Protease of improved type obtained from microorganisms, providing milk clotting

FIELD: biotechnology.

SUBSTANCE: protease is presented which has enhanced milk clotting activity, containing amino acid sequence at least 80 % identical to SEQ ID NO: 3, where the said protease has at least one mutation selected from the group consisting of: (a) substitution of glutamine, corresponding to glutamine at a position of 265 in SEQ ID NO: 3, with amino acid; and (b) replacement of glutamine, corresponding to glutamine at a position of 266 in SEQ ID NO: 3, with amino acid. DNA is described which encodes the said protease, the expression vector containing the said DNA, and the cell transformed with the said vector, designed for expression of the said protease. The method of production of protease having enhanced milk clotting activity is proposed, comprising culturing the said transformed cell in the cultural medium and isolation of protease from the cultural medium.

EFFECT: invention enables to obtain the protease with enhanced milk clotting activity.

16 cl, 2 dwg, 4 tbl

 

The technical FIELD

The present invention relates to obtained from a microorganism of the protease with improved activity against clotting of milk. The protease is preferably used for the production of cheese.

PRIOR art

For a long time rennet from the stomach of calves used as a milk-clotting enzyme for the production of cheese. Milk-clotting activity of rennet from the stomach of calves, mainly associated with chymosin, which is an acidic protease and has site-specific proteolytic activity against casein milk with low site-specific activity (specifically cleaves the peptide bond between the phenylalanine at position 105 and the methionine at position 106 in the amino acid sequence of κ-casein). Assume that non-specific proteolytic activity leads to decreased production of cheese and obtain the peptide with a bitter taste in the maturation process. For this reason, the chymosin is an excellent milk-clotting enzyme.

However, the reduction of slaughter calves and the increase in the demand for cheese has created difficulties in the supply of rennet enzyme from the stomach of calves. Currently, milk-clotting enzyme derived from microorganisms, such as the Rhizomucor mieheiandRhizomucor pusillusand recombinant chymosin obtained by introduction of a gene of calf chymosin in fungi or yeast, widely used as a milk-clotting enzymes.

The above is obtained from a microorganism milk-clotting enzyme, compared with calf chymosin or recombinant chymosin has a higher non-specific proteolytic activity. The problem lies in the fact that the ratio C/P (the ratio of milk-clotting activity to proteolytic activity), which is an important property of a milk-clotting enzyme, remains low. To overcome this obstacle inRhizomucor pusilluscarried out the expression and analysis of a modified gene milk-clotting enzyme, obtained using site-specific mutagenesis method of genetic engineering. In this embodiment, the ratio C/P was improved compared with the wild type by replacing the glutamic acid to alanine at position 19 the amino acid sequence of the milk-clotting enzyme (document 1, non-patent).

However, due to the fact that the activity of the modified milk-clotting enzyme from the replacing amino acids was decreased by about 40 percent, the practical application of this enzyme has proven to be difficult. Therefore clicks the zoom, it is desirable to obtain microorganism milk-clotting enzyme, in which the ratio C/P is still high, maintaining or increasing milk-clotting activity.

In addition, using a dicarboxylic anhydride was attempted acylation of milk-clotting enzyme derived from microorganisms such asRhizomucor pusillusandRhizomucor mieheito increase the ratio C/P (patent document 1). Using this method it was obtained a slight increase; however, this is not yet satisfactory.

[Patent document 1] Patent No. 2-18834B, Japan.

[Document 1, non-patent]J. Biochem.129, 791-794, 2001.

The INVENTION

The aim of the present invention to provide a protease suitable for milk clotting activity (hereinafter referred to in this document also referred to as "non-specific protease activity") which, in relation to the cleavage of the peptide bond that is different from the relationship between the phenylalanine at position 105 and the methionine at position 106 in the amino acid sequence of κ-casein remains low, and milk-clotting activity is maintained or increased.

The authors of the present invention have carefully studied the ways of overcoming the problems described above, and among the mutant strains of microorganisms, producing malakasiotis the Mering enzyme, allocated producing milk-clotting enzyme mutant strain with increased C/P, which is associated with reduced non-specific protease activity; isolated a gene milk-clotting enzyme improved type; determined its nucleotide sequence; carried out the gene expression and measured milk-clotting activity and the ratio C/P milk-clotting enzyme of an improved type, completing thus the present invention.

Thus, the present invention relates to obtained from a microorganism of the protease with milk-clotting activity, with preserved or increased milk-clotting activity and increased C/P, and also provides DNA encoding the protease, the vector containing the DNA, and transformed cells in which the introduced vector.

One aspect of the present invention to provide a protease advanced type, which contains an amino acid sequence that is at least 75% identical to SEQ ID NO:3, where this protease advanced type has at least one mutation selected from the group consisting of:

(A) replacement of glutamine, corresponding glutamine in position 265 in SEQ ID NO:3, an acidic amino acid and

(B) a substitution of glutamine at position 266 SQ ID NO:3 in an acidic amino acid, where this protease advanced type has a milk-clotting activity.

Another aspect of the present invention to provide a protease advanced type, as described above, which is selected from the group consisting of:

(A) a protein containing the amino acid sequence of SEQ ID NO:3 or 43, except that the glutamine at position 265 and/or glutamine at position 266 replaced by an acidic amino acid;

(B) a protein containing the amino acid sequence of SEQ ID NO:3 or 43, except that the glutamine at position 265 and/or glutamine at position 266 replaced by(s) acidic amino acid, and not more than 10 amino acids, preferably not more than 5 amino acids, more preferably not more than 3 amino acids, even more preferably not more than 2 amino acids) in provisions other than 265 and 266, replaced deleterows, inserted or added, and where the specified protease advanced type has a milk-clotting activity.

Another aspect of the present invention to provide a protease advanced type, as described above, in which the acidic amino acid is glutamic acid or aspartic acid.

Another aspect of the present invention to provide a protease advanced type, as described you the e, in which glutamic acid at position 19 is replaced with valine, alanine, isoleucine, or leucine.

Another aspect of the present invention to provide a protease advanced type, as described above, in which the threonine at position 81 is replaced by glutamine or aspartic acid.

Another aspect of the present invention to provide a DNA that encodes a protease advanced type, as described above.

Another aspect of the present invention is to obtain expression vector containing the DNA as described above.

Another aspect of the present invention is to obtain transformed cells, which enter the above expression vector.

Another aspect of the present invention is to obtain transformed cells as described above, where this transformed cell is aSaccharomyces cerevisiae.

Another aspect of the present invention is to provide a method of producing a protease advanced type, with milk-clotting activity, which includes the stage of culturing the above transformed cells in culture medium and collecting protease advanced type in culture medium.

Because milk-clotting activity saved or is lucchina, and the ratio C/P is high, expect a higher volume of cheese production with the use of protease advanced type according to the present invention. In addition, increased ratio C/P, as a rule, involves the reduction of bitter taste of the cheese during ripening, thus, with this enhanced enzyme can be produced cheeses higher quality.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows the structure of the expression vector JS4.

Figure 2 shows the sequence alignment of the protease derived fromRhizomucor pusillus(RMPP), and protease derived fromRhizomucor miehei(RMMP).

DESCRIPTION of embodiments of the INVENTION

The present invention is explained below.

1. Protease advanced type (milk-clotting enzyme) according to the present invention

Protease advanced type according to the present invention contains an amino acid sequence that is at least 75% identical to SEQ ID NO:3 and has at least one mutation selected from the group consisting of:

(A) replacement of glutamine, corresponding glutamine in position 265 in SEQ ID NO:3, an acidic amino acid and

(B) a substitution of glutamine at position 266 in SEQ ID NO:3 in an acidic amino acid with milk-clotting activity.

Examples of the above kalyaninagar include glutamic acid and aspartic acid.

Protease advanced type according to the present invention preferably has a homology of at least 90%, more preferably not less than 95%, with a complete amino acid sequence of SEQ ID NO:3.

In one of the embodiments of the invention the protease advanced type according to the present invention can be obtained by introducing mutation(s) in the protease wild-type derived fromRhizomucor miehei(SEQ ID NO:3). In this embodiment of the invention the protease improved type of the present invention is selected from the group consisting of:

(A) a protein containing the amino acid sequence of SEQ ID NO:3, except that the glutamine at position 265 and/or glutamine at position 266 replaced by(s) on an acidic amino acid;

(B) a protein containing the amino acid sequence of SEQ ID NO:3, except that the glutamine at position 265 and/or glutamine at position 266 replaced by(s) on an acidic amino acid, and not more than 10 amino acids in positions other than positions 265 and 266, replaced deleterows, inserted or added, with a milk-clotting activity.

Figure 2 shows the sequence alignment of the protease derived fromRhizomucor pusillus, and protease derived fromRhizomucor miehei. In both amino acid sequences at positions 265 and 266 are the same, therefore, the protease mustache is chenstohova type according to the present invention can also be obtained by introducing mutations in the protease wild-type from the Rhizomucor pusillus(SEQ ID NO:43). Thus, in another embodiment of the invention the protease advanced type according to the present invention may be a protein containing the amino acid sequence of SEQ ID NO:43, except that the glutamine at position 265 and/or glutamine at position 266 replaced by(s) on an acidic amino acid. In addition, this enhanced protease type can contain another mutation (substitution, deletion, insertion, or addition of not more than 10 amino acids), other than the substitution of glutamine at position 265 and/or glutamine at position 266, the condition of preservation of milk-clotting activity.

