Collagenase mutant, gene segment, recombinant plasmid, recombinant expression system and application thereof, and method for preparing collagenase mutant and collagen tripeptide

Abstract

The invention discloses a collagenase mutant, a gene segment, a recombinant plasmid, a recombinant expression system and application thereof, and a method for preparing the collagenase mutant and collagen tripeptide, and belongs to the field of gene engineering and fermentation engineering. The invention provides a collagenase mutant KT231 capable of realizing high-efficiency secretory expression in streptomyces S.lividans TK24 and a preparation method of the collagenase mutant KT231. The collagenase mutant KT231 can be used for efficiently carrying out enzymolysis on the skin of the narrow gadus at 60 DEG C, has excellent heat resistance and higher enzyme activity, and can be used for efficiently preparing the collagen tripeptide from the skin of the narrow gadus. The collagen peptide is compounded with commercially available papaya chymotrypsin, so that the proportion of collagen tripeptide in the fish skin enzymatic hydrolysate can be remarkably increased, and the fish skin enzymatic hydrolysate has no bitter taste. The method has wide application prospects in preparation of products, tissue engineering materials and wound healing accelerators for resisting wrinkles, preserving moisture, repairing damaged skin and improving bone and joint health.

Description

Technical Field

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

[0002] Collagen is the most abundant protein in animals and is the main component of skin, bones, tendons, teeth and other tissues. Collagenase is an enzyme that can specifically degrade collagen. It acts on the chain structure of collagen, cuts the interchain bonds, and hydrolyzes collagen into low molecular weight collagen peptides, including the highly-regarded collagen tripeptide. The typical structure of collagen tripeptide is "Gly-XY", where X is mostly proline and Y is mostly hydroxyproline. Collagen tripeptide is widely used and has become a research hotspot in the field of 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 is complex, costly, and has a potential risk of disease transmission, which limits large-scale industrial applications. To overcome these limitations, researchers have begun to explore the production of collagenase by microbial fermentation in recent years. Microbial fermentation has a wide source of raw materials, low cost, and easy control, and has become an important development direction for collagenase production. This method has great potential, but it 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 enzymes by screening highly active collagenase mutants and mutants with high substrate binding specificity.

[0004] Therefore, it is of great research value to develop 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 its use, and a method for preparing a collagenase mutant and a collagen tripeptide.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides a collagenase mutant, wherein the amino acid sequence of the collagenase mutant has at least 99% sequence identity with the sequence shown in SEQ ID NO: 3.

[0007] Furthermore, the collagenase mutant has an amino acid sequence as shown in SEQ ID NO: 3.

[0008] The present invention also provides a gene fragment encoding a collagenase mutant, the nucleotide sequence of the gene fragment is shown in SEQ ID NO: 4 or SEQ ID NO: 6.

[0009] The present invention also provides a recombinant plasmid, which comprises the above gene fragment. The present invention also provides a recombinant expression system, which contains a host bacterium and the recombinant plasmid.

[0010] The present invention also provides a method for preparing the above collagenase mutant, the method comprising 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.

[0011] Furthermore, the vector plasmid in step (1) is pSET152, the Escherichia coli is DH5α; the host bacteria in step (2) is Streptomyces S. lividans TK24; The transfer method is conjugation transfer.

[0012] The present invention also provides the use of the above-mentioned collagenase mutant, gene fragment, recombinant plasmid and recombinant expression system in the preparation of collagen tripeptide.

[0013] The present invention also provides a method for preparing collagen tripeptide, which comprises the following steps: taking collagen as raw material, and subjecting it to enzymatic hydrolysis by the above-mentioned collagenase mutant and chymosin in sequence to obtain collagen tripeptide.

[0014] Furthermore, the chymosin is chymopapain.

