Subtilisin bacillus variant (variants), dna encoding the same, expression vector and cleaning composition

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

 

Related applications

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 inherent in at least two genetically distinct families of enzymes: 1) subtilisins and 2) related to the disease of man 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 two 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 in large quantities are secreted variety and species of Bacillus and other microorganisms. Protein sequence of subtilisin are defined at least nine different species of Bacillus. Markland. F.S., 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 sitespecifically mutagenesis (Wells and Estell (1988) TIBS 13:291-297).

A brief statement of the substance of the invention

The object of this invention which is a variant of a protease with the substitution of amino acids at positions one or more residues, the relevant provisions of the residues selected from the group comprising positions 62, 212, 230, 232, 252 and 257 of subtilisin Bacillus amyloliquefaciens.

Although it may have any combination of the above substitutions of amino acids, the preferred enzyme variant protease of the present invention can be substituted amino acid residues in the following combinations provisions. All these provisions residues correspond to positions of the subtilisin of Bacillus amyloliquefaciens:

(1) a variant of a protease with substitutions of amino acid residues in position 62 and one or more of the following provisions: 103, 104, 109, 159, 213, 232, 236, 245, 248 and 252;

(2) a variant of a protease with substitutions of amino acid residues in position 212 and one or more of the following provisions: 12, 98, 102, 103, 104, 159, 232, 236, 245, 248 and 252;

(3) a variant of a protease with substitutions of amino acid residues in position 230 and one or more of the following provisions: 68, 103, 104, 159, 232, 236 and 245;

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

(5) a variant of a protease with substitutions of amino acid residues in position 232 and one or more of the following provisions: 103, 104, 236 and 245;

(6) a variant of a protease with substitutions of amino acid OST the Cove at position 232 and 103, and in one or more of the following provisions: 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 205, 209, 210, 212, 213, 217, 230, 236, 245, 248, 252, 257, 260, 270 and 275;

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

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

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

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

(11) a variant of a protease with substitutions of amino acid residues at position 252, and one or more of the following provisions: 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;

(12) a variant of a protease with substitutions of amino acid residues at position 252, and one or more SL is blowing positions: 103, 104, 236 and 245;

(13) a variant of a protease with substitutions of amino acid residues at positions 252 and 103, and one or more of the following provisions: 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;

(14) a variant of a protease with substitutions of amino acid residues at positions 252 and 104, and one or more of the following provisions: 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;

(15) a variant of a protease with substitutions of amino acid residues at positions 252 and 236, and one or more of the following provisions: 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;

(16) a variant of a protease with substitutions of amino acid residues at positions 252 and 245, and in one or more of the following provisions: 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270;

(17) a variant of a protease with substitutions of amino acid residues at positions 252, 103, 104, 236 and 245, and in one or more of the following provisions: 1, 9, 12, 61, 62, 68, 97, 98, 101, 102, 103, 104, 109, 130, 131, 159, 183, 185, 210, 212, 213, 217, 232, 236, 245, 248 and 270; and

(18) a variant of a protease with substitutions of amino acid residues at position 257 and in one or more of the following provisions 68, 103, 104, 205, 209, 210, 232, 236, 245 and 275.

More preferred protease variants have a set of substitutions selected from the group including provisions residues subtilis is on Bacillus amyloliquefaciens, the relevant provisions specified in table 1.

The most preferred protease variants have a set of substitutions selected from the group including provisions residues of subtilisin Bacillus amyloliquefaciens, consistent with the requirements specified in table 2.

Another object of this invention are DNA sequences encoding such variant proteases, as well as 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 for the production of protease variants intracellular or extracellular.

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

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

This invention relates also to compositions for treatment of tissue containing the variant protease of the present invention.

Brief description of drawings

On figa-In 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 is basic residues 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 number refers to the amino acid sequence of subtilisin from Bacillus amyloliquefaciens (which is sometimes defined as subtilisin BPN’). The second number refers to the amino acid sequence of subtilisin from Bacillus subtilis. The third number refers to the amino acid sequence of subtilisin from B.licheniformis. The fourth number refers to the amino acid sequence of subtilisin from Bacillus lentus (which is also referred to as the subtilisin 309 in PCT WO 89/06276). The symbol * means the absence of specific amino acid residues in comparison with subtilisin BPN’.

Detailed description of the invention

Proteases are carbonyl hydrolases, which usually 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 peptidylarginine, 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 enzymes, Protea is s, 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 the derived amino acid sequence of this variant. 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 these 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 variantarray 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 protease 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, deletion 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 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". Protease precursor are natural and recombinant proteases. Amino acid sequence variants of the protease derived from the amino acid sequence of the protease-predecessor by substitution, division 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-predecessor", the coding amino acid sequence of the protease-predecessor, and not the manipulation of enzyme protease predecessor per se. Acceptable ways such modifications of the DNA sequence of the predecessor include the 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.).