In the protease advanced type according to the present invention glutamic acid at position 19 and threonine at position 81 in the amino acid sequence of SEQ ID NO:3 or 43 can be replaced by other amino acids. Glutamic acid at position 19 is preferably substituted with valine, alanine, isoleucine or leucine, while the threonine at position 81 is preferably substituted with glutamine or aspartic acid.

Really izobreteniya "position 265", "position 266", "regulation 19 and regulation 81" does not necessarily indicate the absolute position from the N-Terminus of the protease, but indicate the relative position when compared with the amino acid sequence SEQ ID NO:3 or 43. For example, protease, containing the amino acid sequence SEQ ID NO:3 or 43, in the case of a deletion of one amino acid at a position closer to the N-terminal region from position 265 the above position 265 becomes the position 264. Even in this case, the amino acid at position 264, counted from the N-terminal residue is the amino acid at position 265" of the present invention. The absolute position of the amino acids is determined using an alignment of the amino acid sequence described protease with an amino acid sequence of SEQ ID NO:3 or 43. The amino acid denoted by the term "corresponding position", also refers to the amino acid at relative position compared to the amino acid sequence of SEQ ID NO:3 or 43.

SEQ ID NO:3 and SEQ ID NO:43 is the amino acid sequence of the Mature form of the protease. Protease advanced type according to the present invention may include amino acid sequence of the signal peptide, propeptide and so on.

Using the method described in the examples of this description of the invention, the protease advanced type according to the present invention can be obtained from cells of mutant strain or from the appropriate culture medium with selection of a mutant strain that produces protease advanced type with high respect to the observed C/P, from a microorganism that produces protease wild-type, with milk-clotting activity with a relatively low C/P, and cultivating the mutant strain in the culture medium.

Examples of microorganisms which produce protease wild-type with a relatively low C/P, include strains of wild-typeRhizomucor miehei(ATCC16457),Rhizomucor pusillus(ATCC16458) and the strains derived from these strains. These strains can be purchased at the American type culture collection (ATCC; P.O. Box 1549 Manassas, VA 20108 USA). Protease advanced type according to the present invention can also be obtained by using DNA that encodes a protease improved type of the above mutant strain, and the expression of this DNA.

In addition, protease advanced type according to the present invention can also be obtained by using the DNA that encodes the amino acid sequence of SEQ ID NO:3 or 43, of the strains of wild-typeRhizomucor miehei(ATCC16457),Rhizomucor pusillus(ATCC16458) or from strains derived from these strains, and modification of DNA by site-specific mutagenesis so that DNA encodes the protease improved type of the present invention, with subsequent expression of the modified DNA.

The expression of the above DNA can osushestvljali by constructing expression vector, containing the above DNA, and introducing it into the cell of the host. Although the host-cell can be prokaryotic, and eukaryotic cells, is preferred eukaryotic cell. Examples of eukaryotic cells are yeast cell, a cell of a fungus and a plant cell. Yeast cell is preferred, and the cell isSaccharomyces cerevisiaeit is especially preferred.

In addition, the expression of the above DNA can be performed in a cell-free system.

The ratio C/P of protease improved type of the present invention is higher than the ratio C/P of the corresponding protease wild-type (SEQ ID NO:3 or 43). The ratio C/P of protease improved type of the present invention is preferably not less than 1.2 times, more preferably not less than 1.5 times, more preferably not less than 2.0 times the ratio C/P of protease wild-type (SEQ ID NO:3 or 43).

Hereafter in this document the ratio C/P denotes [milk-clotting activity (MCA)]/[proteasa activity (PA)]. Measuring PA and MCA can be made by the methods set forth hereinafter. With regard to the measurement of MCA, despite the fact that there is an international Standard Method (described in ISO15174, IDF176; first edition 2002-09-01, Self-imposed Specifications for Food Additives), the value of MCA in the present description must count ivali on the way, specified below (hereinafter in this document identified as a way Meito).

[1] Measurement of PA

Casein derived from milk (manufactured Wako Pure Chemical Industries, Ltd.), dissolved in 0,05M solution disubstituted hydrogen phosphate sodium and pH adjusted to 6.0 by adding a standard solution of hydrochloric acid (1 mol/l)to obtain a 0.6% solution of casein substrate. The sample (0.2 ml), diluted appropriately, add to 1 ml of this substrate solution. The mixture is left at 37°C for 10-30 minutes to complete the reaction, which is then stopped by adding 1 ml of the solution, stopping the reaction (a mixed solution consisting of 0.11 mol/l trichloroacetic acid, 0.21 mol/l of anhydrous sodium acetate and 0.33 mol/l acetic acid). The supernatant get through centrifugation and 0.4 ml of supernatant was added 1 ml of 0.55 mol/l of anhydrous sodium carbonate, and then add 0.2 ml of the diluted twice phenol reagent production Wako Pure Chemical Industries, Ltd. (reagent Polina-Ciocalteu). The mixture was incubated for 30 minutes at 37°C for completion of the reaction, and then measure the absorption of light at 660 nm (optical path length 1 cm). Separately, 1 ml of the substrate solution, add 1 ml of solution to stop the reaction, followed by the addition of 0.2 ml of the sample. Then get the mixture using the same methods and solution the resulting, used as a control. The value obtained by subtracting the absorption of the control of the absorption of the sample, transfer in the amount of free tyrosine to calculate the values of PA. Units PA are U/ml, This 1 IU denotes the amount of enzyme that causes a strengthening of the coloring substances phenol reagent, equivalent to 1 µmol of tyrosine for 1 minute according to the method specified above. In addition, the receive correlation ratio between the amount of tyrosine and colouring substances phenol reagent using a calibration curve of tyrosine as described below.

Calibration curve of tyrosine

Tyrosine, which is the standard (molecular weight 181,2, production Wako Pure Chemical Industries, Ltd.) dried at 105°C for 3 hours. Then 0,050 g standard, accurately weighed and dissolved in the test solution of hydrochloric acid (0.2 mol/l) so, to get exactly 50 ml of the final solution. 1, 2, 3, and 4 ml of this solution to accurately measure and to each of them add a standard solution of hydrochloric acid (0.2 mol/l) to obtain a volume of 100 ml Accurately measure two ml of each solution. Then add 5 ml of 0.55 mol/l standard solution of sodium carbonate and 1 ml of phenol reagent diluted twice. Immediately after this, the solution of paramashiva the t by shaking and incubated for 30 minutes at 37±0.5°C. From the resulting solution is taken exactly 2 ml of the obtained solution and measure the absorption of A1, A2, A3, and A4 at a wavelength of 660 nm, and the absorption of the control solution prepared in the same way. Delaying the absorption values A1, A2, A3, and A4 on the vertical axis and the number of tyrosine (in µmol) in 2 ml of each solution on the horizontal axis, receive a calibration curve for determining the amount of tyrosine (in µmol) when the difference in absorption equal to 1.

[2] the Method of measurement (MCA method Meito)

Skimmed milk powder for use as a substrate, it is preferable production CHR.HANSEN, (10%) dissolved in 0,01M calcium chloride (pH of 6.0). A solution of the sample (0.5 ml)prepared in the concentration at which the formation of caseous fragments within 2-5 minutes, preferably 2 minutes and 30 seconds, add 5 ml of this substrate, and the mixture is incubated at 35°C. the Formation of caseous fragments observed when mixing the mixture with a glass rod. MCA is determined using the calculation: how many times more substrate can roll the unit number of the sample per unit of time compared with the value for the standard for which MCA is known and calculated in the same way. The formula for calculation is as follows:

MCA (Mu/ml) = S×(Ts×Ws)/(T×W),

Where S is the specific aktivnosti milk-clotting enzyme standard (Mu/g);

Ts -the clotting time of milk standard solution (second);

Ws -the number of standard in 1 ml of a standard solution (g);

T - clotting time of milk solution sample (second);

W - the number of the sample in 1 ml of sample (ml).

In addition, the MCA can be calculated in terms of a single protein with the help estimate the total amount of protein contained in the sample. In example 13, described later, MCA counting on 1 mg of protein (Mu/mg protein).

The MCA is calculated by the above method, correlates with the value of MCA, calculated using the International Standard (described in ISO 15174, IDF176; first edition 2002-09-01, Self-imposed Specifications for Food Additives). This correlation can be represented by the following formula:

1 unit of international standard (IMCU/ml) ~ 1 unit Meito (Mu/ml)/100.

MCA protease of the present invention preferably is equal to or greater than MCA protease wild-type. When the protease of the present invention and protease wild-type (SEQ ID NO:3 and 43) are obtained in the same conditions for comparison MCA, MCA protease improved type of the present invention is preferably not less than 0.8, more preferably not less than 0.9, more preferably not less than 1.0 on the value of MCA protease d is anyone type.

