known to be invariant in subtilizing and determine the character sequence. For example, figure 2 shows conservative residues, which are common to subtilisin .amyloliquefaciens and .lentus. After performing a comparative analysis of conservative residues, allowing to determine the necessary insertion and division while maintaining the primary structure (i.e. in beanie delete conservative residues in the case of accidental deletions and insertions), determine the residues equivalent to certain amino acids in the primary sequence of subtilisin from Bacillus amyloliquefaciens. Comparison of conservative residues should preferably detect the presence of 100% of such residues. However, the presence of more than 75% or at least 50% conservative residues is sufficient to determine equivalent residues. The catalytic triad Asp32/His64/Ser221 must 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 subtilase or subtilizing serine protease.

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

Thus, these conserved residues can be used to determine appropriate equivalent amino acid residues of subtilisin illus amyloliquefaciens in other subtilisins, such as subtilisin from Bacillus lentus (PCT publication no WO 89/06279 dated July 13, 1989), a preferred protease enzyme predecessor or subtilisin, defined as RV (European patent No. 0328299), which is homologous preferred subtilisin Bacillus lentus. On figa and 3B shows a comparative analysis of amino acid sequences of some of these subtilisin sequence of subtilisin from Bacillus amyloliquefaciens, allowing to identify the maximum homology conservative residues. As can be seen, in the sequence of Bacillus lentus there are a number of deletions in comparison with subtilisin Bacillus amyloliquefaciens. So, for example, amino acids for Val165 in subtilisin Bacillus amyloliquefaciens equivalent to isoleucine in other subtilisin .lentus and .licheniformis.

"Equivalent residues" can also be set by determining homology at the level of tertiary structure for protease predecessor, the tertiary structure is studied 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 particular amino acid residue of the protease-predecessor and subtilisin Bacillus amyloliquefaciens (N covers N, SA overlaps SA, overlaps With and overlaps) after ordering are within 0.13 nm and preferably in the range of 0.1 nm. The ordering of the primary structure is achieved when the orientation and the location of locamoda provides the maximum overlap of atomic coordinates of the protein atoms, are not hydrogen at the considered protease and subtilisin Bacillus amyloliquefaciens. The best model is the crystallographic model, which allows to obtain the lowest R-factor for the experimental diffraction data at the highest resolution.

Equivalent residues which are functionally analogous to a specific residue of subtilisin Bacillus amyloliquefaciens, defined as amino acid protease predecessor, which may adopt a conformation in which they can change, modify or stimulate the formation of protein structure, binding to the substrate or catalysis similarly particular and peculiar to a specific residue of subtilisin Bacillus amyloliquefaciens. In addition, these residues are residues of the protease-predecessor (tertiary structure established by x-ray crystallography), occupying a similar position at which the atomic coordinates of at least two atoms in the side chain of residue located at a distance of 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 criteria of equivalence from the point of view of homology position. The coordinates of the three-dimensional structure of subtilisin Bacillus amyloliquefaciens shown in public, the tion of EPO No. 0251446 (corresponding to U.S. patent No. 5182204, included in this description by reference), and, as indicated above, can be used to determine equivalent residues on the level of tertiary structure.

Some of the substituted residues are conservative residues, while other residues are not. In the case of residues that are not conservative substitution of one or several amino acids is limited to substitutions, allowing you to get the variant having the amino acid sequence that does not correspond to the natural sequence. In the case of conservative residues such substitution shall not result in the formation of the natural sequence. Variants of the protease of the present invention include the Mature form of the protease variants, as well as Pro - and prepratory such protease variants. Prepratory are the preferred design, as they facilitate the expression, secretion and maturation of the protease variants.

"Proposedvalue" means an amino acid sequence that is associated with the N-terminal part of the Mature form of the protease, the removal of which causes the appearance of a Mature form of the protease. Many proteolytic enzymes found in nature in the form of products broadcast proferment, and in the absence of post-translational processing they are expressed in such widebrimmed proposedvalue to obtain variants of the protease is estimated Proposedvalue subtilisin Bacillus amyloliquefaciens, although you can use other proposedvalue protease.