[0015] The present invention has achieved the following beneficial effects: The present invention provides a method for S. lividans The collagenase mutant KT231 is highly secreted and expressed in TK24. The collagenase mutant KT231 can efficiently enzymatically hydrolyze the skin of haddock at 60°C, and has excellent heat resistance and high enzyme activity, and can be used for the efficient preparation of collagen tripeptides from haddock skin. The combination with the commercially available papain chymosin 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 and tissue engineering materials for anti-wrinkle and moisturizing, repairing damaged skin, improving bone and joint health, and wound healing promoters.

[0016] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0017] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the SDS-PAGE image of collagenase KT16 and KT231.

[0019] Figure 2 The pH curves of collagenase KT16 and KT231.

[0020] Figure 3 Shown are the temperature curves of collagenase KT16 and KT231.

[0021] Figure 4 This is the enzyme activity curve of collagenase KT16 and KT231.

[0022] Figure 5 This is a liquid chromatogram of oligopeptides of different molecular weight in the enzymatic hydrolysate of haddock skin.

[0023] Figure 6 This is a liquid chromatogram of the collagen tripeptide content in the enzymatic hydrolyzate of haddock skin. DETAILED DESCRIPTION

[0024] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0025] The formula of the culture medium involved in the embodiments of the present invention is as follows, % refers to mass percentage, and g / L refers to grams per liter: LB liquid medium: 1% tryptone, 0.5% yeast powder, 0.5% sodium chloride; LB solid medium: 1% tryptone, 0.5% yeast powder, 0.5% sodium chloride, 1.5% agar; Skim milk powder medium: 1% tryptone, 0.5% yeast powder, 0.5% sodium chloride, 1% skim milk powder, 1.5% agar; MS solid medium: soybean powder 20.0 g / L, mannitol 20.0 g / L, calcium carbonate 3.0 g / L, agar 20.0 g / L; TSB medium for Streptomyces seed culture: 1.5% tryptone, 0.5% soy peptone, 0.5% sodium chloride; Streptomyces fermentation medium: glucose 10.0 g / L, tryptone 5.0 g / L, yeast extract 5.0 g / L, casamino acids 2.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, magnesium sulfate heptahydrate 0.5 g / L.

[0026] Example 1: Gene cloning and expression plasmid construction Construction process of the recombinant expression plasmid pSET152-KT16 of wild-type collagenase KT16: chemical synthesis of genes kt16 The expression cassette DNA fragment is shown in SEQ ID NO. 5, which contains the strong promoter KasO*p of Streptomyces, gene kt16 The full-length coding sequence collagenase - kt16 , strong terminator Ter-SCO0356. Among them, collagenase collagenase - kt16 The amino acid sequence is shown in SEQ ID NO. 1; kt16 The encoding nucleotide sequence is shown in SEQ ID NO. 2.

[0027] Plasmid pSET152 was digested with restriction endonuclease BamHI, and the linearized pSET152 DNA was recovered by gel electrophoresis; the above two DNA fragments (i.e., gene kt16 The expression cassette DNA fragment was seamlessly cloned with the linearized pSET152 DNA and 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.

[0028] Construction process of the recombinant expression plasmid pSET152-KT231 of collagenase mutant KT231: Chemical synthesis gene kt231 The expression cassette DNA fragment is shown in SEQ ID NO. 6, which contains the strong promoter KasO*p of Streptomyces, gene kt231 The full-length coding sequence collagenase - kt231 , strong terminator Ter-SCO0356. Among them, collagenase collagenase - kt231 The amino acid sequence is shown in SEQ ID NO. 3; kt231 The encoding nucleotide sequence is shown in SEQ ID NO. 4.

[0029] Plasmid pSET152 was digested with restriction endonuclease BamHI, and the linearized pSET152 DNA was recovered by gel electrophoresis; the above two DNA fragments (i.e., gene kt231The expression cassette DNA fragment was seamlessly cloned with the linearized pSET152 DNA and transformed into Escherichia coli DH5α; plasmids were extracted from the positive clones and the correct expression plasmid pSET152-KT231 was obtained by sequence determination.