Examples of receiving protease advanced type according to the present invention and protease wild-type under the same conditions include the introduction of DNA encoding each of the proteases in the same vector for gene expression, the introduction of each of these expression vectors into a cell of the same strain in the same conditions and culturing the cells in the same culture conditions to obtain a cell culture containing the protease solution. The obtained culture can be concentrated in the same way or to clean the same way for use.

2. DNA encoding a protease advanced type according to the present invention

The DNA of the present invention is DNA encoding a protease of the advanced type of the present invention. Specific examples of the DNA of the present invention include a DNA containing a nucleotide from 208 to 1290 in SEQ ID NO:1 and DNA containing a sequence that hybridizes with a nucleotide sequence complementary to the nucleotides from 208 to 1290 in SEQ ID NO:1 under strict conditions; and encoding the protease advanced type with the above described properties. Specific examples of the DNA of the present invention also includes a DNA containing the nucleotide sequence of SEQ ID NO:42, and DNA containing the sequence that hybridizes is with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:42, in simple terms; and encoding the protease advanced type with the above described properties. Strict conditions indicate conditions, which formed the so-called specific hybrids, while non-specific hybrids are not formed. Although conditions vary from the sequence of nucleotides, or its length, examples of conditions include conditions in which DNA with high homology, for example, DNA homology of at least 75%, preferably not less than 90%, more preferably not less than 95%, mutually hybridize, while the DNA with lower homology not hybridized, or conditions of hybridization, which is normal for washing by hybridization to Southern, at 60°C in 1×SSC, 0,1% SDS, preferably of 0.1×SSC and the salt concentration equivalent to 0.1% SDS.

DNA encoding the protease of the present invention, can be isolated from a mutant strain containing the above protease advanced type, using the standard method of gene cloning. For example, it can be selected by pressing the selection of DNA from a genomic library of the above mutant strain using hybridization with a synthetic oligonucleotide probe based on the nucleotide sequence of SEQ ID NO:1 or 42.

In addition, DNA encoding the protease advanced type on the present and the finding, can be obtained by designing primers based on the nucleotide sequence of the known genomic DNA or cDNA of the gene of the protease wild-type and DNA amplification from genomic DNA library and cDNA library above mutant strain using these primers.

DNA obtained by introducing site-directed mutations in the DNA of wild-type, also included in the DNA encoding the protease of the present invention.

For example, DNA encoding the protease advanced type according to the present invention can be easily obtained by using the DNA that encodes the amino acid sequence of SEQ ID NO:3 from strainRhizomucor mieheiwild-type (ATCC16457), or derived from this strain and introducing site-directed mutations. DNA encoding the protease advanced type according to the present invention can also be obtained by using the DNA that encodes the amino acid sequence of SEQ ID NO:43 from strainRhizomucor pusilluswild-type (ATCC16458) or derived from a strain and introducing site-directed mutations.

The introduction of site-directed mutations can be made by methods known to experts in this field. For example, mutations can be entered using fusion primers containing the cleavage site for the restriction enzyme on the one hand and containing CA is t mutation on the other hand, and as a result of replacement of the relevant part of the gene without the mutation fragment of the gene with a mutation (way cassette mutations).

As a way of introducing site-directed mutations, for example, known methods include a method Gapped duplex ("torn" duplex") and the kunkel method. The kunkel method is based on the principle that gene without the mutation clone in single-stranded phage; complementary chain synthesized using as primers the synthetic DNA containing the error mating grounds in point mutations, then get a new phage and replicated DNA, using as matrix obtained complementary circuit containing the mutation. Site-specific mutagenesis can be performed using commercially available kit.

3. The expression vector of the present invention

For expression of the protease advanced type according to the present invention using the expression vector of the present invention. He may have a structure in which the sequence of the promoter regulating expression of the DNA in the region of the 5'end is adjacent to the DNA that encodes the protease advanced type according to the present invention. In addition, in the region of the 3'-end of the DNA can join the terminator.

When the host-cell isE. colias the above promoters can use the ü trp,lac,taq, λPLor similar. If the host-cell is a yeast, the preferred promoters are GAL7, ADH, TPI, or PHO5 or the like, and among them GAL7 is preferable, because it increases gene expression (Y. Nogiet al.Nucl. Acids Res.11, 8555-8568 (1983)).

Examples of terminators include TPI, GAPDH and GAL10. Thus, the expression vector of the present invention can be constructed by attaching the above promoter, DNA encoding the protease improved type of the present invention, and the above terminator in order, from the region which is above the 5'-region towards the region lying below the 3'region, and inserts the resulting construction in the vector.

As a vector that can replicate in yeast, you can use any type of plasmids from the so-called YIp, YRp, YEp and YCp plasmids. From the point of view of copy number and stability, it is preferable type YEp. Since these plasmids usually contain unnecessary sequence, it is preferable to deletion of unnecessary sequences, based on considerations of the stability of plasmids or to facilitate modification of the plasmid.

In the expression vector according to the present invention it is also possible to insert a gene marker selection for breeding or recombinant rap is Chernogo gene to verify the expression of the introduced gene. Examples of the gene marker selection include genes for resistance to hygromycin, genes for resistance to kanamycin genes for resistance to ampicillin. Examples of the reporter gene include genes beta-glucuronidase (GUS), genes chloramphenicol, acetyltransferase (CAT), genes luciferase (LUC) and GFP genes. In addition, in the expression vector according to the present invention it is possible to insert additional sequence to expression of protease advanced type according to the present invention secretory type, or to facilitate purification of expressed protease. In this case, the protease of the present invention is expressed in the form of a fused protein with the protein or peptide encoded by the additional sequence. Examples of additional sequences include a nucleotide sequence encoding a signal peptide or propeptide, and the nucleotide sequence encoding a His-tag or GST-tag.

4. The transformed cell according to the present invention

Transformed cell of the present invention is a cell in which the introduced expression vector of the present invention, and this cell is capable of producing protease advanced type according to the present invention. Despite the fact that the cell can be a prokaryotic cell and eukaryotic, Cleco is, preferred is a eukaryotic cell.

Examples of eukaryotic cells include a yeast cell, a cell of fungi and the plant cell. Yeast cell is preferred, andSaccharomyces cerevisiaeit is especially preferred.

Examples ofSaccharomyces cerevisiaeinclude strains SHY3, D13-1A and MC16.

The method of introducing the expression vector into the cell host may be selected appropriately depending on the type of host cell. These methods are methods known to experts in this field. Transformed cellSaccharomyces cerevisiaefor example, can be obtained as follows.

CultureSaccharomyces cerevisiaecultured in the medium for cultivation of YPD (1% yeast extract (production Difco), 2% Bactopeptone (Proizvodstvo Difco) and 2% glucose) overnight, inoculated in a ratio of 10% by volume on the media for cultivation of YPD and grown for 4 hours at 30°C. the resulting culture (1.5 ml) weakly centrifuged in a tabletop centrifuge to precipitate the cells. Cells are washed with 0,2M LiSCN (production Kanto Chemical Co., Inc.) and dissolved in 0.02 ml of 1M LiSCN.

Then mix 0.01 ml of a solution containing expression vector (approximately from 1 to 10 mcg), and 0.03 ml of 70% PEG 4000, this mixture is then incubated for 1 hour at 30°C. the resulting mixture was diluted by the addition of 0.14 ml of trilinos water and then plated on two tablets SDah (0,67% basics nitrogen agar for yeast without amino acids, 2% glucose, 0,002% adenine sulfate, 0,002% L-histidine-HCl, 2% agar). After incubation at 30°C for 2-3 days, you can obtain the transformant.

5. A method of producing a protease advanced type, with milk-clotting activity, according to the present invention

Protease advanced type according to the present invention can be obtained by culturing the transformed cells of the present invention, and in the expression of protease advanced type according to the present invention in the form of a fused protein with a signal peptide for the implementation of secretions, protease advanced type according to the present invention can accumulate in the environment. When using the inducible promoter, the induction is preferably carried out in the environment. Despite the fact that the method of cultivation of transformed cells varies with cell type, you can use the standard methods.

The following describes an example of a method of cultivation of transformantSaccharomyces cerevisiae.

To implement the proliferation of yeast cells transformed cultured with shaking at 30°C for two days in 50 ml of medium for cultivation in YPD Sakaguchi flask with a volume of 500 ml Culture medium centrifuged at 1000×g for 5 minutes to precipitate the cells. ZAT is m cells are again suspended in 100 ml of culture medium YPGal (1% yeast extract, 2% of bactopeptone, 4% galactose (manufactured Wako Pure Chemical Industries, Ltd.)), and cultivated in shake a Sakaguchi flask 500 ml at 30°C for three days.

Protease with milk-clotting activity, which is secreted into the environment, can be used in the form in which it exists in the supernatant environment, and can also be used after concentration of the supernatant medium. Protease with milk-clotting activity, which is secreted into the environment, you can clear or partially clear. Purification or partial purification can be performed using the General method of purification protein. You can use, for example, a method including chromatography, such as ion-exchange chromatography, gel filtration, salting out using ammonium sulfate or the deposition of organic solvent.

The purified enzyme also can be concentrated by lyophilization, membrane ultrafiltration, precipitation with an organic solvent or similar methods.

