A collagenase mutant, gene fragment, recombinant plasmid, recombinant expression system and their uses, a method for preparing a collagenase mutant and collagen tripeptide

Abstract

The present invention discloses a collagenase mutant, a gene fragment, a recombinant plasmid, a recombinant expression system and their uses, and a method for preparing the collagenase mutant and collagen tripeptide, belonging to the fields of genetic engineering and fermentation engineering. The present invention provides a collagenase mutant KT231 that can be efficiently secreted and expressed in Streptomyces S. lividans TK24. The collagenase mutant KT231 can efficiently hydrolyze Alaska pollock fish skin at 60 °C, and has excellent heat resistance and high enzyme activity, and can be used for the efficient preparation of collagen tripeptide from Alaska pollock fish skin. When combined with commercially available papain, it can significantly increase the proportion of collagen tripeptide in the fish skin hydrolysate and has no bitter taste. It has broad application prospects in the preparation of products for anti-wrinkle and moisturizing, repairing damaged skin, improving bone and joint health, tissue engineering materials, and wound healing promoters.

Description

Technical Field

[0001] The present invention belongs to the fields of genetic engineering and fermentation engineering, and specifically relates to a collagenase mutant, a gene fragment, a recombinant plasmid, a recombinant expression system and their uses, and a method for preparing a collagenase mutant and collagen tripeptide. Background Art

[0002] Collagen is the most abundant protein in animals and is the main component of tissues such as skin, bone, tendon, and teeth. Collagenase is an enzyme that can specifically degrade collagen, acting on the collagen chain structure, cleaving the inter-chain bonds, and hydrolyzing collagen into low-molecular-weight collagen peptides, including the much-concerned collagen tripeptide. The typical structure of collagen tripeptide is "Gly-X-Y", where X is mostly proline and Y is mostly hydroxyproline. Collagen tripeptide has a wide range of applications and has become a research hotspot in the field of big health. It can be used for anti-wrinkle and moisturizing, repairing damaged skin, improving bone and joint health, tissue engineering materials, wound healing promoters, and so on.

[0003] Traditional collagenase mainly relies on extraction from animal tissues (such as pancreas, stomach, etc.). This method has complex processes, high costs, and potential risks of disease transmission, which limits large-scale industrial applications. To overcome these limitations, in recent years, researchers have begun to explore the production of collagenase by microbial fermentation. Microbial fermentation has wide raw material sources, low costs, and is easy to control, and has become an important development direction for collagenase production. This method has great potential, but also faces challenges in practical applications, such as low fermentation enzyme activity and high production costs. It is necessary to promote the efficient production and application of the enzyme by screening collagenase mutants with high activity and mutants with high substrate binding specificity.

[0004] Therefore, it is of great research value to develop a collagenase with excellent enzymatic hydrolysis performance. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the purpose of the present invention is to provide a collagenase mutant, a gene fragment, a recombinant plasmid, a recombinant expression system and their uses, and a method for preparing a collagenase mutant and collagen tripeptide.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] The present invention provides a collagenase mutant, and the amino acid sequence of the collagenase mutant has at least 99% sequence identity with the sequence shown in SEQ ID NO: 3.

[0008] Further, the collagenase mutant has the amino acid sequence shown in SEQ ID NO: 3.

[0009] The present invention also provides a gene fragment encoding a collagenase mutant, and the nucleotide sequence of the gene fragment is as shown in SEQ ID NO: 4 or SEQ ID NO: 6.

[0010] The present invention also provides a recombinant plasmid, and the plasmid includes the above-mentioned gene fragment.

[0011] The present invention also provides a recombinant expression system, and the recombinant expression system contains a host bacterium and the above-mentioned recombinant plasmid.

[0012] The present invention also provides a method for preparing the above-mentioned collagenase mutant, and the method includes the following steps:

[0013] (1) Cloning the DNA fragment as shown in SEQ ID NO: 4 or SEQ ID NO: 6 into a vector plasmid, transforming Escherichia coli to obtain an expression plasmid;

[0014] (2) Transferring the expression plasmid into a host bacterium, collecting colony spores, culturing, and collecting the fermented supernatant enzyme solution to obtain the collagenase mutant.