[0030] The target DNA sequences provided in the examples as shown in SEQ ID NO. 5 and SEQ ID NO. 6 can be obtained by PCR amplification and chemical synthesis, and the examples are not limited thereto.

[0031] Example 2: Heterologous expression of collagenase and preparation of fermentation supernatant enzyme solution Activate Streptomyces conjugative transfer donor host on LB solid plates containing 25 µg / mL kanamycin and 25 µg / mL chloramphenicol E. coli The expression plasmids pSET152-KT16 and pSET152-KT231 provided in the above examples were respectively transferred into E. coli ET12567 / pUZ8002, and screen the positive clones on plates containing 100 µg / mL apramycin, 25 µg / mL kanamycin, and 25 µg / mL chloramphenicol. Then, the positive clones containing the expression plasmid pSET152-KT16 or pSET152-KT231 were transferred into Streptomyces by conjugation transfer. S. lividans TK24. Mg used in the joint transfer process 2+ The concentration was 10 mmol / L, the pre-germination time of Streptomyces was 3 h, the final concentration of apramycin was 1.25 mg / ml, and the final concentration of nalidixic acid was 0.5 mg / ml. After the conjugation transfer was completed, the MS plate was cultured at 28°C. After 5-8 days, a single clone of the conjugation transfer was picked and streaked once on a medium containing 25 mg / L of nalidixic acid and 100 µg / mL of apramycin to obtain the Streptomyces expression strains of wild-type collagenase KT16 and mutant KT231.

[0032] Collect freshly activated wild-type collagenase KT16 and mutant KT231 Streptomyces expression strains, inoculate them into 30 mL TSB seed medium, culture at 30°C, 220 r / min for 24 h. Take 3 mL of seed solution and transfer to 50 mL fermentation medium, culture at 30°C, 220 r / min for 96 h, centrifuge at 8000g for 10 min to collect the supernatant, and obtain proteinase KT16 and KT231. Use SDS-PAGE to analyze the molecular weight of proteinase KT16 and KT231. The results are as follows: Figure 1 shown.

[0033] With reference to GB / T 23527.1-2023 Enzyme Preparation Quality Requirements Part 1: Protease Preparations, the degradation activity of collagenase KT16 and collagenase mutant KT231 on substrate casein was determined by spectrophotometry at 45°C and pH 9.0. Enzyme activity is defined as the amount of enzyme required to hydrolyze casein to produce 1 µg of tyrosine per minute under specific conditions, which is 1 unit of enzyme activity. The expressed enzyme activity of the wild enzyme KT16 is 5231.56 U / mL, while the expressed enzyme activity of the mutant enzyme KT231 is 6602.12 U / mL, which is increased to 1.26 times. The above results show that the secretion expression level of the 4-site mutant KT231 obtained in the embodiment of the present application is significantly improved compared with that of the wild enzyme KT16, which is conducive to further improving the enzymatic efficiency and reducing the application cost.

[0034] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0035] Experimental Example 1: pH stability of the mutant enzyme of the present invention Prepare 50mmol / 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, 12.0, and dilute collagenase KT16 and KT231 enzyme solutions to 10-15 U / mL with the above buffer solutions with different pH values. Determine the enzyme activities of collagenase KT16 and KT231 on the substrate casein at 50°C and corresponding pH conditions. The highest enzyme activity is defined as 100%, and the corresponding pH is the optimal enzymatic pH of the enzyme. Calculate the relative enzyme activities under other pH conditions. The results are as follows: Figure 2 As shown, the optimal action pH of the wild-type KT16 and mutant KT231 on the substrate casein is 8.5. Both are alkaline proteases, and the 4-site mutation has no obvious effect on the pH properties of the enzyme.

[0036] Experimental Example 2: Thermal stability of the mutant enzyme of the present invention According to the optimal action pH determined in Experimental Example 1, under the condition of pH 8.5, the casein hydrolysis activity of collagenase KT16 and KT231 was measured at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C, respectively.