EXAMPLES

In this document, the present invention is described specifically as examples, but the technical field of the present invention is not limited to these examples. All genetic manipulations can be performed as described inMolecular Cloning(Cold Spring Harbor Laboratory Press (1989)).

Example 1

Received the e mutant strain of Rhizomucor mieheithat produces a protease with an increased ratio C/P

The parent strain ofRhizomucor miehei(CBS182-67 (derived strain from ATCC16457)), which produces protease, were subjected to the process of mutagenesis, was thus given mutant strain that secretes a protease with an increased ratio C/P. the Details below.

(1) Process of mutagenesis

The parent strain ofRhizomucor mieheigrown on tablets with malt (2% of malt extract, 2% glucose, 0.1% peptone, 2% agar) and left on time from 3 days to 1 week at 37°C to provide education dispute. Data spores suspended in sterile water using the device for the suspension of glass.

To this suspension containing spores was added nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine produced by SIGMA CHEMICAL CO.) to a final concentration of 200 μg/ml and the Mixture was left at room temperature for 5-20 minutes, so the mortality rate was 90%. A suitable quantity of this mixture was placed on the tablet with the malt and the resulting tablet was kept at 37°C. the next day, each cluster obtained a small fungal hyphae were made in 8 ml of medium for culturing YPD (1% yeast extract, 2% peptone, 2% glucose) and culture supernatant after cultivation at 37°C for 4 days was used as a sample for serenityactual protease (PA) and milk-clotting activity (MCA). Cells were stored at -80°C.

(2) Search protease advanced type

In the measurement result MCA and PA using the above method has been enhanced protease type PA is much smaller than PA parent strain, and the ratio C/P (MCA/PA) is significantly higher than 4.6 times compared with the parent strain.

Example 2

The selection of the gene of the protease mutant strain

(1) preparation of chromosomal DNA of the mutant strain and the parent strain

Obtained in example 1 mutant strain and the parent strain were grown on the tablet with malt and kept at 37°C for from three days to one week to provide education dispute. The obtained spores suspended in sterile water using the device for the suspension of glass. The resulting suspension containing spores were seeded into 200 ml of YPD liquid medium in a Sakaguchi flask 500 ml, so that each vial contained approximately 1×108spores were cultured for two days at 37°C. when the cells formed a debris size from approximately 0.5 to 2 mm, the medium was filtered to remove excess moisture and thus received approximately 5 g wet weight of cells.

After freezing with liquid nitrogen cells was transferred into a pre-chilled mortar and added 3 g of sea sand (from 850 to 1400 microns). In terms of the s cooling with liquid nitrogen, the mixture was ground into a fine powder with a pestle and mortar. The resulting mixture was suspended in 15 ml of a solution containing 0,05M EDTA pH 8.5 and 0.2% SDS solution which was pre-heated to 68°C and the resulting mixture for 15 minutes and kept at 68°C. Further, it is left to stand and allowed to cool to room temperature, and the turbid supernatant was collected by centrifugation. Once added to the collected solution 1/10 volume of 3M sodium acetate, the mixture was gently mixed and collected the supernatant by centrifugation. Further adding to the collected supernatant 15 ml isopropanol and careful mixing appeared tangle genomic DNA c proteins. After the precipitate was washed with 70% ethanol and dried under reduced pressure, it was dissolved in 400 μl TE and 10 μl of a solution of RNase (10 mg/ml). The mixture was left at 37°C for 1 hour. After processing by RNase spent processing the phenol/chloroform and chloroform, followed by precipitation with ethanol, resulting in the obtained genomic DNA.

(2) the Allocation of protease gene from the chromosomal DNA of the mutant strain and the parent strain

Gene protease was isolated by PCR using the chromosomal DNA obtained from a mutant strain obtained as described above, and the parent strain as a matrix. Based on the sequence of the protease of theRhizomucor miehei, registered the Anna in the genomic library (DDBJ access number: E01264), received the primers SEQ ID NO:5 and SEQ ID NO:6. The PCR conditions consisted of (a) at 94°C for 2 minutes; (b) 28 cycles at 94°C for 30 seconds at 55°C for 30 seconds at 72°C for 3 minutes; and (c) at 72°C for 5 minutes. As the polymerase used TaKaRa Ex Taq (manufactured by Takara Bio Inc.). As thermal cycler used PCR TaKaRa thermal cycler Dice Gradient (manufactured by Takara Bio Inc.). By determining the nucleotide sequence of the DNA fragment obtained by PCR, revealed that the amino acid sequence encoded by the DNA amplified from the chromosomal DNA of the parent strain as a matrix, contained the amino acid sequence of SEQ ID NO:3. Amino acid sequence encoded by the DNA amplified from the chromosomal DNA of the mutant strain as a matrix, contained the amino acid sequence of SEQ ID NO:4, where the amino acid at position 19 was replaced with valine, and the amino acid at position 266 was replaced by glutamic acid.

Later in this document protease obtained from a parent strain ofRhizomucor miehei,call RMMP wild-type and protease advanced type called "advanced RMMP" and the gene encoding protease of the advanced type, called "enhanced gene RMMP".

Example 3

Construction of the plasmid is the sector JS4 for the expression of a foreign protein using as host cells budding yeast ( Saccharomyces cerevisiae) MC16

To construct a plasmid vector JS4 as source material used JS5 (described in the patent No. 3012377 [0109], Japan).

First, PCR was performed using DNA primers of SEQ ID nos:7 and 8, using JS5 as matrix thus obtained PCR product length is 0.55 TPN containing the promoter region GAL7. The PCR conditions consisted of (a) at 94°C for 2 minutes; (b) 30 cycles at 98°C for 10 seconds at 52°C for 30 seconds at 72°C for 1 minute; and (c) at 72°C for 5 minutes. As the polymerase used TaKaRa Ex Taq (manufactured by Takara Bio Inc.). As thermal cycler used PCR TaKaRa thermal cycler Dice Gradient (manufactured by Takara Bio Inc.).

Then the obtained PCR product was subjected to cleavage with enzymesEcoR and IBamH I and introduced into pUC18, which was also subjected to cleavage byEcoR and IBamH I. the resulting plasmid was introduced inE. coliDH5α cells were distributed on the tablet with LB agar containing 100 μg/ml of ampicillin, 0.1 mm IPTG and 0.04 mg/ml X-GAL, and incubated at 37°C for 16 hours. Appeared a white colony was cultured with shaking in LB liquid medium containing 100 μg/ml ampicillin, at 37°C for 14 to 16 hours. From the transformants, collected by centrifugation, the plasmid was isolated using a kit QIAprep Minprep kit (QIAGEN, later in this document, all of the plasmids were isolated with the help of this set). The built-in fragment was subjected to sequencing to confirm that unwanted mutations were not introduced.

Then plasmid containing an embedded fragment was digested withEcoR and IBamH I to obtain a DNA fragment with a length of 0.55 TPN and then spent ligation of this DNA fragment and the DNA fragment length of approximately 6 TPN obtained by splitting JS5 withBamH I, followed by partial cleavage withEcoR I. the resulting plasmid was introduced inE. coliDH5α and transformedE. colicultivated in LB medium with agar containing 100 μg/ml ampicillin, at 37°C for 16 hours. The colony was cultured with shaking in the same liquid medium at 37°C for 14 to 16 hours, and then from the transformants, collected by centrifugation, were isolated plasmid. Using enzymesEcoR I,BamH I andPstI this plasmid was digested to confirm the pattern of migration using electrophoretic analysis on an agarose gel. Thus obtained expression vector JS4.

As source material for constructing this plasmid vector, in addition to JS5, it is also possible to use, for example, JS52 (access number FERM BP-3898), described in p is regrate 0112 patent No. 3012377, Japan.

Example 4

Construction of plasmid vectors for ekspressirovali gene RMMP wild-type and gene RMMP advanced type

PCR was performed using primers (SEQ ID nos:9 and 10), designed to contain a restriction site forBamH I from both ends of the nucleotide sequence containing the DNA encoding RMMP wild-type or RMMP advanced type, with the mutations Glu19Val/Gln266Glu, which was obtained in example 2. The resulting PCR product was digested withBamH I, was introduced in JS4, which has also been splitting withBamH I, dephosphorylation, and the resulting vector was introduced inE. coliDH5α.

Using direct primer, which can gibridizatsiya with the GAL7 promoter and a reverse primer that can gibridizatsiya with 3'-end of the gene RMMP (SEQ ID NO:11 and 10)was subjected to PCR with bacterial colonies. The transformantE. coliwith plasmid vector, which confirmed the correct orientation of the introduced gene were subjected to cultivation in liquid medium as described above. The plasmids were isolated and subjected to sequencing to confirm the absence of unwanted errors thus obtained plasmid vectors for gene expression RMMP wild-type and gene RMMP advanced type.

Example 5

Construction of expression century the ora genes RMMP advanced type, entered site-directed mutation (I)

The PCR product of the gene RMMP wild type, obtained using the method described in example 4, which contains the sequence for preprally and websites forBamH I on each of the end sections, were digested withBamH I and introduced into pUC18, which was carried out by splitting withBamH I and dephosphorylation. The obtained plasmid was introduced inE. coliDH5α, and then from the resulting transformants were isolated plasmid and confirmed its nucleotide sequence, and thus gained the pRMMP-wt.