[0015] Further, in step (1), the vector plasmid is pSET152, and the Escherichia coli is DH5α; in step (2), the host bacterium is Streptomyces S. lividans TK24; the transfer method is conjugation transfer.

[0016] The present invention also provides the uses of the above-mentioned collagenase mutant, gene fragment, recombinant plasmid, and recombinant expression system in the preparation of collagen tripeptide.

[0017] The present invention also provides a method for preparing collagen tripeptide, and the method includes the following steps: using collagen as a raw material, and sequentially performing enzymatic hydrolysis with the above-mentioned collagenase mutant and chymotrypsin to obtain collagen tripeptide.

[0018] Further, the chymotrypsin is papain chymotrypsin.

[0019] The present invention has achieved the following beneficial effects:

[0020] The present invention provides a method in Streptomyces S. lividansThe collagenase mutant KT231 that achieves efficient secretory expression in TK24. The collagenase mutant KT231 can efficiently enzymatically hydrolyze Alaska pollock fish skin at 60 °C, and has excellent heat resistance and high enzyme activity, and can be used for the efficient preparation of collagen tripeptides from Alaska pollock fish skin. When combined with commercially available papain, it can significantly increase the proportion of collagen tripeptides in the fish skin hydrolysate and has no bitter taste. It has broad application prospects in the preparation of products for anti-wrinkle and moisturizing, repairing damaged skin, improving bone and joint health, tissue engineering materials, and wound healing promoters.

[0021] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0022] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0023] Figure 1 It is the SDS-PAGE diagram of collagenases KT16 and KT231.

[0024] Figure 2 It is the pH curve of collagenases KT16 and KT231.

[0025] Figure 3 It is the temperature curve of collagenases KT16 and KT231.

[0026] Figure 4 It is the enzyme activity curve of collagenases KT16 and KT231.

[0027] Figure 5 It is the liquid chromatography diagram of oligopeptides with different molecular weights in the hydrolysate of enzymatically hydrolyzed Alaska pollock fish skin.

[0028] Figure 6 It is the liquid chromatography diagram of the content of collagen tripeptides in the hydrolysate of enzymatically hydrolyzed Alaska pollock fish skin. Detailed Description of the Embodiments

[0029] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0030] The formula of the culture medium involved in the embodiments of the present invention is as follows, where % refers to mass percentage and g / L refers to grams per liter:

[0031] LB liquid culture medium: tryptone 1%, yeast extract 0.5%, sodium chloride 0.5%;

[0032] LB solid medium: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar;

[0033] Skim milk powder medium: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1% skim milk powder, 1.5% agar;

[0034] MS solid medium: 20.0 g / L soybean powder, 20.0 g / L mannitol, 3.0 g / L calcium carbonate, 20.0 g / L agar;

[0035] TSB medium for Streptomyces seed culture: 1.5% tryptone, 0.5% soy peptone, 0.5% sodium chloride;

[0036] Streptomyces fermentation medium: 10.0 g / L glucose, 5.0 g / L tryptone, 5.0 g / L yeast extract, 2.0 g / L casamino acids, 2.5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate.

[0037] Example 1: Gene cloning and construction of expression plasmid

[0038] Construction process of recombinant expression plasmid pSET152-KT16 of wild-type collagenase KT16: Chemically synthesize the expression cassette DNA fragment of the gene, as shown in SEQ ID NO. 5, which contains the strong Streptomyces promoter KasO*p, the full-length coding sequence of the gene kt16 - kt16 and the strong terminator Ter-SCO0356. Among them, the amino acid sequence of collagenase collagenase - kt16 is as shown in SEQ ID NO. 1; the coding nucleotide sequence of the gene collagenase - kt16 is as shown in SEQ ID NO. 2. kt16 The plasmid pSET152 was digested with the restriction endonuclease BamHI, and the linearized pSET152 DNA was recovered by gel electrophoresis; the above two DNA fragments (i.e., the expression cassette DNA fragment of the gene