In this description of the invention identified specific substitutions of amino acids at positions one or more residues corresponding to the positions of the residues selected from the group comprising positions 62, 212, 230, 232, 252 and 257 of subtilisin Bacillus amyloliquefaciens.

The preferred options are options with a combination of C is of the sites in terms of residues, the relevant provisions of the subtilisin of Bacillus amyloliquefaciens in table 1.

Preferred variants are variants with combinations of substitutions in positions of residues corresponding to positions of the subtilisin of Bacillus amyloliquefaciens in table 2.

Other preferred variants are variants with combinations of substitutions in positions of residues corresponding to positions of the subtilisin of Bacillus amyloliquefaciens in table 3.

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 the particular 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 B.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 the WMD residue or portion of that residue in the subtilisin of Bacillus amyloliquefaciens (i.e. has the same or similar functional capacity to connect, to respond 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 Bacillus amyloliquefaciens and B.lentus. After performing a comparative analysis of conservative residues, allowing to identify insertions and deletions required to preserve the primary structure (i.e. to avoid deleting a conservative residues in 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, 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 who have 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 (carlsbergensis) 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 B.lentus) is presented in figure 2.

Thus, these conserved residues can be used to determine appropriate equivalent amino acid residues of subtilisin Bacillus amyloliquefaciens in other subtilisins, such as subtilisin from Bacillus lentus (PCT publication no WO 89/06279 dated July 13, 1989), the 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 Vall65 in subtilisin Bacillus amyloliquefaciens equiv the build-isoleucine in other subtilisin .lentus and B.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 location of the best models provides the maximum overlap of atomic coordinates of the protein atoms that 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 Bacillus amyloliquefaciens, defined as amino acid protease predecessor, which may adopt a conformation in which they can change, modify or stimulate the contraction in the Finance protein structure, the binding of 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 described in EPO publication No. 0251446 (corresponding to U.S. patent No. 5182204, which is 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. 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 this form. The preferred 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 the protease that may be involved in 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, involved in CE the SIP 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 subtilis, 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 that is operable linked to suitable regulatory sequence capable of 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 applicable to the th 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 methods described in U.S. patent No.'RE 34606, with the result that they lose the ability to secrete enzymatically active 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 subtilis I168 (which are also described in U.S. patent No. 34606 and 5264366 included in this description by reference), as well as any acceptable strain of Bacillus, such as B.licheniformis, B.lentus etc.

Cell host transformed or transferout vectors, sconst fravannini 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, the coding pre - 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, 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 the splitting of 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 PR is tease, and screening these libraries in relation to 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, operatively associated with the considered genome (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 stability of the 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 that enables constant cultivation 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 chrome is somnig restrictions. 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 microorganisms 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 sequence 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. An example of constructing a synthetic gene shown in example 3 of U.S. patent No. 5204015, which is 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 m is Magenta, 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 (deletion, 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 downregulation of state 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 convenient restriction site that is not redundant 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 changed nor the reading frame or the encoded amino acids. Mutation of the gene to change its placenta is, even so, to match the desired sequence, carried out using primer M13 in accordance with known methods. The task of localization acceptable flanking regions and identify changes needed to get two easy 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 the cognate restriction enzymes and are ligated in several gene cassettes oligonucleotides, complementary to the terminal areas. 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 which are economical measurement of proteolytic activity (K.M.Kalisz, "Microbial Proteinases", Advances in Biochemical 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 asm, 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 this invention, the aim is to obtain the variant protease with altered, preferably improved wetting characteristics compared with protease-precursor, at least one detergent composition and/or at least one wash cycle.

Washing conditions, the effect of which may be a variant protease, are very diverse, including different detergents, different amounts of the washing water, varying the temperature of the washing water and the duration of washing. For example, detergent compositions used in different regions, characterized by different concentrations of detergent components in the wash water. For example, detergents, used in Europe, usually contain about 4,500 to 5,000 ppm of detergent to the of mponents in the washing water, while the detergents used in Japan, usually contain approximately 667 ppm of detergent components in the wash water. In North America, particularly in the United States, detergents usually contain about 975 ppm of detergent components in the wash water.

System with a low concentration of detergent includes detergents, characterized by the presence of less than 800 ppm of detergent components in the wash water. 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 from about 800 to about 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 contain approximately 975 ppm of detergent components in the wash water. In Brazil commonly used detergents containing about 1500 ppm of detergent components in the wash water.

System with a high concentration of detergent includes detergents, characterized by the presence of more than 2000 ppm of detergent components in the wash water. European detergents are generally considered systems with a high concentration is of emergent, because they contain approximately 4,500 to 5,000 ppm of detergent components in the wash water.