"Signal sequence" or "proposedvalue" means any amino acid sequence that is associated with the N-terminal part of the protease or with the N-terminal part of proprotein, which may participate in the secretion of the Mature form or Pro forma protease. This definition signal sequence is functional and includes all of the amino acid sequence encoded N-terminal part of the protease gene, which participates in the secretion of the protease in vivo. Such sequences used in the present invention to implement the secretion described here protease variants. One acceptable signal sequence contains the first seven amino acid residues of the signal sequence selected from subtilisin Bacillus subtills, merged with the rest of the signal sequence, subtilisin Bacillus lentus (ATCC 21536).

"Shall" form of a variant protease contains Mature form of the protease with proposedvalue, operable associated with aminocom.com protease, and "pre" or "signal" sequence, operable associated with aminocom.com proposedvalue.

"Expressing vector" means a construct based on DNA containing a DNA sequence, which operas is belino linked to suitable regulatory sequence, able to Express the indicated DNA in an acceptable host. Such regulatory sequences include a promoter for transcription, an optional operator sequence to control transcription, a sequence encoding the binding sites of the ribosome with acceptable mRNA, and sequences that control the termination of transcription and translation. The vector may be a plasmid, fagboy particle or simply potential genomic insert. After the introduction of an acceptable host, the vector may replicate and function independently of the genome of the host, and in some cases it may integrate into the genome itself. In this description, the terms "plasmid" and "vector" are sometimes used interchangeable in value, as the plasmid is the most commonly used form of vector at the present time. However, in the scope of this invention includes other forms expressing vectors which serve equivalent functions and known in this field.

"Cell bosses"used in the present invention, are typically prokaryotic or eukaryotic hosts, which are preferably treated in accordance with the methods described in U.S. patent No.'RE 34606, with the result that they lose the ability to secrete enzyme Akti is strong endoprotease. The preferred cell is a host for expression of the protease is a strain BG2036 Bacillus, which has no enzymatic active neutral protease and alkaline protease (subtilisin). Construction of strain BG2036 described in detail in U.S. patent No. 5264366. Other cells-hosts for expression of the protease are Bacillus subtills I168 (also described in U.S. patent No.'RE 34606 and 5264366, which are included in the description of the invention as a reference), as well as any acceptable strain of Bacillus, such as .licheniformis, B.lentus etc.

Cell host transformed or transferout vectors, constructed by the methods of recombinant DNA. Such transformed cell hosts capable of replicating vectors encoding the protease variants, or to Express the desired option protease. In the case of vectors that encode Pro - or prereform variant proteases, such options being expressed, usually are secreted from the host cell in a medium containing cells of the host.

The term "operable linked"used to describe the relationship between two regions of DNA, simply means that they are functionally related to each other. For example, proposedvalue operable linked to a peptide, if it acts as a signal sequence that is involved in the secretion of the Mature form of the protein, causing rossalini the specified signal sequence. A promoter operable linked to the coding sequence if it controls the transcription of the sequence; binding site of the ribosome operable linked to the coding sequence if it makes it possible to broadcast.

The genes encoding the natural protease predecessor, can be obtained by conventional methods known in this field. These methods typically include the synthesis of labeled probes having imaginary sequence encoding the region of interest protease, the creation of genomic libraries of microorganisms expressing the protease, and the screening of these libraries against the desired gene by hybridization with probes. Then positively hybridizers clones Carteret and is sequenced.

The cloned protease used for transformation of a host cell with the purpose of expression of the protease. Gene protease then are ligated into a plasmid with a large number of copies. This plasmid is replicated to the owners, because it contains the well-known elements necessary for replication of plasmids: the promoter, operatable associated with this gene (which may be introduced in the form of a homologous promoter of the gene, if it does, there Transcriber, host), the region of the termination of transcription and the region polyadenylation (which is necessary to ensure the us is ascioti mRNA, transcribed by the owner of the protease gene in certain eukaryotic cells-owners), which is exogenous or is supported by the endogenous terminator region of the gene of the protease, and it is desirable breeding gene, in particular the gene of resistance to antibiotics, which makes it possible to always maintain in culture infected with plasmid host cells by cultivation in media containing antibiotics. Plasmids with a large number of copies have also a replication source to the host, which allows to obtain large quantities of the plasmid in the cytoplasm without chromosomal restriction. However, the present invention relates also to integrate multiple copies of the protease gene in the host genome. This is made easier by prokaryotic and eukaryotic microorganisme that are particularly exposed to homologous recombination.