[0039] and the linearized pSET152 DNA) were subjected to seamless cloning, and then transformed into Escherichia coli DH5α; the plasmid was extracted from the positive clone, and the correct expression plasmid pSET152-KT16 was obtained by sequence determination. kt16 Construction process of recombinant expression plasmid pSET152-KT231 of collagenase mutant KT231: Chemically synthesize the gene

[0040] ​kt231 The expression cassette DNA fragment, as shown in SEQ ID NO. 6, which contains the strong promoter KasO*p of Streptomyces, the gene kt231 full-length coding sequence collagenase - kt231 , and the strong terminator Ter-SCO0356. Among them, the collagenase collagenase - kt231 amino acid sequence is as shown in SEQ ID NO. 3; the coding nucleotide sequence of the gene kt231 is as shown in SEQ ID NO. 4.

[0041] The plasmid pSET152 was digested with the restriction endonuclease BamHI, and the linearized pSET152 DNA was recovered by gel electrophoresis; the above two DNA fragments (i.e., the expression cassette DNA fragment of the gene kt231 and the linearized pSET152 DNA) were subjected to seamless cloning, and Escherichia coli DH5α was transformed; the plasmid was extracted from the positive clone, and the correct expression plasmid pSET152-KT231 was obtained by sequence determination.

[0042] The target DNA sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6 provided in the examples can be obtained by PCR amplification and chemical synthesis, and the examples are not limited.

[0043] Example 2: Heterologous expression of collagenase and preparation of fermentation supernatant enzyme solution

[0044] The Streptomyces conjugation transfer donor host bacterium E. coli ET12567 / pUZ8002 was activated on an LB solid plate containing 25 μg / mL of kanamycin and 25 μg / mL of chloramphenicol. The expression plasmids pSET152-KT16 and pSET152-KT231 provided in the above examples were respectively transferred into E. coli ET12567 / pUZ8002, and positive clones were screened on a plate containing 100 μg / mL of apramycin, 25 μg / mL of kanamycin, and 25 μg / mL of chloramphenicol. Then, the positive clones containing the expression plasmid pSET152-KT16 or pSET152-KT231 were respectively transferred into Streptomyces S. lividans TK24 by conjugation transfer. Mg used in the conjugation transfer process 2+The concentration is 10 mmol / L, the pre-germination time of Streptomyces is 3 h, the final concentration of apramycin is 1.25 mg / ml, and the final concentration of nalidixic acid is 0.5 mg / ml. After the conjugation transfer is completed, the MS plate is statically cultured at 28 °C. After 5-8 days, monoclonal conjugation transferants are picked and streaked and passaged once on a medium containing 25 mg / L of nalidixic acid and 100 μg / mL of apramycin to obtain Streptomyces expression strains of wild-type collagenase KT16 and mutant KT231.

[0045] Collect the freshly activated Streptomyces expression strains of wild-type collagenase KT16 and mutant KT231, and inoculate them into 30 mL of TSB seed medium respectively. Incubate at 30 °C and 220 r / min for 24 h. Take 3 mL of the seed liquid and transfer it to 50 mL of fermentation medium. Incubate at 30 °C and 220 r / min for 96 h, and centrifuge at 8000g for 10 min to collect the supernatant to obtain proteases KT16 and KT231. Use SDS-PAGE to analyze the molecular weights of proteases KT16 and KT231. The results are as Figure 1 shown.