The detergents used in Latin America, are usually vysokoparnymi, modified phosphate detergents, so they can be classified as systems with medium and high concentrations of detergent, as they are characterized by the presence of from 1500 to 6000 ppm of detergent components in the wash water. As mentioned above, the Brazilian detergents usually contain 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 containing up to 6000 ppm of detergent components in the wash water.

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 ppm ("regions of application systems with an average concentration of detergent"), for example, about 975 parts per million in the U.S. and about 1500 parts is a million in Brazil, and 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.

As another example of use is different in different regions of the washing temperature. The temperature of the washing water in Japan are generally lower than in Europe.

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

Another object of this invention is the substitution of amino acids at positions one or more residues corresponding to the positions of the residues selected from the group comprising positions 62, 212, 230, 232, 252 and 257 of subtilisin Bacillus amyloliquefaciens, which is important for improving the Oia of the cleansing action of this enzyme.

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.

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 variants of enzymes.

Many variants of the protease according to this invention are useful for various cleaning compositions or personal care products 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.Anderson, 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 options FR the basics of this invention can be used in any area, using 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 defined as the best spot cleaning is 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 wt.% (preferably from 0.1% to 0.5%). These cleansing composition with a detergent may also contain other enzymes, in particular 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, applicable to the s for a given detergent, acceptable for 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 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 not exhaustive 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 this 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 described in publications such as RD 216034, European patent # 134267, U.S. patent No. 4533359 and European patent No. 344259.

Further, the present invention is illustrated in the examples which do not limit the scope 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 using methods well known in this area. All mutations made in the subtilisin GG36 Bacillus lentus. These options are shown in table 4.

EXAMPLE 2

A large number of options protease obtained in example 1 was 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 comosition".

Table 5 presents the analyzed variants of the protease and the test results in two different detergents. In column a the data for filtered detergent Ariel Ultra (Procter & Gamble, Cincinnati, USA) with a concentration of 0.67 g/l in a solution containing 42,78 g/m3(3 grain/gallon) mixed hardness CA2+/MD2+and 0.3 ppm enzyme in each tank at 20°C. In the column In the data for filtered detergent Ariel Futur (Procter & Gamble, Cincinnati, USA) with a concentration 3,38 g/l 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°C.

EXAMPLE 3

Table 6 presents the analyzed variants of the protease of example 1 and the results of tests in four different detergents. These options proteases were subjected to the same tests on the efficiency, as in example 2, using the following detergents. In column a the data for filtered detergent Ariel Ultra (Procter & Gamble, Cincinnati, USA) with a concentration of 0.67 g/l in a solution containing 42,78 g/m3(3 grain/gallon) mixed hardness CA2+/MD2+and 0.3 ppm enzyme in each tank at 20°C. In the column In the data for filtered detergent Arie Futur (Procter & Gamble, Cincinnati, USA) with a concentration 3,38 g/l 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°C. In the column With the data for detergent with a concentration of 3.5 g/l HSP1 (Procter & Gamble, Cincinnati, USA) in a solution containing 114,08 g/m3(8 Grand per gallon) mixed hardness CA2+/MD2+and 0.3 ppm enzyme in each tank at 20°C. In column D shows the data for detergent concentration 1.5 g/l Tide CT (Procter & Gamble, Cincinnati, USA) in a solution containing 42,78 g/m3(3 grain/gallon) mixed hardness CA2+/MD2+and 0.3 ppm enzyme in each tank at 20°C.

1. Variant of subtilisin Bacillus, which is the result of a combination of substitutions, one of which is a replacement of the amino acid residue of the native form of the enzyme in a position corresponding to position 232 of subtilisin Bacillus amyloliquifaciens, valine, and showing increased compared with the combination of substitutions N76D/S103A/V104I the effectiveness of actions, where this option is the result of one of the combinations of substitutions presented in table 6.

2. Variant of subtilisin Bacillus, which is the result of a combination of substitutions, one of which is a replacement of the amino acid residue natively the shape of the enzyme in position, corresponding to the position 212 of subtilisin Bacillus amyloliquifaciens, Proline, and showing increased compared with the combination of substitutions N76D/S103A/V104I the effectiveness of actions, where this option is the result of a combination of substitutions selected from the group consisting of

N76D/S103A/V104I/S212P/E271V;

N76D/S103A/V104I/S212P/G258R;

N76D/S103A/V104I/S212P/V268F/E271 V, and

N76D/S87R/S103A/V104I/S212P/E271V.

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

4. DNA encoding a variant of subtilisin Bacillus, which is characterized by a nucleotide sequence in accordance with the genetic code determines the amino acid sequence of the variant subtilisin according to any one of claims 1 to 3.

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 Bacillus according to any one of claims 1 to 3.

The conventional priority of claims 1 to 6 is selected from 23.10.1997 according to the date of filing 08/956,323, 08/956,324 and 08/956,564 in the U.S. Patent office.



 

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