[0037] The results are as follows Figure 3 As 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 4-site mutant KT231 has better heat resistance and is more suitable for high-temperature enzymatic hydrolysis conditions in industrial practice to obtain higher enzymatic hydrolysis efficiency.

[0038] Experimental Example 3: Collagenase hydrolysis performance of the mutant enzyme of the present invention Under high temperature conditions, fish skin collagen is converted into soluble gelatin, and gelatin can be used as a substrate to measure collagenase activity. Referring to Zhang's method (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 substrate gelatin was determined by the ninhydrin colorimetric method. The enzyme activity unit is defined as the amount of enzyme required to degrade 1 mg of gelatin per minute under specific conditions to produce 1 µmol of glycine.

[0039] The gelatin degradation enzyme activity of enzymes KT16 and KT231 in the temperature range of 30℃-80℃ was determined. The enzyme activity determination method at 30℃ was as follows: 2 mg / mL gelatin solution was prepared with Tris·HCl buffer solution (50 mmol / L Tris·HCl, 5 mmol / L CaCl2, pH8.5) at pH 8.5. 100 μL of the fermentation supernatant of KT16 and KT231 prepared according to the steps of Example 2 were taken, 2.5 mL of gelatin solution was added, mixed, reacted at 30℃ for 1 h, and 2.5 mL of stop solution (25 mmol / L EDTA, 12% (w / v) PEG6000) was added to terminate the reaction. 100 μL of the reaction solution after the termination reaction was taken, 900 μL of ultrapure water was added, mixed with 2.5 mL of ninhydrin colorimetric solution, boiled and heated for 10 min, cooled, 3 mL of ultrapure water was added, mixed evenly, and the absorbance was measured at 570 nm. The blank control group was composed of 100 μL of the diluted enzyme solution inactivated by boiling and reacted with 2.5 mL of gelatin solution, and the other steps were synchronized with the experimental group. 100 μL of glycine solution of different concentrations was reacted with ninhydrin-sodium citrate solution, and the standard curve was drawn with the absorbance value at 570 nm.

[0040] The method for measuring gelatin-degrading enzyme activity at 35°C-80°C is similar to the above steps.

[0041] The results are as follows Figure 4As shown in the figure, the enzymatic activity of the enzyme on the substrate gelatin changes with temperature, which is consistent with the enzymatic activity of the enzyme on the substrate casein. Under pH 8.5 and 40℃, enzyme KT16 has the highest enzymatic activity on the substrate gelatin, while the optimal enzymatic temperature of KT231 for gelatin is increased to 60℃. Moreover, at the corresponding optimal enzymatic temperature, the collagenase activity of the mutant enzyme KT231 is 9254.62 U / mL, and the collagenase activity of the wild enzyme KT16 is 7238.31U / mL. The mutant enzyme is significantly higher than the wild enzyme, which is 1.28 times that of the wild enzyme. The above results once again confirm that the mutant enzyme KT231 is more suitable for enzymatic hydrolysis of collagen under high temperature conditions and is more in line with the requirements of industrial applications.

[0042] Experimental Example 4: Preparation of collagen tripeptide by enzymatic hydrolysis of haddock skin using the mutant enzyme of the present invention The pretreated fresh haddock fish skin was rinsed with running water, cut into small pieces, put into a masher, and 4-5 times the volume of water was added to crush the fish skin into a uniform and fine slurry. The temperature of the fish skin mixture was raised to 60°C, the pH was adjusted to 8.5, and the collagenase mutant KT231 was added according to the enzyme / substrate ratio of 400 U / g, 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 the commercially available papain chymosin was added according to the enzyme / substrate ratio of 400 U / g, 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, and the collagen tripeptide aqueous solution (i.e., fish skin enzymatic hydrolyzate) prepared by enzymatic hydrolysis of haddock fish skin was obtained. During the preparation process, the collagen tripeptide aqueous solution prepared by the combined action of the commercially available Novozymes peptidase Alcalase and papain chymosin was used as a control.