[0046] Referring to "GB / T 23527.1-2023 Quality Requirements for Enzyme Preparations - Part 1: Protease Preparations", the spectrophotometric method is used to measure the degradation enzyme activities of collagenase KT16 and collagenase mutant KT231 on the substrate casein at 45 °C and pH 9.0. The enzyme activity is defined as: under specific conditions, the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute is 1 enzyme activity unit. The expressed enzyme activity of wild enzyme KT16 is 5231.56 U / mL, while the expressed enzyme activity of mutant enzyme KT231 is 6602.12 U / mL, which is increased to 1.26 times. The above results show that the 4-site mutant KT231 obtained in the examples of this application has a significant improvement in the secretion and expression level compared with the wild enzyme KT16, which is beneficial to further improving the enzymatic hydrolysis efficiency and reducing the application cost.

[0047] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0048] Experimental Example 1: pH Stability of the Mutant Enzyme of the Present Invention

[0049] Prepare 50 mmol / L boric acid-borax-sodium hydroxide buffer solutions with pH values of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, and 12.0. Dilute the enzyme solutions of collagenase KT16 and KT231 to 10-15 U / mL with the buffer solutions of the above different pH values respectively. Measure the enzyme activities of collagenase KT16 and KT231 on the substrate casein at 50 °C and the corresponding pH conditions. Define the highest enzyme activity as 100%, and the corresponding pH is the optimal enzymatic hydrolysis pH of the enzyme. Calculate the relative enzyme activities under other pH conditions. The results are as Figure 2 shown. The optimal pH values for the wild-type KT16 and the mutant KT231 on the substrate casein are both 8.5. Both of them are alkaline proteases, and the mutation at site 4 has no obvious effect on the pH properties of the enzyme.

[0050] Experimental Example 2: Thermal stability of the mutant enzyme of the present invention

[0051] According to the optimal pH for action determined in Experimental Example 1, measure the casein hydrolysis activities of collagenase KT16 and KT231 at 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, and 80 °C under the condition of pH 8.5.

[0052] The results are as Figure 3 shown. The optimal enzymatic hydrolysis temperature of the wild enzyme KT16 is 40 °C, and the optimal enzymatic hydrolysis temperature of the mutant enzyme KT231 is increased to 60 °C. This shows that the mutant KT231 at site 4 has better heat resistance and is more suitable for the high-temperature enzymatic hydrolysis conditions in industrial practice to obtain higher enzymatic hydrolysis efficiency.

[0053] Experimental Example 3: Collagen enzymatic hydrolysis performance of the mutant enzyme of the present invention

[0054] Under high temperature conditions, fish skin collagen is transformed into soluble gelatin, and gelatin can be used as a substrate to determine the activity of collagenase. Referring to the method of Zhang (Zhang Y, Fu Y, Zhou S, et al. A straightforward ninhydrin-based method for collagenase activity and inhibitor screening of collagenase using spectrophotometry[J]. Analytical Biochemistry, 2013, 437(1):46-48.), the activity of enzymes KT16 and KT231 on the substrate gelatin was determined by the ninhydrin colorimetric method. The enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of glycine per minute per milligram of gelatin degraded under specific conditions, which is one enzyme activity unit U.

[0055] The gelatin-degrading activities of enzymes KT16 and KT231 were determined in the temperature range of 30°C - 80°C. The method for determining the enzyme activity at 30°C is as follows: Prepare a 2 mg / mL gelatin solution with Tris·HCl buffer solution (50 mmol / L Tris·HCl, 5 mmol / L CaCl 2 , pH 8.5) at pH 8.5. Respectively take 100 μL of the fermentation supernatants of KT16 and KT231 prepared according to the steps of Example 2, add 2.5 mL of the gelatin solution, mix well, react at 30°C for 1 h, and add 2.5 mL of the termination solution (25 mmol / L EDTA, 12% (w / v) PEG6000) to terminate the reaction. Take 100 μL of the reaction solution after terminating the reaction and add 900 μL of ultrapure water, mix with 2.5 mL of the ninhydrin colorimetric solution, boil and heat for 10 min, cool, add 3 mL of ultrapure water, mix evenly, and measure the absorbance at 570 nm. The blank control group is the reaction of 100 μL of the diluted enzyme solution inactivated by boiling with 2.5 mL of the gelatin solution, and other steps are the same as those of the experimental group. React different concentrations of 100 μL glycine solutions with the ninhydrin-sodium citrate solution, and draw a standard curve with the absorbance values at 570 nm.