Next, using PCRusing as matrix pRMMP-wt primer pairs of SEQ ID NO:12 and 13, SEQ ID NO:4 and 15, SEQ ID NO:16 and 17, SEQ ID NO:18 and 19, SEQ ID NO:20 and 21, SEQ ID NO:22 and 23, or SEQ ID NO:24 and 25 and set PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc., later in this document for brevity, referred to as "set") was introduced mutations so that one of residue or a glutamic acid at position 19 or glutamine at position 266 in SEQ ID NO:3 was replaced with another amino acid. Designing primers for introducing mutations and PCR were performed according to the manual attached to this set. Experiments with mutagenesis was performed according to the manual.

Obtained PCR products were introduced inE. coliDH5α cells were applied to the tablet with LB agar containing 100 μg/ml ampicillin, and incubated at 37°C in techenie hours thereby obtaining transformants. As described above from the data transofrmation plasmids were isolated and subjected their sequencing to confirm the absence of unwanted mutations.

Using these methods got the genes encoding the enhanced RMMP with mutation Glu19Val, Glu19Ala, Glu19Ile, Glu19Leu, Glu19Phe, Gln266Glu or Gln266Asp. Data of plasmid vectors containing the enhanced gene RMMP, called pRMMP-E19V, pRMMP-E19A, pRMMP-E19I, pRMMP-E19L, pRMMP-E19F, pRMMP-Q266E and pRMMP-Q266D respectively.

Next, using pRMMP-Q266E or pRMMP-Q266D as a matrix, pairs of primers of SEQ ID nos:12 and 13, SEQ ID NO:14 and 15, SEQ ID NO:16 and 17, or SEQ ID NO:18 and 19, as well as the above set, PCR was performed. Obtained PCR products were introduced inE. coliDH5α, and then from the obtained transformants in the same manner as described above, were isolated plasmids and sequencing was performed, was thus given to the genes encoding the RMMP advanced type, with the mutations Glu19Val/Gln266Asp, Glu19Ala/Gln266Glu, Glu19Ala/Gln266Asp, Glu19Ile/Gln266Glu, Glu19Ile/Gln266Asp or Glu19Leu/Gln266Glu. Data of plasmid vectors containing the gene RMMP advanced type, called pRMMP-E19VQ266D, pRMMP-E19AQ266E, pRMMP-E19AQ266D, pRMMP-E19IQ266E, pRMMP-E19IQ266D and pRMMP-E19LQ266E respectively.

Thus, the obtained plasmid vectors were digested withBamH I, the resulting fragments were introduced in JS4 using the above method, thereby obtaining expressionvisitor for each of the above genes RMMP advanced type.

Example 6

Construction of expression vector of genes RMMP advanced type, which made the site-directed mutation (II)

Next, PCR was performed using as matrix pRMMP-wt using a pair of primers of SEQ ID nos:26 and 27, SEQ ID NO:28 and 29, SEQ ID NO:30 and 31, SEQ ID NO:32 and 33, SEQ ID NO:34 and 35, or SEQ ID NO:36 and 37, as well as the above set. Obtained PCR products were introduced inE. coliDH5α and then in the same way as described above, from the obtained transformants were isolated plasmids and sequencing was performed, therefore, received the genes encoding the RMMP advanced type, with the mutations Gln265Glu, Gln265Asp, Gln265Glu/Gln266Glu, Gln265Glu/Gln266Asp, Gln265Asp/Gln266Glu or Gln265Asp/Gln266Asp. Data of plasmid vectors containing the gene RMMP advanced type, called pRMMP-Q265E, pRMMP-Q265D, pRMMP-Q265EQ266E, pRMMP-Q265EQ266D, pRMMP-Q265DQ266E and pRMMP-Q265DQ266D respectively.

Genes RMMP advanced type was obtained by cleavage of the thus obtained plasmid vector using theBamH I and the introduction of the above in JS4, was thus given expression vectors of the above genes RMMP advanced type.

Example 7

Construction of expression vectors gene RMMP advanced type, which made the site-directed mutation (III)

PCR was performed using as matrix pRMMP-E19V, pRMMP-E19A or pRMMP-E19I, pairs of primers SQ ID NO:30 and 31, SEQ ID NO:32 and 33, SEQ ID NO:34 and 35, or SEQ ID NO:36 and 37, as well as the above set. Obtained PCR products were introduced inE. coliDH5α, and then in the same way as described above, from the obtained transformants were isolated plasmids and sequencing was performed, therefore, received the genes encoding the enhanced RMMP with mutations Glu19Val/Gln265Glu/Gln266Glu, Glu19Val/Gln265Glu/Gln266Asp, Glu19Val/Gln265Asp/Gln266Glu, Glu19Val/Gln265Asp/Gln266Asp, Glu19Ala/Gln265Glu/Gln266Glu, Glu19Ala/Gln265Glu/Gln266Asp, Glu19Ala/Gln265Asp/Gln266Glu, Glu19Ala/Gln265Asp/Gln266Asp, Glu19Ile/Gln265Glu/Gln266Glu, Glu19Ile/Gln265Glu/Gln266Asp, Glu19Ile/Gln265Asp/Gln266Glu or Glu19Ile/Gln265Asp/Gln266Asp. Data of plasmid vectors containing the gene RMMP advanced type, called pRMMP-E19VQ265EQ266E, pRMMP-E19VQ265EQ266D, pRMMP-E19VQ265DQ266E, pRMMP-E19VQ265DQ266D, pRMMP-E19AQ265EQ266E, pRMMP-E19AQ265EQ266D, pRMMP-E19AQ265DQ266E, pRMMP-E19AQ265DQ266D, pRMMP-E19IQ265EQ266E, pRMMP-E19IQ265EQ266D, pRMMP-E19IQ265DQ266E and pRMMP-E19IQ265DQ266D respectively.

Genes RMMP advanced type was obtained by cleavage of the thus obtained plasmid vectors using theBamH I and introduced into JS4 using the above method, therefore, received expression vectors of the above genes RMMP advanced type.

Example 8

The transformation of the budding yeast MC16 using an expression vector containing the gene RMMP wild-type or advanced type

Expression vectors, obtained as described above was introduced into budding yeast MC16 (MATα,leu2,his4 ,ade2) according to the method of Gietz and Schiestl (1995) and the cells were distributed on the tablet SDah and incubated at 30°C for 3 days, thus obtained transformants.

Example 9

Expression of RMMP wild and enhanced types with subsequent expression

Transformants obtained as described above were cultured with shaking at 200 rpm in 100 ml of YPD liquid medium, which was previously received in the Erlenmeyer flask with the tabs inside volume of 500 ml at 30°C for 24 hours. Yeast cells are collected by centrifugation, resuspendable in the double volume YPGal liquid medium, transferred into a sterile Erlenmeyer flask with the tabs inside and then cultured with shaking in the same way within 72 to 96 hours to obtain expression and subsequent secretion. After culturing medium for cultivation centrifuged, the thus obtained culture supernatant containing the above RMMP.

Example 10

Measurement of MCA and PA and evaluation relations C/P

In respect of culture supernatant containing RMMP, measured MCA and PA for calculating the ratio C/P. the Results are presented in table 1.

Table 1
CONCENTRATION: not found failed to register a milk-clotting activity and protease activity).

The ratio C/P for RMMP with mutation Glu19Val/Gln266Glu derived fromRhizomucor miehei(mutant strain) 3.8 times the ratio C/P for RMMP wild-type.

For RMMP with mutation Glu19Val, Glu19Ala and Glu19Ile, was shown a higher ratio C/P. OfRhizomucor pusilluswas already known mutation Glu19Ala (J. Biochem.129, 791-794, 2001).

In RMMP wild type, as indicated in SEQ ID NO:3, the two amino acids at positions 265 and 266 are glutamine. Confirmed that the ratio C/P for RMMP with the only substitution of glutamine for an acidic amino acid at position 265, Gln265Glu and Gln265Asp (advanced type 14 and 15 in table 1) in both cases was higher compared with the ratio C/P for the wild type. Similarly, the ratio C/P for RMMP with the only substitution of glutamine for an acidic amino acid at position 266, Gln266Glu and Gln266Asp (enhanced type 6 and 7 in table 1), in both cases it was higher, compared with a C/P for the wild type.

In the present invention first shown that the ratio C/P is increased due to the substitution of glutamine at position 265, or 266.

In addition, confirmed that when combined substitution of glutamine at position 266 in an acidic amino acid and replacement glutaminolytic in regulation 19 (advanced type from 8 to 13 in table 1) the ratio C/P is increased compared with the ratio of C/P in the case when it was replaced only glutamine at position 266.

Next, the ratio C/P for RMMP with mutation Gln265Glu/Gln266Glu, Gln265Glu/Gln266Asp, Gln265Asp/Gln266Glu and Gln265Asp/Gln266Asp in which amino acids at positions 265 and 266 simultaneously replaced by an acidic amino acids (advanced types from 16 to 19 in table 1)was significantly higher compared with the ratio C/P for RMMP with mutation, in which the acidic amino acids replaced by glutamine at position 265 or only glutamine at position 266.