[0043] The extraction efficiency of haddock skin collagen was characterized by the concentration ratio of hydroxyproline in the enzymatic hydrolysate. The extraction efficiency calculation formula is as follows: Extraction efficiency (%) = m1 × dilution factor × 11.1 / m0 × 100 Wherein, m1 is the hydroxyproline content, g; 11.1 is the coefficient of converting hydroxyproline to collagen; m0 is the total amount of protein in haddock skin.

[0044] Referring to GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food", the total amount of protein in the skin of Alaska pollock was determined by the Kjeldahl method. The hydroxyproline content in the enzymatic hydrolysate was determined by 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 standard curve was drawn with the mass concentration of L-hydroxyproline standard as the horizontal axis and the absorbance value at 560 nm as the vertical axis. The regression equation was y=0.5332x-0.0007 (R 2 =0.99993). The above regression equation was used to calculate the hydroxyproline content in the fish skin hydrolysate prepared by enzyme KT231 or enzyme Alcalase. Under 60℃ conditions, the extraction rate of haddock skin collagen was 81.14% after combined enzymatic hydrolysis by enzymes Alcalase and chymopapain in the control group; after combined enzymatic hydrolysis by enzymes KT231 and chymopapain, the extraction rate was 96.26%, which was 118.63% of that in the control group, significantly improving the extraction efficiency of haddock skin collagen.

[0045] The molecular weight and proportion of peptides in the above-mentioned haddock skin hydrolysate were determined by high performance liquid chromatography. Figure 5 As shown in Table 1. In the fish skin hydrolysate prepared by the enzyme Alacase and chymopapain, the proportion of peptides with a molecular weight less than 1000 Da was 88.56%, and the proportion of peptides with a molecular weight less than 500 Da was 69.32%. In the fish skin hydrolysate prepared by the enzyme KT231 and chymopapain, the proportion of peptides with a molecular weight less than 1000 Da increased to 96.76%; among them, the proportion of peptides with a molecular weight less than 500 Da reached 80.26%, which was 115.8% of the control group.

[0046] The above results show that the mutant enzyme KT231 of the present invention has a higher enzymatic hydrolysis efficiency for haddock fish skin, and the proportion of small molecular weight peptides in the fish skin enzymatic hydrolysate is greatly increased, which is beneficial to maintain the excellent biological activity of the prepared peptides.

[0047] Table 1 Proportion of oligopeptides of different molecular weight in fish skin hydrolysate The content of collagen tripeptide (CTP) in the enzymatic hydrolysate was determined by HPLC using collagen tripeptide GPH (glycine-proline-hydroxyproline) as the standard. Figure 6As shown in Table 2, the content of collagen tripeptide in the fish skin hydrolysate of the control group was 24.13%, while the content of collagen tripeptide in the fish skin hydrolysate prepared by the mutant collagenase mutant KT231 reached 38.62%, which was increased by 1.60 times, achieving significant results. And the collagen tripeptide prepared by the mutant collagenase mutant KT231 has no bitter taste.

[0048] Table 2 Content of collagen tripeptide in fish skin enzymatic hydrolysate In summary, the present invention provides a method for S. lividans The collagenase mutant KT231 is highly secreted and expressed in TK24. The collagenase mutant KT231 can efficiently enzymatically hydrolyze the skin of haddock at 60°C, and has excellent heat resistance and high enzyme activity, and can be used for the efficient preparation of collagen tripeptides from haddock skin. The combination with the commercially available papain chymosin 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 and tissue engineering materials for anti-wrinkle and moisturizing, repairing damaged skin, improving bone and joint health, and wound healing promoters.