[0056] The method for determining the gelatin-degrading activity at 35°C - 80°C refers to the above steps.

[0057] The results are as Figure 4As shown, the trend of the enzymatic hydrolysis activity of the enzyme towards the substrate gelatin with temperature is consistent with that of the enzymatic hydrolysis activity of the enzyme towards the substrate casein with temperature. Under the conditions of pH 8.5 and 40 °C, the enzyme KT16 has the highest enzymatic hydrolysis activity towards the substrate gelatin, while the optimal enzymatic hydrolysis temperature of KT231 for gelatin is increased to 60 °C. Moreover, at the corresponding optimal enzymatic hydrolysis temperature, the collagenase activity of the mutant enzyme KT231 is 9254.62 U / mL, and the collagenase activity of the wild enzyme KT16 is 7238.31 U / mL. The mutant enzyme is significantly higher than the wild enzyme, being 1.28 times that of the wild enzyme. The above results once again confirm that the mutant enzyme KT231 is more suitable for hydrolyzing collagen under high-temperature conditions and better meets the requirements of industrial application.

[0058] Experimental Example 4: Preparation of collagen tripeptides from the skin of Alaska pollock using the mutant enzyme of the present invention

[0059] The pre-treated fresh Alaska pollock skin was rinsed clean with flowing water, cut into small pieces, placed in a blender, and 4 - 5 times the volume of water was added. The fish skin was ground into a uniform and delicate slurry. The temperature of the fish skin mixture was raised to 60 °C, the pH was adjusted to 8.5, and according to the enzyme / substrate ratio of 400 U / g, the collagenase mutant KT231 was added, and the reaction was stirred at 60 °C for 2 h. After the above reaction, the pH of the reaction solution was adjusted to 6.0 with phosphoric acid, and according to the enzyme / substrate ratio of 400 U / g, commercially available papain was added, and the reaction was stirred at 60 °C for 2 h. The temperature of the reaction solution was raised to 90 °C and treated for 30 minutes to inactivate the protease, obtaining an aqueous solution of collagen tripeptides prepared by enzymatic hydrolysis of Alaska pollock skin (i.e., fish skin enzymatic hydrolysate). During the preparation process, an aqueous solution of collagen tripeptides prepared by the combined action of commercially available Novozymes peptidase Alcalase and papain was used as a control.

[0060] The extraction efficiency of the collagen in the Alaska pollock skin was characterized by the concentration ratio of hydroxyproline in the enzymatic hydrolysate. The calculation formula for the extraction efficiency is as follows:

[0061] Extraction efficiency (%) = m 1 × dilution factor × 11.1 / m 0 × 100

[0062] In the formula, m 1 is the content of hydroxyproline, g; 11.1 is the coefficient for converting hydroxyproline to collagen; m 0 is the total protein content of the Alaska pollock skin.

[0063] Referring to GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Foods", the total protein content in Alaska pollock fish skin was determined by the Kjeldahl method. Referring to the method of Guo (Guo Hengbin, Zeng Qingzhu. Determination of Hydroxyproline Content in Fish Skin by Spectrophotometry. Food Research and Development, 2007 (28): 145-147.), the hydroxyproline content in the enzymatic hydrolysate was determined. Using the mass concentration of L-hydroxyproline standard as the abscissa and the absorbance value at 560 nm as the ordinate, a standard curve was plotted, and the regression equation was y = 0.5332x - 0.0007 (R 2 = 0.99993). Using the above regression equation, the hydroxyproline content in the fish skin enzymatic hydrolysate prepared by enzyme KT231 or enzyme Alcalase was calculated.

[0064] Under the condition of 60 °C, after the control group was hydrolyzed by the composite enzymes of Alcalase and papain, the extraction rate of Alaska pollock fish skin collagen was 81.14%; after being hydrolyzed by the composite enzymes of KT231 and papain, the extraction rate was 96.26%, which was 118.63% of the control group, and the extraction efficiency of Alaska pollock fish skin collagen was significantly improved.