The gene may be natural genome B.lentus. Alternatively, you can get a synthetic gene that encodes a natural or mutant protease predecessor. In this case, determine the DNA and/or amino acid sequence of the protease-predecessor. Then synthesize multiple overlapping fragments of the synthetic single-stranded DNA after hybridization and ligation to form a synthetic DNA encoding the protease predecessor. The example design Sint the political gene shown in example 3 of U.S. patent No. 5204015, included in this description by reference.

After cloning, natural or synthetic protease gene predecessor perform a number of modifications aimed at enhancing the synthesis of the gene compared with the natural protease predecessor. Such modifications include the production of recombinant proteases, as described in U.S. patent No.'RE 34606 and in EPO publication No. 0251446 and production described here protease variants.

You can use the following way cassette mutagenesis, which facilitates the design of protease variants of the present invention, although it is possible to use other methods. First get the natural gene encoding the protease, which is sequenced completely or partially. Then the sequence is scanned to find the point at which it is desirable to produce a mutation (division, insertion or substitution) of one or several amino acids in the encoded enzyme. The sequences flanking this point, examine for the presence of restriction sites to replace the short segment of the gene pool of oligonucleotides that during the expression must encode different mutants. These restriction sites are preferably unique sites in the protease gene, facilitating the replacement of a segment of a gene. However, you can use any easy the first restriction site, which is not excessive in the protease gene, provided that the fragments of the gene, resulting restrictively, can be reassembled in the proper 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 the nucleotides in a gene so that the final design has not been altered reading frame and the encoded amino acids. Mutation of the gene to change its sequence so that it meets the required sequence, carried out using primer M13 in accordance with known methods. The task of localization acceptable flanking regions and identify changes needed to achieve two convenient sequence at the restriction site is greatly facilitated due to the presence of redundant genetic code, restriction map of the enzyme gene and a large number of different restriction enzymes. It should be noted that when there is a convenient flanking restriction site above method should be used only when flanking region which does not have customers.

After cloning the natural or synthetic DNA restriction sites flanking intended for mutation position, split sibling who passed restriction enzymes and are ligated in several gene cassettes oligonucleotides, complementary terminal regions. This method allows to simplify mutagenesis, as all the oligonucleotides can be synthesized so that they have the same restriction sites, and to create restriction sites do not require synthetic linkers.

Used here is the term "proteolytic activity" means the rate of hydrolysis of peptide bonds in milligrams of the active enzyme. There are many methods for measuring proteolytic activity (K.M.Kalisz, "Microbial Proteinases", Advances in Biochenical Engineering/Biotechnology, A.Fiechter ed., 1988). In addition or alternatively, a modified proteolytic activity of the variants of the enzymes of the present invention can be modified by other properties, such as Km, kcatthe relation kcat/Kmthe specificity to the substrate and/or the activity profile depending on pH. These enzymes can be designed for a specific substrate, which would be expected to occur, for example, upon receipt of the peptides or hydrolytic processes applied in the wash.

In one aspect of the invention the aim is to obtain the variant protease with altered proteolytic activity compared to protease predecessor, as the increase in such activity (in numeric terms) allows more efficient use of the data is the enzyme on the substrate target. In addition, of particular interest are the variant enzymes with altered stability and/or altered specificity for the substrate in comparison with the predecessor. In some cases it may be desirable lower proteolytic activity, such as decreased proteolytic activity necessary when using the synthesizing activity of proteases (for example, for the synthesis of peptides). The need to reduce proteolytic activity may occur in cases where 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) resistance options, such as resistance to alkali or heat. Increase or decrease these kinetic parameters, kcat, Kmor kcat/Kmthat is, depending on substrate.

