Search

CN-121991934-A - Collagenase mutant and preparation method and application thereof

CN121991934ACN 121991934 ACN121991934 ACN 121991934ACN-121991934-A

Abstract

The invention discloses a collagenase mutant and a preparation method and application thereof. Compared with the amino acid sequence of the wild collagenase, the collagenase mutant has the mutation of tryptophan at position 23 into histidine, the mutation of isoleucine at position 26 into glutamine, the mutation of glutamic acid at position 289 into histidine and the mutation of tryptophan at position 486 into alanine. The collagenase mutant provided by the invention can effectively degrade collagen, and has important practical significance for application of collagenase and effective utilization of collagen resources.

Inventors

  • WEI HONGKUI
  • Huo Qiyuan
  • PENG JIAN
  • ZHOU YUANFEI

Assignees

  • 华中农业大学

Dates

Publication Date
20260508
Application Date
20260306

Claims (10)

  1. 1. A collagenase mutant is characterized in that compared with the amino acid sequence of wild collagenase, tryptophan at position 23 is mutated to histidine, isoleucine at position 26 is mutated to glutamine, glutamic acid at position 289 is mutated to histidine, and tryptophan at position 486 is mutated to alanine; the amino acid sequence of the wild collagenase is shown as SEQ ID NO. 1.
  2. 2. The collagenase mutant according to claim 1, which has an amino acid sequence shown in SEQ ID NO. 2.
  3. 3. A gene encoding the collagenase mutant as set forth in claim 1, wherein the nucleotide sequence of the collagenase mutant gene is shown in SEQ ID NO. 4.
  4. 4. A method for producing a collagenase mutant according to claim 1, comprising the steps of: (1) Obtaining a gene sequence of a collagenase mutant, respectively adding NdeL and HindIII enzyme cutting sites at two ends of the gene, and carrying out double enzyme cutting on the gene by NdeL and HindIII to obtain a collagenase mutant gene fragment; (2) Carrying out double digestion on the vector pET30a to obtain a vector fragment; (3) Connecting the collagenase mutant gene fragment and the vector fragment to obtain a collagenase recombinant expression vector; (4) Transferring the collagenase recombinant expression vector into host bacteria, culturing in a shake flask to perform protein expression, breaking bacteria to obtain crude enzyme liquid, and purifying the crude enzyme liquid to obtain collagenase mutants.
  5. 5. The method according to claim 4, wherein the ligase is T4 ligase and the host bacterium is E.coli.
  6. 6. A collagenase recombinant expression vector, which is characterized by comprising the gene of claim 3, wherein the expression vector is pET30a.
  7. 7. A collagenase expression strain, characterized in that the strain comprises the vector of claim 6, and the host strain of the strain is Escherichia coli BL21.
  8. 8. Use of any one of the following in catalyzing collagen hydrolysis, characterized by: 1) The collagenase mutant of claim 1; 2) The vector of claim 6; 3) The strain of claim 7.
  9. 9.A method of producing glycyl-prolyl-alanine using a collagenase mutant as claimed in claim 1, wherein the glycyl-prolyl-alanine is produced by mixing a fish skin gelatin solution with the collagenase mutant and incubating the mixture.
  10. 10. The application of the method according to claim 9, wherein the concentration of the fish skin gelatin solution is 15-25 g/L, and the enzyme activity of the collagenase mutant added into the fish skin gelatin solution is 1-1.1U/ml; the incubation condition is 30-55 ℃ and the pH value is 4-9.

Description

Collagenase mutant and preparation method and application thereof Technical Field The invention relates to the technical field of biotechnology and protein engineering, in particular to a collagenase mutant, a preparation method and application thereof. Background Collagen is a biological high molecular protein, is a main component of extracellular matrix, not only provides a flexible scaffold for cells, but also regulates vital cell processes including differentiation, cell growth, survival, migration and the like, and is one of key materials in biotechnology industry, and has wide application in medical treatment, food, daily chemicals and the like. Collagen has a complex molecular structure and good stability, and is difficult to degrade by common proteases, while matrix metalloproteinase (matrix metalloproteinase, MMP) and microbial collagenase (collagenase) are currently known to be few enzymes capable of degrading collagen in situ in a physiological environment, and can maintain higher enzyme activity under the environment to exert degradation function. The microbial collagenase has the advantages of capability of being secreted outside cells, convenience in separation and extraction, larger molecular mass, more complex structural domain, stronger adaptability of substrates, no need of generating enzyme activity by activating zymogen and the like. Among them, the Col H collagenase, col G collagenase and Col A collagenase derived from Clostridium perfringens are called "true" collagenase because they can cleave triple-helical collagen, and in addition, col A collagenase extracted from Bacillus cereus has been added to the collection of "true" collagenases. At present, commercial collagenase is mainly extracted from clostridium histolyticum, and is recognized as a standard enzyme for identifying novel collagenase, but the problems of high preparation cost and pathogenicity of bacterial strains exist, so that the application safety and the large-scale production of the collagenase are limited. Bacillus cereus and members of its kindred family are gram-positive, spore-forming facultative anaerobes that are ubiquitous in the environment, but they act as opportunistic pathogens and have become causative factors for food-borne gastrointestinal diseases and non-gastrointestinal diseases, and there is a safety risk in direct application. In view of the problems, the construction of a collagenase production strain with high expression and no pathogenicity has important significance, and the limited expression quantity of the collagenase in engineering strains currently restricts the practicability. Disclosure of Invention The invention aims to overcome the defects of the prior art and provides a collagenase mutant and a preparation method and application thereof. The invention optimizes the performance of ColA collagenase from two key dimensions by mutating the amino acid sequence of the collagenase, namely improving the soluble expression quantity and enhancing the enzyme activity of hydrolyzed collagen. So as to realize high-efficiency soluble expression and high enzyme activity of ColA and provide a solution for safe and low-cost collagenase production. In order to achieve the above purpose, the technical scheme of the invention is as follows: the invention provides a collagenase mutant, which is characterized in that compared with the amino acid sequence of wild collagenase, the 23 rd tryptophan is mutated to histidine, the 26 th isoleucine is mutated to glutamine, the 289 th glutamic acid is mutated to histidine, the 486 th tryptophan is mutated to alanine, and the amino acid sequence of the wild collagenase is shown as SEQ ID NO. 1. Further, the amino acid sequence of the collagenase mutant is shown as SEQ ID NO. 2. The invention also provides a gene for encoding the collagenase mutant, and the nucleotide sequence of the gene of the collagenase mutant is shown as SEQ ID NO. 4. The invention also provides a preparation method of the collagenase mutant, which comprises the following steps: (1) Obtaining a gene sequence of a collagenase mutant, respectively adding NdeL and HindIII enzyme cutting sites at two ends of the gene, and carrying out double enzyme cutting on the gene by NdeL and HindIII to obtain a collagenase mutant gene fragment; (2) Carrying out double digestion on the vector pET30a to obtain a vector fragment; (3) Connecting the collagenase mutant gene fragment and the vector fragment to obtain a collagenase recombinant expression vector; (4) Transferring the collagenase recombinant expression vector into host bacteria escherichia coli, culturing in a shake flask to perform protein expression, breaking bacteria to obtain crude enzyme liquid, and purifying the crude enzyme liquid to obtain collagenase mutants. Further, the linked ligase is T4 ligase and the host bacterium is Escherichia coli. The invention also provides a collagenase recombinant expression vector, which comprises the gene, and th