[0065] The molecular weight and proportion of peptide segments in the above-mentioned Alaska pollock fish skin enzymatic hydrolysate were determined by high performance liquid chromatography, and the results Figure 5 are shown in Table 1. In the fish skin enzymatic hydrolysate prepared by Alacase and papain, the proportion of peptide segments with a molecular weight less than 1000 Da was 88.56%, and the proportion of peptide segments with a molecular weight less than 500 Da was 69.32%. In the fish skin enzymatic hydrolysate prepared by KT231 and papain, the proportion of peptide segments with a molecular weight less than 1000 Da increased to 96.76%; among them, the proportion of peptide segments with a molecular weight less than 500 Da reached 80.26%, which was 115.8% of the control group.

[0066] The above results show that the mutant enzyme KT231 of the present invention has a higher enzymatic hydrolysis efficiency for Alaska pollock fish skin, and the proportion of small molecular weight peptide segments in the fish skin enzymatic hydrolysate is greatly increased, which is beneficial to maintaining the excellent biological activity of the prepared peptides.

[0067] Table 1 Proportion of Oligopeptides with Different Molecular Weights in Fish Skin Enzymatic Hydrolysate

[0068]

[0069] Using collagen tripeptide GPH (glycine-proline-hydroxyproline) as the standard product, the content of collagen tripeptide (CTP) in the enzymatic hydrolysate was determined by high performance liquid chromatography. The results are as Figure 6As shown in Table 2, the content of collagen tripeptide in the control group of fish skin hydrolysate was 24.13%, while in the fish skin hydrolysate prepared with the mutant collagenase mutant KT231, the content of collagen tripeptide reached 38.62%, which was increased to 1.60 times, achieving significant results. Moreover, the collagen tripeptide prepared by enzymatic hydrolysis with the mutant collagenase mutant KT231 had no bitter taste.

[0070] Table 2 Content of collagen tripeptide in fish skin hydrolysate

[0071]

[0072] In summary, the present invention provides a collagenase mutant KT231 that can be efficiently secreted and expressed in Streptomyces S. lividans TK24. The collagenase mutant KT231 can efficiently hydrolyze Alaska pollock fish skin at 60 °C, and has excellent heat resistance and high enzyme activity, and can be used for the efficient preparation of collagen tripeptide from Alaska pollock fish skin. When combined with commercially available papain, it can significantly increase the proportion of collagen tripeptide in the fish skin hydrolysate, and has no bitter taste. It has broad application prospects in the preparation of products for anti-wrinkle and moisturizing, repairing damaged skin, improving bone and joint health, tissue engineering materials, and wound healing promoters.

[0073] Sequence Listing

[0074] SEQ ID NO. 1:

[0075] VQKNRLVRTLQKLAAAGAVALAALSLQPVSSATAAPNPVVGGTRAAQGEFPWMVRLSMGCGGSLISPQVVLTAAHCVGATGNNTSITATAGVVDLQSSSAIKVRSTKIYRAPGYNGKGKDWALIKLASPITSLPTLKLAETTAYNSGTFTVAGWGAAREGGGQQRYLLKANVPFVSDASCQASYGSDLVPSEEICAGYPQGGVDTCQGDSGGPMFRKDNAGAWVQVGIVSWGQGCARPDYPGVYTEVSTFAAAIKSAAATL.