Next, the ratio C/P for proteases RMMP with mutation Glu19Val/Gln265Glu/Gln266Glu, Glu19Val/Gln265Glu/Gln266Asp, Glu19Val/Gln265Asp/Gln266Glu and Glu19Val/Gln265Asp/Gln266Asp in which amino acids at positions 265, 266 and 19 simultaneously replaced by an acidic amino acids (advanced type from 20 to 23 in table 1)was significantly higher (to a value approximately five times) in comparison with RMMP wild type. Confirmed that the protease of an improved type in the highest degree works pretty well as a milk-clotting enzyme.

Example 11

Construction of expression vector of genes RMMP advanced type, which make a site-directed mutation (IV)

Next, the received expression vector of the gene RMMP with mutation, which leads to the substitution of threonine at position 81 the amino acid sequence SEQ ID NO:3 glutamine is whether aspartic acid.

PCR was performed using as matrix pRMMP-wt or pRMMP-Q265EQ266E, the primers of SEQ ID nos:38 and 39, as well as the above set. Obtained PCR products were introduced inE. coliDH5α, and then in the same way as described above, from the obtained transformants were isolated plasmids and sequencing was performed, was thus given to the genes encoding the RMMP advanced type, with the mutations Thr81Gln and Thr81Gln/Gln265Glu/Gln266Glu. Data of plasmid vectors containing genes RMMP advanced type, called pRMMP-T81Q and pRMMP-T81QQ265EQ266E respectively.

Next, PCR was performed using as matrix pRMMP-Q265EQ266D, DNA primer of SEQ ID NO:40 and 41, and the above set. The resulting PCR product was inserted in theE. coliDH5α, and then in the same way as described above, from the obtained transformants were isolated plasmids and sequencing was performed, therefore, received the genes encoding the RMMP advanced type, with the mutations Thr81Asp/Gln265Glu/Gln266Asp. The plasmid vector containing enhanced gene RMMP, called pRMMP-T81DQ265EQ266D.

Next, PCR was performed using as matrix pRMMP-E19VQ265EQ266E, pRMMP-E19VQ265EQ266D, pRMMP-E19VQ265DQ266E, pRMMP-E19VQ265DQ266D, pRMMP-E19AQ265EQ266E, pRMMP-E19AQ265EQ266D, pRMMP-E19IQ265EQ266E, pRMMP-E19IQ265EQ266D, pRMMP-E19IQ265DQ266E or pRMMP-E19IQ265DQ266D, the primers of SEQ ID nos:38 and 39, as well as the above set. Obtained PCR products were introduced inE. coliDH5α, and then in the same way as you described is e, from the obtained transformants were isolated plasmids and sequencing was performed, was thus given to the genes encoding the RMMP advanced type, with the mutations Glu19Val/Thr81Gln/Gln265Glu/Gln266Glu, Glu19Val/Thr81Gln/Gln265Glu/Gln266Asp, Glu19Val/Thr81Gln/Gln265Asp/Gln266Glu, Glu19Val/Thr81Gln/Gln265Asp/Gln266Asp, Glu19Ala/Thr81Gln/Gln265Glu/Gln266Glu, Glu19Ala/Thr81Gln/Gln265Glu/Gln266Asp, Glu19Ile/Thr81Gln/Gln265Glu/Gln266Glu, Glu19Ile/Thr81Gln/Gln265Glu/Gln266Asp, Glu19Ile/Thr81Gln/Gln265Asp/Gln266Glu, or Glu19Ile/Thr81Gln/Gln265Asp/Gln266Asp. Data of plasmid vectors containing genes RMMP advanced type, called pRMMP-E19VT81QQ265EQ266E, pRMMP-E19VT81QQ265EQ266D, pRMMP-E19VT81QQ265DQ266E, pRMMP-E19VT81QQ265DQ266D, pRMMP-E19AT81QQ265EQ266E, pRMMP-E19AT81QQ265EQ266D, pRMMP-E19IT81QQ265EQ266E, pRMMP-E19IT81QQ265EQ266D, pRMMP-E19IT81QQ265DQ266E and pRMMP-E19IT81QQ265DQ266D respectively.

Genes RMMP advanced type was obtained by cleavage of the thus obtained plasmid vectors using theBamH I and introduction to JS4 through the above-described method, and thus obtained expression vectors of the above genes RMMP advanced type.

Example 12

In accordance with the methods described in examples 8 to 10, the expression vectors obtained in example 11, was introduced in budding yeast MC16 and transformants were subjected to cultivation in liquid culture thus obtained culture supernatant containing RMMP advanced type. In relation to the culture supernatant, which contained RMMP, measured MCA and PA for the Vacha the comprehension of the relations C/P. The results are presented in table 2.

Table 2

As shown in table 2, confirmed that RMMP with substitutions of threonine at position 81 glutamine or aspartic acid and the substitution of glutamine at positions 265 and 266 in acidic amino acids (advanced type 33 and 34 in table 2) showed a higher C/P, than the ratio C/P for RMMP wild type. In particular, the ratio C/P for RMMP with mutation Thr81Asp/Gln265Glu/Gln266Asp, increases to the amount, 4.4 times the value of the ratio C/P for RMMP wild-type.

In the case of a combination of replacement of the glutamic acid at position 19 with the above replacement ratio C/P was increased in comparison with the ratio C/P RMMP wild-type (advanced type 35 to 44 in table 2). In particular, the ratio C/P for RMMP with mutation Glu19Val/Thr81Gln/Gln265Asp/Gln266Glu, increases to the amount 4.8 times greater than the value of the ratio C/P for RMMP wild-type.

The results presented in table 1 and 2 indicate that when replacing a glutamine at positions 265 and/or 266 amino acid sequence of SEQ ID NO:3 acidic amino acid ratio C/P is increased compared with RMMP wildly what about the type, and when combined replacement(s) of the amino acids at positions 19 and/or 81, the ratio C/P is additionally increased.

Example 13

Cleaning RMMP wild-type and advanced type and analysis of the purified enzyme

Budding yeast MC16 containing expression vector containing the gene RMMP wild-type or gene RMMP advanced type, with the mutations Glu19Val/Gln266Glu, Glu19Val, Glu19Ala, Gln266Glu, Gln266Asp, Glu19Ala/Gln266Glu, Gln265Glu, Gln265Asp, Gln265Glu/Gln266Glu, Gln265Glu/Gln266Asp, Gln265Asp/Gln266Glu, Gln265Asp/Gln266Asp, Glu19Val/Gln265Glu/Gln266Asp or Glu19Val/Gln265Asp/Gln266Asp, were cultured according to the method described above, promote the expression of RMMP with subsequent secretion. The culture supernatant was collected by centrifugation, were applied to a column Packed with HiTrap Q HP (manufactured by GE Healthcare), pre-equilibrated with 50 mm buffer of sodium acetate, pH 5.5, to absorb protein RMMP. After washing the column with the same buffer, elution was performed protein with 0,3M buffer of NaCl. Two µl fractions were placed on the tablet with skim milk (1% lowfat milk (production Difco), 100 mm buffer of acetic acid, pH 5.2, and 1% agar) and incubated at 37°C for 10 minutes, the active fraction was determined by the appearance of a turbid halo.

The obtained active fraction was concentrated using ultrafiltration membranes and then purified by high performance liquid is cromatografia using a column for gel filtration Super SW3000 (production Tosoh Corporation). When cleaning fractions using analysis LTO-PAG was observed only one lane.

The measurement results for MCA received and cleared so RMMP presented in table 3. Quantification of proteins was performed using reagent for protein analysis BCA (Pierce manufacturing).

Table 3

From the obtained results, MCA is reduced due to the replacement of only the glutamic acid at position 19, while MCA increases due to the substitution of glutamine at position 265, or 266. MCA also increases due to substitutions in positions 19 and 266 and substitutions at positions 19, 265 and 266.

Example 14

Measuring the weight of dry matter in whey

Cheese production is one of the important properties of commercial use of milk-clotting enzyme. Measuring the weight of dry substance in the serum is an effective measure to estimate the production of cheese. Low weight of dry matter in whey milk is a sign of increased production of cheese.

Next will be described a method of measuring the weight of dry matter in whey using RMMP wild-type and E19V/Q266E RMMP, both of which expressed the yeast.

Budding yeast MC16 containing expression vector containing the gene RMMP wild-type or gene RMMP advanced type, with the mutations Glu19Val/Gln266Glu, were cultured using the above method, allowing the expression of RMMP with subsequent secretion. The culture supernatant collected by centrifugation, concentrated using ultrafiltration membranes and the resulting material was used as a milk-clotting enzyme.