Another aspect of the invention is the discovery that protease variants with substitutions of amino acids in the position of one or more residues, are made so that this substitution alters the charge at that position, making it more negative or less positive compared to the protease predecessor, are more the effect of the main at low concentrations of detergent, than protease-predecessor.

Another feature of this invention is the discovery that protease variants with substitutions of amino acids in the position of one or more residues, are made so that this substitution alters the charge at that position, making it more positive or less negative compared to the protease predecessor, are more effective at high concentration of detergent than protease-predecessor.

In addition, applicants have found that many variants of the protease, which is effective in a system with a low concentration of detergent and/or in a system with a high concentration of detergent, to effectively operate the system with an average concentration of detergent.

These substitutions are preferably made in the subtilisin Bacillus lentus (recombinant or native type), though this replacement can be performed in any protease of Bacillus, preferably in subtilisin Bacillus.

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

Many variants of the protease in which the invention is useful for obtaining different detergent compositions or personal hygiene, such as shampoos or lotions. In the compositions containing the mutant protease according to this invention, it is possible to use a number of known compounds, which is acceptable surfactants. These substances are non-ionic, anionic, cationic or zwitterionic detergents are described in U.S. patent No. 4404128 issued Barry J.Andersen, and in U.S. patent No. 4261868 issued by Jiri Flora et al. Acceptable detergent composition described in example 7 of U.S. patent No. 5204015 (previously included in this description by reference). In this area known different compositions that can be used as cleaning compositions. It is clear that in addition to conventional cleaning compositions variants of the protease according to this invention can be used in any area where there are native protease or protease wild-type. So, these options can be used, for example, when the applications of solid or liquid soap, compositions for dishwashing, solutions or products for cleaning contact lenses, compositions for the hydrolysis of peptides, waste management, textile processing, as well as digestive enzymes in obtaining proteins, etc. Variants of the present invention can inform the cleaning composition of higher efficiency (compared to the predecessor). High efficiency detergent is determined it is best to removing stains, susceptible to a specific enzyme, such as stains of vegetable origin or blood spots, according to the method of the usual evaluation after a standard wash cycle.

Protease according to this invention may comprise the known powdered and liquid detergents with a pH from 6.5 to 12.0 and concentration of from about 0.01 wt%. up to about 5% weight. (preferably from 0.1% to 0.5%). These cleansing composition with a detergent may also contain other enzymes, such as the known protease, amylase, cellulase, lipase or endoglycosidase, as well as additives and stabilizers.

Introduction proteases according to this invention in conventional cleaning compositions does not involve any special restrictions. In other words, any temperature and pH acceptable for this detergent is acceptable also for the compositions of the present invention, if the pH is within the above range and the temperature below the denaturation temperature of the described protease. In addition, protease according to this invention can be used in cleansing compositions without detergents alone or in combination with additives and stabilizers.

The present invention relates to cleansing compositions containing protease variants in this invention. The cleaning compositions can optionally contain additives which are usually used which are in such compositions. These additives include, but are not limited to, bleaches, surfactants, modifying components, enzymes and catalysts whitening. Specialist in this field should be obvious that the additives are selected based on their suitability for this composition. This list is illustrative and should only be considered as examples of acceptable additives. The specialist in this area should also be clear that you must use only those additives that are compatible with enzymes and other components of the composition, for example with surface-active substance.

The amount of additive present 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%.

Variants of the protease of the present invention can be introduced into animal feed as part of the additives, as described, for example, in U.S. patent No. 5612055, 5314692 and 5147642.

One aspect of the invention is a composition for treatment of tissue, including variants of the protease of the present invention. This song can be used for processing, such as silk or wool, as it is described in such publikacii as RD 216034, European patent # 134267, U.S. patent No. 4533359 and European patent No. 344259.

Gave the e present invention is illustrated in the examples, not limited the scope of the following claims.

All of the listed publications and patents are fully incorporated in this description by reference.

EXAMPLE 1.