[0076] SEQ ID NO. 2:

[0077] gtgcagaagaaccgactcgtccgcaccctgcagaaactcgccgccgccggcgccgtcgcgctcgccgcgctcagcctccagcccgtctccagcgccaccgcggcccccaacccggtcgtcggcggcacccgcgccgcccagggcgagttcccctggatggtccggctctccatgggctgcggcggctccctgatctccccgcaggtcgtcctcaccgccgcccactgcgtcggcgcgaccggcaacaacacctcgatcaccgccaccgccggcgtcgtggacctccagtccagcagcgccatcaaggtccgctccaccaagatctaccgagcccccggctacaacggcaagggcaaggactgggcgctcatcaagctcgccagccccatcacctcgctgcccaccctgaagctcgccgagaccaccgcgtacaacagcggcaccttcaccgtggccggctggggcgcggcccgcgagggcggcggccagcagcgctacctgctcaaggcgaacgtccccttcgtctcggacgcctcctgccaggcctcgtacggcagcgacctcgtgccgtccgaggagatctgcgccggctacccccagggcggcgtcgacacctgtcagggcgactccggcggcccgatgttccgcaaggacaacgccggcgcctgggtccaggtcggcatcgtgagctggggccagggctgcgcgcggcccgactaccccggcgtctacacggaggtctcgaccttcgccgccgcgatcaagtccgccgcggccacgctg.

[0078] SEQ ID NO. 3:

[0079] VQKNRLVRTLQKLAAAGAVALAALSLQPVSSATAAPNPVVGGTRAAQGEFPWMVRLSMGCGGSLISPQVVLTAAHCVGATGNNTSITATAGVVDLQSSSAIKVRSTKIYRAPGYNGKGKDWALIKLASPITSLPTLKLAETTAYNSGTFTVAGWGAAREGGAQQRYLLKANVPFVSDASCQASYGSSLVPSEEICAGYPQGGVDTCQGDSGGPMFRKDNAGAWIQVGIVSWGQGCARPNYPGVYTEVSTFAAAIKSAAATL.

[0080] SEQ ID NO. 4:

[0081] gtgcagaagaaccgcctcgtccgcaccctccagaagctggccgccgccggcgcggtcgcgctggccgccctgtccctgcagcccgtctcctccgccaccgccgccccgaacccggtcgtgggcggcacccgcgccgcccagggcgagttcccgtggatggtccgcctgtccatgggctgcggcggctcgctgatctccccccaggtcgtgctgaccgccgcccactgcgtcggcgccaccggcaacaacacctccatcaccgccaccgcgggcgtcgtggacctgcagtcctcctcggccatcaaggtccgctccaccaagatctaccgcgcccccggctacaacggcaagggcaaggactgggccctgatcaagctggcgagccccatcacctccctcccgaccctgaagctggccgaaacgaccgcctacaactccggcaccttcaccgtcgccggctggggcgccgcccgcgagggcggcgcccagcagcgctacctgctgaaggccaacgtgccgttcgtgtccgacgccagctgccaggcctcctacggctcctccctggtcccctccgaggagatctgcgccggctacccgcagggcggcgtggacacctgccagggcgactccggcggccccatgttccggaaggacaacgccggcgcctggatccaggtcggcatcgtctcctggggccagggctgcgcgcggcccaactacccgggcgtctacaccgaagtcagcaccttcgccgccgccatcaagtccgcggcggccaccctg.

[0082] SEQ ID NO. 5: Promoter - Signal Peptide - KT16 Coding Sequence - Terminator Ter - SCO0356:

[0083] tgttcacattcgaacggtctctgctttgacaacatgctgtgcggtgttgtaaagtcgtggccaggagaatacgacagcgtgcaggactgggggagttgtgcagaagaaccgactcgtccgcaccctgcagaaactcgccgccgccggcgccgtcgcgctcgccgcgctcagcctccagcccgtctccagcgccaccgcggcccccaacccggtcgtcggcggcacccgcgccgcccagggcgagttcccctggatggtccggctctccatgggctgcggcggctccctgatctccccgcaggtcgtcctcaccgccgcccactgcgtcggcgcgaccggcaacaacacctcgatcaccgccaccgccggcgtcgtggacctccagtccagcagcgccatcaaggtccgctccaccaagatctaccgagcccccggctacaacggcaagggcaaggactgggcgctcatcaagctcgccagccccatcacctcgctgcccaccctgaagctcgccgagaccaccgcgtacaacagcggcaccttcaccgtggccggctggggcgcggcccgcgagggcggcggccagcagcgctacctgctcaaggcgaacgtccccttcgtctcggacgcctcctgccaggcctcgtacggcagcgacctcgtgccgtccgaggagatctgcgccggctacccccagggcggcgtcgacacctgtcagggcgactccggcggcccgatgttccgcaaggacaacgccggcgcctgggtccaggtcggcatcgtgagctggggccagggctgcgcgcggcccgactaccccggcgtctacacggaggtctcgaccttcgccgccgcgatcaagtccgccgcggccacgctgtgataaagcccgcatcgccgcgcggccgaggcgcccgtgcgaggacgggtccctcggccgcttcgtggtgcttcct.