(1) Method of coagulation of milk

Commercially available pasteurized dehomogenization milk cows (Takanashi milk products Co. Ltd.) (500 g) were placed in a chemical beaker and heated to 32°C. when the temperature of the milk cows reached 32°C, was added 0.4 g of D-(+) glucono-1,5-lactone (D-gluconic acid δ-lactone, production Wako Pure Chemical) was mixed and then slowly added calcium chloride (manufactured Wako Pure Chemical) to a final concentration of 1 mm and mixed. After adding the reagents were added milk-clotting enzyme (2,000 Mu), was stirred for 1 minute and left at 32°C. After 30 minutes after adding the milk-clotting enzyme set the time of rennet coagulation. Coagulated milk is cut into cubes of size from 1 to 1.5 cm and left for 10 minutes. After that, the cubes gently broke. Kubie and left at 32°C for 20 minutes, gently stirring occasionally. Then a chemical beaker was transferred into an incubator at 37°C, and the internal temperature was increased to 37°C (0.5°C for 1 minute). When the crud reached 37°C, it was left for another 30 minutes, gently stirring occasionally. After that, curdled milk and serum were separated using gauze. The obtained coagulated milk was wrapped in gauze and placed in a mold for direct production of cheese. Next, while maintaining pressure (5 MPa for 90 minutes) serum was separated, and collected. All the collected milk serum was mixed and filtered through qualitative filter paper No.1 (ADVANTEC). The resulting serum was used as total serum.

(2) measurement of the weight of dry matter in whey

Chemical glass was pre-dried in a drying Cabinet at 105°C. is Not less than 30 chemical glass were removed from the oven, placed in a desiccator and measured weight. Approximately 25 g of whey obtained as described above were placed in a chemical beaker and dried in a drying Cabinet at 105oC for 12 to 15 hours or more. After drying chemical beaker was placed in a desiccator. Not less than 30 minutes was measured by weight. The value obtained at the preliminary subtraction of the weight of chemical glass, felt the weight of dry matter.

According to described what use method dry matter content of 15 lots serum and the total weight of dry matter was measured twice, and the results are presented in table 4.

Table 4

The total amount of dry matter in whey RMMP wild-type and RMMP Glu19Val/Gln266Glu was 28,4810 g and 28,0511 g, respectively. A significant difference was confirmed using t-student test (two-sided test). Between them were found significant differences (p<0,01). That is, it was found that when using RMMP Glu19Val/Gln266Glu can be increased cheese production compared using RMMP wild-type, and it is possible to produce approximately 1.51% more cheese than using RMMP wild type. This is equivalent to 85,97 kg in the case of cheese production using 100 tons of milk.

1. Protease with improved milk-clotting activity, which contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 3, where this protease with improved milk-clotting activity, has at least one mutation selected from the group consisting of:
(A) replacement of glutamine, corresponding glutamine in position 265 in SEQ ID NO: 3, an acidic amino acid and
(C) replacing glutamine, the corresponding glutamine in position 266 in SEQ ID NO:3, an acidic amino acid.

2. The protease according to claim 1, selected from the group consisting of:
(A) a protein containing the amino acid sequence of SEQ ID NO:3 or 43, except that the glutamine at position 265 and/or glutamine at position 266 replaced by(s) on an acidic amino acid;
(B) a protein containing the amino acid sequence of SEQ ID NO:3 or 43, except that the glutamine at position 265 and/or glutamine at position 266 replaced by(s) on an acidic amino acid, and not more than 10 amino acids in positions other than 265 and 266, replaced deleterows, inserted or added.

3. The protease according to claim 1 or 2, in which the acidic amino acid is glutamic acid or aspartic acid.

4. The protease according to claim 1 or 2, in which glutamine corresponding to glutamine in position 266, replaced by an acidic amino acid, and glutamine, corresponding glutamine in position 265, not substituted for an acidic amino acid.

5. The protease according to claim 1 or 2, in which glutamine corresponding to glutamine in position 265, and glutamine, corresponding glutamine in position 265 is replaced with an acidic amino acid.

6. The protease according to claim 4, in which the acidic amino acid is glutamic acid or aspartic acid.

7. The protease according to claim 5, in which the acidic amino acid is put mirovoi acid or aspartic acid.

8. The protease according to claim 4, where the glutamic acid at position 19 is replaced by valine, alanine, isoleucine, or leucine.

9. The protease according to claim 5, where the glutamic acid at position 19 is replaced by valine, alanine, isoleucine, or leucine.

10. Protease under item 5, where the threonine at position 81 is replaced by glutamine or aspartic acid.

11. The protease according to claim 5, where the glutamic acid at position 19 is replaced by valine, alanine, isoleucine, or leucine, and threonine at position 81 is replaced by glutamine or aspartic acid.

12. DNA encoding a protease with improved milk-clotting activity according to any one of claims 1 to 11.

13. The expression vector containing the DNA according to item 12.

14. Transformed cell in which you entered the expression vector according to item 13, for expression of the protease with improved milk-clotting activity.

15. The transformed cell according to 14, where this transformed cell is aSaccharomyces cerevisiae.

16. A method of producing a protease with improved milk-clotting activity, including the stage of culturing the transformed cells under 14 or 15 in the culture medium and the allocation of protease with improved milk-clotting activity of the culture medium.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: method includes a stage of yeast cultivation, transformed by a vector containing a DNA sequence, determined by the formula X-B-Y-A, coding the precursor of insulin glargine, where X is a sequence of leader peptide, containing at least one amino acid. B is a B1-B30 sequence of amino acids of B-chain of the insulin glargine molecule. Y is a linker peptide containing at least two amino acids. A is an A1-A21 sequence of amino acids of an A-chain of a molecule insulin glargine, a stage of extraction of an expressing precursor of insulin glargine, a stage of crystallisation of the extracted precursor of insulin glargine, a stage of completion of fermentative conversion of insulin glargine precursor crystals at pH from 8 to 10 in presence of tripsin or tripsin-like ferment and water soluble organic dissolvents at the ratio from 40% to 60% of the reaction mix with formation of insulin glargine, containing at least one related admixture. Then the stage of insulin glargine treatment by reverse phase highly efficient liquid chromatography is carried out on a chromatographic matrix, using a polar organic buffer dissolvent in a water phase, containing a buffer based on organic acid, in which the matrix is first balanced with 10% acetonitrile in 250 mM of acetic acid with further elution of insulin glargine in the specified acetonitrile. Then the matrix is again balanced with 10% acetonitrile in the buffer on the basis of organic acid in concentration from 20 mM to 200 mM at pH from 3 to 8.5 with subsequent elution of insulin glargine in the specified acetonitrile, and further repeatedly the matrix is balanced with 6% ethanol in the buffer on the basis of organic acid in concentration from 10 mM to 50 mM with subsequent elution of the specified insulin glargine in the specified ethanol. Further the treated insulin glargine is deposited by means of addition of the buffer on the basis of citric acid and zinc chloride at pH from 6 to 8.

EFFECT: invention makes it possible to produce insulin glargine with high purity and low content of glycolised admixtures.

12 cl, 9 dwg, 8 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: plasmid genetic structure pOL-DsRed2 is produced, being built on the basis of a plasmid vector pIRES (Clontech), where fragments of cDNA of human genes OCT4 and LIN28 are placed, being connected with a nucleotide sequence coding P2A-peptide and gene cDNA, coding fluorescent protein DsRed2.

EFFECT: invention provides for simultaneous translation of human proteins OCT4 and LIN28 and fluorescent protein DsRed2 in production of induced pluripotent stem cells of a human being and animals in medicine and veterinary science.

3 cl, 1 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of isolating and purifying recombinant human growth hormone which is secreted by Saccharomyces cerevisiae yeast during fermentation thereof in suitable conditions. The target protein is precipitated in biomass-free culture fluid by either acidification to pH 2.9-4.0 or adding polyethylene glycol with molecular weight of 3000-6000 Da. The obtained precipitate is then dissolved in a suitable solvent. Preliminary purification of the target protein is carried out either by anion-exchange chromatography at pH 5.6 or by diafiltration in the presence of 0.1-0.5 M sodium chloride. Main purification of the target protein is then carried out by anion-exchange chromatography at pH not below 7.3 and gel filtration.

EFFECT: invention enables to obtain a growth hormone which is free from parent proteins, host-producer protein and other impurities such as pigments, with output of up to 60%.

8 ex

FIELD: biotechnology.

SUBSTANCE: method provides for culturing mammalian cells expressing the recombinant protein of interest in a culture medium containing anti-aging compound selected from carnosine, acetyl-carnosine, homo-carnosine, anserine, and K-alanine and their combinations, under conditions and for a time sufficient for expression of the protein.

EFFECT: invention enables to improve the performance of the cell culture selected from higher titer, of increased specific cell productivity, increased cell viability, increased integrated viable cell density, decreased accumulation of high molecular aggregates, decreased accumulation of acidic molecules, and their combinations.

44 cl, 14 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: method consists in expression of a gene of a human plasminogen fragment from 453 to 543 amino acid within E.coli cells with subsequent extraction and treatment of a finished product from cell periplasm. Expression is carried out in cells E.coli BL21 (DE3), transformed by plasmid DNA pEK5 or pEK5H with physial maps represented in figure 2, containing a gene of target polypeptide fused with a gene of signal peptide OmpA, origin of replication pUC ori, a gene of resistance to kanamycin and a gene coding lac-repressor under control of T7-promotor. At the same time the recombinant plasmid DNA pEK5H additionally contains between genes OmpA and target polypeptide the sections coding amino acid sequences HHHHHH and DDDDK.