[0049] Sequence Listing SEQ ID NO. 1: VQKNRLVRTLQKLAAAGAVALAALSLQPVSSATAAPNPVVGGTRAAQGEFPWMVRLSMGCGGSLISPQVVLTAAHCVGATGNNTSITATAGVVDLQSSSAIKVRSTKIYRAPGYNGKGKDWALIKLASPIT SLPTLKLAETTAYNSGTFTVAGWGAAREGGGQQRYLLKANVPFVSDASCQASYGSDLVPSEEICAGYPQGGVDTCQGDSGGPMFRKDNAGAWVQVGIVSWGQGCARPDYPGVYTEVSTFAAAIKSAAATL. SEQ ID NO. 2: gtgcagaagaaccgactcgtccgcaccctgcagaaactcgccgccgccggcgccgtcgcgctcgccgcgctcagcctccagcccgtctccagcgccaccgcggcccccaacccggtcgtcggcggcacccgcgccgcccagggcgagttcccctggatggtccggctctccatgggctgcggcggctccctgatctccccgcaggtcgtcctcaccgccgcccactgcgtcggcgcgaccggcaacaacacctcgatcaccgccaccgccggcgtcgtggacctccagtccagcagcgccatcaaggtccgctccaccaagatctaccgagcccccggctacaacggcaagggcaaggactgggcgctcatcaagctcgccagccccatcacctcgctgcccaccctgaagctcgccgagaccaccgcgtacaacagcggcaccttcaccgtggccggctggggcgcggcccgcgagggcggcggccagcagcgctacctgctcaaggcgaacgtccccttcgtctcggacgcctcctgccaggcctcgtacggcagcgacctcgtgccgtccgaggagatctgcgccggctacccccagggcggcgtcgacacctgtcagggcgactccggcggcccgatgttccgcaaggacaacgccggcgcctgggtccaggtcggcatcgtgagctggggccagggctgcgcgcggcccgactaccccggcgtctacacggaggtctcgaccttcgccgccgcgatcaagtccgccgcggccacgctg. SEQ ID NO. 3: VQKNRLVRTLQKLAAAGAVALAALSLQPVSSATAAPNPVVGGTRAAQGEFPWMVRLSMGCGGSLISPQVVLTAAHCVGATGNNTSITATAGVVDLQSSSAIKVRSTKIYRAPGYNGKGKDWALIKLASPITSLPTLKLAETTAYNSGTFTVAGWGAAREGGAQQRYLLKANVPFVSDASCQASYGSSLVPSEICAGYPQGGVDTCQGDSGGPMFRKDNAGAWIQVGIVSWGQGCARPNYPGVYTEVSTFAAAIKSAAATL. SEQ ID NO. 4: gtgcagaagaaccgcctcgtccgcaccctccagaagctggccgccgccggcgcggtcgcgctggccgccctgtccctgcagcccgtctcctccgccaccgccgccccgaacccggtcgtgggcggcacccgcgccgcccagggcgagttcccgtggatggtccgcctgtccatgggctgcggcggctcgctgatctccccccaggtcgtgctgaccgccgcccactgcgtcggcgccaccggcaacaacacctccatcaccgccaccgcgggcgtcgtggacctgcagtcctcctcggccatcaaggtccgctccaccaagatctaccgcgcccccggctacaacggcaagggcaaggactgggccctgatcaagctggcgagccccatcacctccctcccgaccctgaagctggccgaaacgaccgcctacaactccggcaccttcaccgtcgccggctggggcgccgcccgcgagggcggcgcccagcagcgctacctgctgaaggccaacgtgccgttcgtgtccgacgccagctgccaggcctcctacggctcctccctggtcccctccgaggagatctgcgccggctacccgcagggcggcgtggacacctgccagggcgactccggcggccccatgttccggaaggacaacgccggcgcctggatccaggtcggcatcgtctcctggggccagggctgcgcgcggcccaactacccgggcgtctacaccgaagtcagcaccttcgccgccgccatcaagtccgcggcggccaccctg. SEQ ID NO. 5: Promoter - Signal Peptide - KT16 Coding Sequence - Terminator Ter - SCO0356: tgttcacattcgaacggtctctgctttgacaacatgctgtgcggtgttgtaaagtcgtggccaggagaatacgacagcgtgcaggactgggggagttgtgcagaagaaccgactcgtccgcaccctgcagaaactcgccgccgccggcgccgtcgcgctcgccgcgctcagcctccagcccgtctccagcgccaccgcggcccccaacccggtcgtcggcggcacccgcgccgcccagggcgagttcccctggatggtccggctctccatgggctgcggcggctccctgatctccccgcaggtcgtcctcaccgccgcccactgcgtcggcgcgaccggcaacaacacctcgatcaccgccaccgccggcgtcgtggacctccagtccagcagcgccatcaaggtccgctccaccaagatctaccgagcccccggctacaacggcaagggcaaggactgggcgctcatcaagctcgccagccccatcacctcgctgcccaccctgaagctcgccgagaccaccgcgtacaacagcggcaccttcaccgtggccggctggggcgcggcccgcgagggcggcggccagcagcgctacctgctcaaggcgaacgtccccttcgtctcggacgcctcctgccaggcctcgtacggcagcgacctcgtgccgtccgaggagatctgcgccggctacccccagggcggcgtcgacacctgtcagggcgactccggcggcccgatgttccgcaaggacaacgccggcgcctgggtccaggtcggcatcgtgagctggggccagggctgcgcgcggcccgactaccccggcgtctacacggaggtctcgaccttcgccgccgcgatcaagtccgccgcggccacgctgtgataaagcccgcatcgccgcgcggccgaggcgcccgtgcgaggacgggtccctcggccgcttcgtggtgcttcct. SEQ ID NO. 6: Promoter-Signal Peptide-KT231 Coding Sequence-Terminator Ter-SCO0356: tgttcacattcgaacggtctctgctttgacaacatgctgtgcggtgttgtaaagtcgtggccaggagaatacgacagcgtgcaggactgggggagttgtgcagaagaaccgcctcgtccgcaccctccagaagctggccgccgccggcgcggtcgcgctggccgccctgtccctgcagcccgtctcctccgccaccgccgccccgaacccggtcgtgggcggcacccgcgccgcccagggcgagttcccgtggatggtccgcctgtccatgggctgcggcggctcgctgatctccccccaggtcgtgctgaccgccgcccactgcgtcggcgccaccggcaacaacacctccatcaccgccaccgcgggcgtcgtggacctgcagtcctcctcggccatcaaggtccgctccaccaagatctaccgcgcccccggctacaacggcaagggcaaggactgggccctgatcaagctggcgagccccatcacctccctcccgaccctgaagctggccgaaacgaccgcctacaactccggcaccttcaccgtcgccggctggggcgccgcccgcgagggcggcgcccagcagcgctacctgctgaaggccaacgtgccgttcgtgtccgacgccagctgccaggcctcctacggctcctccctggtcccctccgaggagatctgcgccggctacccgcagggcggcgtggacacctgccagggcgactccggcggccccatgttccggaaggacaacgccggcgcctggatccaggtcggcatcgtctcctggggccagggctgcgcgcggcccaactacccgggcgtctacaccgaagtcagcaccttcgccgccgccatcaagtccgcggcggccaccctgtgatgataaagcccgcatcgccgcgcggccgaggcgcccgtgcgaggacgggtccctcggccgcttcgtggtgcttcct。

Claims

1. A collagenase mutant, characterized in that: The collagenase mutant has an amino acid sequence as 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, the Escherichia coli is DH5α; the host bacteria in step (2) is Streptomyces S. lividans TK24; The transfer-in method is joint transfer.

7. Use of the collagenase mutant according to claim 1, the gene fragment according to claim 2, the recombinant plasmid according to claim 3, and the recombinant expression system according to claim 4 in the preparation of collagen tripeptide.

8. A method for preparing collagen tripeptide, characterized in that: The method comprises the following steps: taking collagen as raw material, and sequentially hydrolyzing it with the collagenase mutant according to claim 1 and chymosin to obtain collagen tripeptide.

9. The method according to claim 8, characterized in that: The chymosin is chymopapain.

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