[0084] SEQ ID NO. 6: Promoter - Signal peptide - KT231 coding sequence - Terminator Ter - SCO0356

[0085] tgttcacattcgaacggtctctgctttgacaacatgctgtgcggtgttgtaaagtcgtggccaggagaatacgacagcgtgcaggactgggggagttgtgcagaagaaccgcctcgtccgcaccctccagaagctggccgccgccggcgcggtcgcgctggccgccctgtccctgcagcccgtctcctccgccaccgccgccccgaacccggtcgtgggcggcacccgcgccgcccagggcgagttcccgtggatggtccgcctgtccatgggctgcggcggctcgctgatctccccccaggtcgtgctgaccgccgcccactgcgtcggcgccaccggcaacaacacctccatcaccgccaccgcgggcgtcgtggacctgcagtcctcctcggccatcaaggtccgctccaccaagatctaccgcgcccccggctacaacggcaagggcaaggactgggccctgatcaagctggcgagccccatcacctccctcccgaccctgaagctggccgaaacgaccgcctacaactccggcaccttcaccgtcgccggctggggcgccgcccgcgagggcggcgcccagcagcgctacctgctgaaggccaacgtgccgttcgtgtccgacgccagctgccaggcctcctacggctcctccctggtcccctccgaggagatctgcgccggctacccgcagggcggcgtggacacctgccagggcgactccggcggccccatgttccggaaggacaacgccggcgcctggatccaggtcggcatcgtctcctggggccagggctgcgcgcggcccaactacccgggcgtctacaccgaagtcagcaccttcgccgccgccatcaagtccgcggcggccaccctgtgatgataaagcccgcatcgccgcgcggccgaggcgcccgtgcgaggacgggtccctcggccgcttcgtggtgcttcct。

Claims

1. A collagenase mutant, characterized in that: The amino acid sequence of the collagenase mutant is shown in SEQ ID NO:

3.

2. A gene fragment encoding a collagenase mutant, characterized in that: The nucleotide sequence of the gene fragment is shown in SEQ ID NO:4 or SEQ ID NO:

6.

3. A recombinant plasmid, characterized in that: The plasmid comprises the gene fragment according to claim 2.

4. A recombinant expression system, characterized in that: The recombinant expression system contains a host bacterium and the recombinant plasmid according to claim 3.

5. A method for preparing the collagenase mutant according to claim 1, characterized in that: The method comprises the following steps: (1) cloning the DNA fragment shown in SEQ ID NO:4 or SEQ ID NO:6 into a vector plasmid, transforming Escherichia coli to obtain an expression plasmid; (2) The expression plasmid is transferred into the host bacteria, the colony spores are collected, cultured, and the fermentation supernatant enzyme liquid is collected to obtain the collagenase mutant.

6. The method according to claim 5, characterized in that: The vector plasmid in step (1) is pSET152, and the Escherichia coli is DH5α; the host bacteria in step (2) is Streptomyces lividans TK24; The transfer method is conjugation transfer.

7. A method for preparing collagen tripeptide, characterized in that: The method comprises the following steps: taking collagen as raw material, enzymolyzing it with the collagenase mutant according to claim 1 and chymopapain in sequence to obtain collagen tripeptide.

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