A large number of options protease obtained and purified by methods well known in this area. All mutations made in the subtilisin Bacillus lentus GG36.

Received protease variants were tested for effectiveness in the two types of detergents and washing conditions using microscopic analysis, described in U.S. patent No. 60/068796 "An improved method of assaying for a preferred enzyme and/or preferred detergent composition".

In tables 1-13 presents analyzed variants of the protease and the test results in two different detergents. All values are given in comparison with the first protease is shown in table (i.e. value of 1.32 corresponds to 132% of the stain removing, compared with 100%, obtained for the first variant in the table).

In column a shows the difference between the charge option. In a column In the data for the filtered washing liquid with a concentration of 0.67 g/l Ariel Ultra (Procter & Gamble, Cincinnati, USA) in a solution containing 42,78 g/m3(3 Grand per gallon) mixed hardness CA2+/MD2+and 0.3 ppm enzyme in each tank at 25° (a system with a low concentration of detergent). In the column With the data to be filtered is on detergent concentration to 3.38 g/l Ariel Futur (Procter & Gamble, Cincinnati, USA) in a solution containing 213,9 g/m3(15 Grand per gallon) mixed hardness CA2+/MD2+and 0.3 ppm enzyme in each tank at 40°system (with a high concentration of detergent).

EXAMPLE 2.

The following options protease obtained and tested as in example 1.

The options are shown in table 14, are variants of the protease with both types of substitutions: substitution alters the charge at the specified position, making it more negative or less positive, and the substitution alters the charge at the specified position, making it more positive or less negative compared to B.lentus GG36 and neutral substitutions that do not alter the charge in the position of this residue. This allows to obtain protease variants that are more effective than standard p is Oteiza, in systems with low concentrations of detergent (column A; 0,67 g/l filtered Ariel Ultra (Procter & Gamble, Cincinnati, USA) in a solution containing 42,78 g/m3(3 Grand per gallon) mixed hardness Ca2+/MD2+and 0.3 ppm enzyme in each tank at 25° (C), and in systems with a high concentration of detergent (column; 3,38 g/l filtered Ariel Futur (Procter & Gamble, Cincinnati, PCs Ohio, USA) in a solution containing 213,9 g/m3(15 Grand per gallon) mixed hardness CA2+/Mg2+and 0.3 ppm enzyme in each tank at 40°).

 
Table 14
           AndIn
N76DS103AV104I        1.001.00
V68AS103AV104IG159DA232VQ236HQ245RN252K    1.411.85
V68AN76DS103AV104IG159DT213RA232VQ236HQ245RTA 1.301.73
V68AS103AV104IG159DA232VQ236HQ245RN248DN252K  2.771.20
V68AS103AV104IN140DG159DA232VQ236HQ245RN252K  2.961.42
N43KV68AS103AV104IG159DA232VQ236HQ245R   2.051.78
N43DV68AS103AV104IG159DA232VQ236HQ245RN252K  2.001.34
V68AN76DS103AV104IG159DA215RA232VQ236HQ245R 1.671.45
Q12RV68AN76DS103AV104IG159DA232VQ236HQ245R  2.161.72
N76DS103AV104IV147IG159DA232VQ236HQ245RN248SK251R 1.351.29
V68AN76DS103AV104IG159DA232VQ236HQ245RS256R  2.011.72
V68AN76DS103AV104IG159DQ206RA232VQ236HQ245R  2.091.62
S103AV104IG159DA232VQ236HQ245RN248DN252K   1.441.41
G20RV68AS103AV104IG159DA232VQ236H/td> Q245RN248DN252K 1.811.72
V68AS103AV104IG159DA232VQ236HQ245RN248DN252KL257R 1.511.41
V68AS103AV104IA232VQ236HQ245RN248DN252K   1.041.50
N76DS103AV104IG159DA232VQ236HQ245RL257V   1.921.09

1. Variant of subtilisin with substitution of the amino acid residue at one or more positions where the specified substitution alters the charge at that position, making it more negative or less positive compared to subtilisin-predecessor, and where the variant subtilisin has a higher efficiency at removing stains than the specified predecessor in the system with a low concentration of detergent having less than about 800 ppm (parts per million) detergent components in the wash water, and in biretta from the group consisting of options presented in tables 2, 5, 8, 9, 10, 11 and 12.