EFFECT: invention provides for secretion of target polypeptide into cell periplasm with high yield.

4 cl, 5 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: fusion peptide is presented for neutralisation and destruction of organophosphorous compounds, comprising a signal peptide TAT3 with amino acid sequence SEQ ID NO: 5, presented in the description, functionally connected to the sequence of organophosphate hydrolase SEQ ID NO: 18, classified as protein EC 3.1.8. The following components are described: extracted polynucleotide, which codes the specified fusion protein; a vector containing the specified polynucleotide; and a procaryotic host cell containing the specified vector and expressing the specified fusion protein. The method is proposed to produce a ferment, which destroys organophosphorous compounds, including expression of the specified polynucleotide in the procaryotic host cell and production of the specified ferment.

EFFECT: invention makes it possible to increase expression of organophosphate hydrolase in a host cell.

13 cl, 1 tbl, 9 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: method of highly effective production of methionine-containing form of staphylokinase (SAK-1) and some of its biologically active analogs in a host cell, providing culturing a bacterial cell transformed by the expression vector which comprises the DNA sequence encoding SAK-1 at a high degree of aeration and reducing the level of oxygen by 5% of atmospheric level with achievement the exponential growth phase.

EFFECT: use of the invention provides a significant increase in the yield of the target product.

11 dwg, 4 ex

FIELD: biotechnology.

SUBSTANCE: method comprises culturing CHO cell in which the gene of taurine transporter (TauT) was artificially transferred, the DNA encoding the desired polypeptide and the DNA encoding dihydrofolate reductase (DHFR) was introduced in the presence of methotrexate concentration at which 90% or more cells, in which TauT was not introduced die within three weeks after subculturing. From the number of surviving CHO cells the CHO cell is selected which is capable to produce a desired polypeptide with a high yield. The cell prepared in this manner produces a desired polypeptide in higher yield than the CHO cell transfected with DNA encoding the desired polypeptide and DNA encoding DHFR, but not the genome of taurine transporter. The method of producing a desired polypeptide comprises culturing the above mentioned cell and isolating the desired peptide.

EFFECT: invention enables to produce the desired polypeptide with a high yield.

9 cl, 10 dwg, 6 ex

FIELD: biotechnology.

SUBSTANCE: proposed enzymatically inactive IgA1 protease with replacement Ser267Ala for use as a component of a polyvalent vaccine designed to protect people against meningococcal infection and other microorganisms, which pathogenicity is caused by IgA1 protease. The invention includes a polynucleotide encoding the said mutant form of IgA1 protease of Neisseria meningitidis of serogroup B, comprising the said polynucleotide, a recombinant plasmid DNA, the strain Escherichia coli - producer of a mutant form of IgA1 protease according to the invention, the method of preparing a recombinant form of the enzyme using a technology of recombinant DNA, and recombinant inactivated IgA1 protease.

EFFECT: increased level of immunogenicity.

6 cl, 1 tbl, 5 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a compound and a pharmaceutically acceptable salt thereof to be used as an antifungal agent, particularly, a therapeutic agent for deep fungal disease. The fungus Acremonium persicinum is collected, and a cyclic compound is recovered from its cultural fluid.

EFFECT: what is presented is the compound applicable as an antifungal agent.

10 cl, 16 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: preparation of thrombolytic and fibrinolytic action is produced by submerged cultivation on a liquid nutrient medium, of a strain of basidium fungus of Coprinus lagopides TI-12, or Coprinus lagopides TI-32, or Coprinus lagopides TI-33 stored in the high basidium fungi culture collection of the microbiological synthesis technology department of the St.-Petersburg State Technical University, separation of a mycelial biomass from a native solution. The prepared solution is concentrated by ultrafiltration with using a semipermeable membrane with a nominal molecular-mass rejection limit up to 20 kDa followed by cool dehumidification in the mode suitable for protein compounds. The invention has allowed detecting Coprinus lagopides from basidium fungi of Coprinus exhibiting high thrombolytic and fibrinolytic activity.

EFFECT: preparation in the low concentration shows a high level of thrombolytic and fibrinolytic activity in vivo.

2 dwg, 4 tbl, 5 ex

FIELD: food industry.

SUBSTANCE: oyster mushroom fruit bodies are homogenised, the obtained homogenate is frozen followed by defrosting and dividing into extract and residue. Residue is mixed with distilled water in mass ratio of 1:2, held at +4-+8°C, the secondary extract is separated and mixed with previously obtained extract. Food acid is added into the obtained mixture to achieve pH 3.8 and it is precipitated by saturation of the solution with sodium chloride at +4-+8°C. The obtained residue is separated and dried by freezing at -18°C. The dried residue is kept in a refrigerator. Before using, it is dissolved in a minimum amount of water and centrifuged.

EFFECT: high milk-clotting activity and increased ratio of milk-clotting and general proteolytic activity.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method of obtaining milk-turning ferment includes deep cultivation of agaric macromycetes Coprinus lagopides on liquid medium, which contains sources of carbon, nitrogen and mineral salts.

EFFECT: ferment with high level of milk-turning activity and low level of general proteolytic activity.

3 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: strain of basidium fungus Trametes hirsuta (Wulfen) Pilat, deposited with collections of the OOO PKF "BIGOR" enumerated as CF-28, is extracted by repeated passages from monoglobular isolates of deep culture of the strain TsNIIMOD 56. The said strain features an active synthesis of extracellular laccase. The laccase activity makes some 20 to 26 ME/ml.

EFFECT: production of the basidium fungus strain with active extracellular laccase synthesis.

2 tbl, 2 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: proposed method includes deep cultivation of the strain of fungi Trametes hirsuta (Wulfen) Pilat, OOO PKF "BIGOR" CF-28 on a nutrient medium, separation of the fungi biomass, membrane concentration of the filtrate. The nutrient medium composition incorporates growth factors in concentration of 0.1-1.02 wt % and wheat flour in concentration of 2.0-4.0 wt % is used as a source of carbon. The cultivation is carried out at 28 to 34°C, pH 4.0 to 4.5, the concentration of dissolved oxygen making at least 1.5 to 2.0 mg·dm-3. The membrane concentration is effected with the membrane element pore sizes varying from 0.1 to 0.5 microns, at a pressure difference of 1.0 to 4.0 MPa and temperature of 20 to 40°C. Laccase activity makes some 140 to 200 ME.

EFFECT: production of fermented laccase preparation with high activity.

2 ex

FIELD: medicine; pharmacology.

SUBSTANCE: substance for dermatological medical products on the basis of a collagenase of a microbic parentage represents an ultrafiltrate allocated from a cultural liquid of Streptomyces lavendulae strain VKPM S-910 with collagenolytic activity of 1800-2500 KEA/ml and proteolytic activity of 120-200 PE/ml.

EFFECT: obtaining of biologically active ultrafiltrate with high collagenolytic and proteolytic activity for an effective utilisation as a part of various wound-healthing preparations and dermatology.

3 ex

FIELD: biotechnology.

SUBSTANCE: disclosed is isolated polypeptide being acid-proof metalloprotease isolated from Thermoascus aurantiacus. Described are strain Thermoascus aurantiacus CGMCC No 0670 for polypeptide production and method for polypeptide production using said strain. Disclosed is method for plant protein treatment to increase digestion value thereof by using said polypeptide.

EFFECT: protease of good acid resistance useful in feed production.

6 cl, 7 ex, 4 tbl

The invention relates to biotechnology and concerning the processing of leather and fur raw materials with the help of a new complex enzyme preparation

The invention relates to biotechnology, in particular, to obtain the strain - producer of complex hydrolytic enzymes, which consists of acidic and weakly acidic protease,- amylases and related enzymes, such as Exo -- glucanase, cicasa, xylanase, and can be applied in microbiological industry for preparation of enzyme preparations for hydrolysis of plant, animal and microbial substrates in the food industry, fermentation industry, agriculture, upon receipt of amino acid mixtures, fermentolizate yeast and other biologically active substances

The invention relates to the medical industry, in particular the microbiological synthesis of enzymes for medical purposes

FIELD: biotechnologies.

SUBSTANCE: recombinant nucleic acid expresses one or several polypeptides of interest, a vector of expression and bacteria, which contain this recombinant nucleic acid. The recombinant nucleic acid contains a natural promotor of a gene of HU-like DNA-binding protein (PhilA) of Lactococcus type with the sequence SEQ TD NO:28, or its homological or functional version, which at least by 95% identical to the promotor with sequence SEQ ID NO:28, functionally linked with one or several open reading frames, heterological for the promotor RhIIA, where the promotor RhIIA is located above one or several open reading frames. The expression vector contains the above recombinant nucleic acid, preferably, the specified vector is produced from pTINX. A bacterium contains the above recombinant nucleic acid or the above vector.

EFFECT: proposed invention makes it possible to increase level of expression of polypeptide genes of interest and therefore produce sufficient number of expressed proteins.

19 cl, 26 dwg, 12 tbl, 9 ex

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