2. Variant subtilisin according to claim 1, obtained from subtilisin Bacillus.

3. Variant subtilisin according to claim 2, obtained from subtilisin Bacillus lentus.

4. DNA encoding a variant of subtilisin according to claim 1.

5. Expressing a vector comprising a DNA sequence according to claim 4, functionally linked to suitable regulatory sequence capable of Express the indicated DNA in the cell strain of Bacillus.

6. A cleansing composition comprising a protease, where this protease is a variant of subtilisin according to claim 1.

7. Animal feed containing as an additive a protease, where this protease is a variant of subtilisin according to claim 1.

8. The composition for treatment of tissue containing the protease, where this protease is a variant of subtilisin according to claim 1.

9. Variant of subtilisin with substitution of the amino acid residue at one or more positions where the specified substitution alters the charge at that position, making it more positive or less negative compared to subtilisin-predecessor, and where the variant subtilisin has a higher efficiency at removing stains than the specified predecessor in the system with a high concentration of detergent, with more than approximately 2000 ppm (parts per mill is he) detergent components in the wash water, and is selected from the group consisting of the options presented in tables 1, 3, 4, 6, 7, and 13.

10. Variant subtilisin according to claim 9, obtained from subtilisin Bacillus.

11. Variant subtilisin of claim 10, obtained from subtilisin Bacillus lentus.

12. DNA encoding a variant of subtilisin according to claim 9.

13. Expressing a vector comprising a DNA sequence according to item 12, functionally associated with suitable regulatory sequence capable of Express the indicated DNA in the cell strain of Bacillus.

14. A cleansing composition comprising a protease, where this protease is a variant of subtilisin according to claim 9.

15. Animal feed containing as an additive a protease, where this protease is a variant of subtilisin according to claim 9.

16. The composition for treatment of tissue containing the protease, where this protease is a variant of subtilisin according to claim 9.

17. Variant of subtilisin with substitution of the amino acid residue at one or more positions where the specified substitution alters the charge at that position, making it more positive or less negative compared to subtilisin-predecessor, and where the variant subtilisin has a higher efficiency at removing stains than the specified predecessor as in the system with a low concentration of detergent with less than approx the sory 800 ppm (parts per million) detergent components in the wash water, and in the system with a high concentration of detergent, with more than approximately 2000 ppm (parts per million) detergent components in the wash water, and is selected from the group consisting of options, presented in table 14.

18. Variant of subtilisin with substitution of the amino acid residue at one or more positions where the specified substitution alters the charge at that position, making it more negative or less positive compared to subtilisin-predecessor, and where the variant subtilisin has a higher efficiency at removing stains than the specified predecessor as in the system with a low concentration of detergent having less than about 800 ppm (parts per million) detergent components in the wash water, and in the system with a high concentration of detergent, with more than approximately 2000 ppm (parts per million) detergent components in the wash water and is selected from the group consisting of options, presented in table 14.

Convention priority is selected from 23.10.1997 in accordance with the date of filing 08/956,323, 08/956,324 and 08/956,564 in the U.S. Patent office.



 

Same patents:

FIELD: gene and protein engineering, in particular substances for detergents and cleaning compositions.

SUBSTANCE: invention relates to mutant forms of subtilisin Bacillus, obtained by certain combination of replacement in origin native enzyme amino acid sequence. Such mutant forms are differ from precursor subtilisin by increased cleaning effect. According to present invention all subtilisin variants are characterized either by residue substitution in position accepted to 232 position of Bacillus amyloliquifaciens amino acid sequence with valine, or by residue substitution in position accepted to 212 position of said subtilisin natural form with proline. To produce disclosed new form of enzyme respective mutant DNA sequences have been expressed in host cells, preferably in strain Bacillus cells with decreased level of proteases activity. Subtilisin variants of present invention are useful as components of any cleaning composition allowing for protease including.

EFFECT: new materials for detergents and cleaning compositions of improved effect.

6 cl, 5 dwg, 6 tbl, 3 ex

The invention relates to non-natural variants of carbonhydrates with altered proteolytic activity, stability, substrate specificity, pH profile and/or physico-chemical properties in comparison with the predecessor of carbonhydrate from amino acid sequence, which amino acid sequence of one or another variant

FIELD: gene and protein engineering, in particular substances for detergents and cleaning compositions.

SUBSTANCE: invention relates to mutant forms of subtilisin Bacillus, obtained by certain combination of replacement in origin native enzyme amino acid sequence. Such mutant forms are differ from precursor subtilisin by increased cleaning effect. According to present invention all subtilisin variants are characterized either by residue substitution in position accepted to 232 position of Bacillus amyloliquifaciens amino acid sequence with valine, or by residue substitution in position accepted to 212 position of said subtilisin natural form with proline. To produce disclosed new form of enzyme respective mutant DNA sequences have been expressed in host cells, preferably in strain Bacillus cells with decreased level of proteases activity. Subtilisin variants of present invention are useful as components of any cleaning composition allowing for protease including.

EFFECT: new materials for detergents and cleaning compositions of improved effect.

6 cl, 5 dwg, 6 tbl, 3 ex

the enzyme" target="_blank">

The invention relates to the field of medicine and biotechnology and relates to DNA sequences encoding human proteins TX and The related converging interleukin-1-beta enzyme

by activity" target="_blank">

The invention relates to the production of biologically active IL-1protease using recombinant DNA technology and can be used in medicine

The invention relates to native proteins schemes complement modified such that the protein is able to form stable C3 to Mac

The invention relates to non-natural variants of carbonhydrates with altered proteolytic activity, stability, substrate specificity, pH profile and/or physico-chemical properties in comparison with the predecessor of carbonhydrate from amino acid sequence, which amino acid sequence of one or another variant

The invention relates to biotechnology and genetic engineering

The invention relates to genetic engineering in particular the production of mutant proteases in Bacillus cells

FIELD: gene and protein engineering, in particular substances for detergents and cleaning compositions.

SUBSTANCE: invention relates to mutant forms of subtilisin Bacillus, obtained by certain combination of replacement in origin native enzyme amino acid sequence. Such mutant forms are differ from precursor subtilisin by increased cleaning effect. According to present invention all subtilisin variants are characterized either by residue substitution in position accepted to 232 position of Bacillus amyloliquifaciens amino acid sequence with valine, or by residue substitution in position accepted to 212 position of said subtilisin natural form with proline. To produce disclosed new form of enzyme respective mutant DNA sequences have been expressed in host cells, preferably in strain Bacillus cells with decreased level of proteases activity. Subtilisin variants of present invention are useful as components of any cleaning composition allowing for protease including.

EFFECT: new materials for detergents and cleaning compositions of improved effect.

6 cl, 5 dwg, 6 tbl, 3 ex

The invention relates to detergents and can be used both for manual and machine washing and soaking of all kinds of textile products, including non-ferrous, and other household needs

The invention relates to biotechnology; multicomponent system for mediated mediated enzymatic oxidation includes (a) an oxidation catalyst chosen from the group margantsovistyh oxidase, (b) an oxidizing agent chosen from the group comprising oxygen and oxygen-containing compounds, b) the mediator from the group of compounds containing Mn ions

The invention relates to the field of dyeing and finishing production and, in particular, to a method for removal of excess dye from printed or dyed fabric or yarn, comprising processing a rinsing solution containing at least one enzyme exhibiting peroxidase or LACCASO activity, in a concentration of 0.005 to 5 mg protein enzyme per 1 l of a solution for rinsing, oxidizing agent, mediator - 1-hydroxybenzotriazole in a concentration of from 1 μm to 1 mm and, optionally, additives

The invention relates to a paste-like compositions of detergent designed for washing and decontamination of clothing from various tissue and polymer materials

The invention relates to multiphase detergent tablets, and how they are used in washing machines
Up!