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CN-121991921-A - Lipase mutant and application thereof

CN121991921ACN 121991921 ACN121991921 ACN 121991921ACN-121991921-A

Abstract

The present disclosure provides a lipase mutant. Corresponding to the amino acid sequence of the wild-type lipase, said lipase mutant has a mutation at one or more of amino acids 10, 40, 45, 47, 156, 159. The lipase mutant disclosed by the invention can effectively improve the catalytic efficiency of the enzyme and the rate of catalytic reaction under the low-temperature condition, can effectively reduce the optimal catalytic reaction temperature, and provides important significance for the application of the lipase in key fields such as food processing, detergent manufacturing and the like.

Inventors

  • YU SHANSHAN
  • WANG SHIPING
  • SU HAO
  • SHENG XIANG

Assignees

  • 中国科学院天津工业生物技术研究所
  • 天工生物科技（天津）有限公司

Dates

Publication Date
20260508
Application Date
20241104

Claims (10)

  1. 1. Lipase mutant, characterized in that it corresponds to the amino acid sequence of a wild-type lipase, which has a mutation at one or more of amino acids 10, 40, 45, 47, 156, 159, wherein the wild-type lipase is wild-type lipase a derived from bacillus subtilis (Bacillus subtilis), preferably the amino acid sequence of the wild-type lipase is shown in SEQ ID No. 1.
  2. 2. The lipase mutant according to claim 1, which has one or more mutations in I10M, W40I, T45N, Y47R, G156S and Y159A, Preferably, the lipase mutant has at least one selected from the following group of mutations: (1) I10M and W40I mutations; (2) I10M and Y47R mutations; (3) I10M and G156S mutations; (4) I10M and G159A mutations; (5) I10M, W I and Y47R mutations; (6) I10M, W I and G156S mutations; (7) The I10M, W I and Y159A mutations; (8) The I10M, W40I, Y R and G156S mutations; (9) The I10M, W40I, Y R and Y159A mutations; (10) The I10M, W40I, T45N, Y47R and G156S mutations; (11) The I10M, W40I, Y47R, G S and Y159A mutations; (12) The I10M, W40I, T45N, Y47R, G S and Y159A mutations; preferably, the lipase mutant has the amino acid sequence shown in one or more of SEQ ID NO. 12-28, or the amino acid sequence shown in one or more of SEQ ID NO. 12-28 has been added, deleted, substituted or modified by one or more amino acids.
  3. 3. Lipase mutant according to claim 1 or 2, characterized in that the reaction temperature of the lipase mutant is 10-35 ℃, preferably 20-30 ℃, and/or The relative activity of the lipase mutant compared to the wild-type lipase is higher than 1.0, preferably higher than 1.3.
  4. 4. A nucleic acid molecule encoding the lipase mutant according to any of claims 1-3.
  5. 5. A recombinant vector comprising the nucleic acid molecule of claim 4.
  6. 6. A host cell comprising the lipase mutant of any one of claims 1-3, the nucleic acid molecule of claim 4, or the recombinant expression vector of claim 5; Preferably, the host cell is a eukaryotic cell or a prokaryotic cell, preferably a bacterium or a fungus.
  7. 7. A method of preparing the lipase mutant according to any of claims 1-3, wherein the method comprises the steps of: culturing the host cell of claim 6, and Optionally, the lipase mutants are isolated from the host cells or from the growth medium or supernatant.
  8. 8. Use of the lipase mutant according to any of claims 1-3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the host cell according to claim 6 or the lipase mutant obtained by the method according to claim 7 for hydrolyzing lipid compounds; preferably, the lipid compound comprises a C8-C30 aromatic carboxylic acid ester, preferably a phenyl C2-C18 carboxylic acid ester.
  9. 9. A method of hydrolyzing a lipid compound comprising the step of contacting the lipase mutant of any of claims 1-3 with a lipid compound; preferably, the lipid compound comprises a C8-C30 aromatic carboxylic acid ester, preferably a phenyl C2-C18 carboxylic acid ester.
  10. 10. The use according to claim 8 or the process according to claim 9, wherein the hydrolysis reaction system has a pH of 6-10, preferably 7-8, more preferably 7-7.5, and/or The hydrolysis temperature is 10-50deg.C, preferably 10-35deg.C, more preferably 20-30deg.C, and/or The time of the hydrolysis is 1 to 24 hours, preferably 0.5 to 8 hours, more preferably 0.5 to 1 hour.

Description

Lipase mutant and application thereof Technical Field The invention belongs to the field of genetic engineering, and in particular relates to a lipase A mutant from bacillus subtilis (Bacillus subtilis) and application thereof in hydrolyzing ester compounds under a low-temperature condition. Background The cold-suitable lipase has the characteristics of extremely high catalytic rate, heat sensitivity and the like at a lower temperature, has lower requirement on a heating program compared with the medium-temperature lipase and the high-temperature lipase, can improve the sustainability and the cost benefit of industrial production, can reduce the energy consumption, and is applied to the aspects of food processing, fabric care, environmental degradation and the like. Therefore, the development of lipases with this property is of great importance to the biotechnology industry. Lipase A derived from Bacillus subtilis belongs to the class of medium temperature lipases, is capable of promoting hydrolysis reactions of fats, has a high degree of specific catalysis on lipid-related substrates, and exhibits high activity and high turnover under mild environmental conditions, making it a candidate enzyme for efficient catalytic reactions. At present, some researches report that the gene of the enzyme is modified by protein engineering technology, and the thermal stability of the lipase A under high temperature condition is improved by introducing specific mutation or modification, so that the lipase A is applied to a process requiring continuous catalytic reaction under high temperature condition. In addition, there are also some studies reporting that a five-site combined mutant enzyme of lipase A is obtained by protein site saturation mutation, and its catalytic activity under low temperature conditions is enhanced by increasing the catalytic activity of the enzyme, although its optimal reaction temperature is not changed, wherein the catalytic efficiency under 10℃conditions is 13mM -1min-1. However, this activity is still difficult to meet in industrial applications. Therefore, there is an urgent need in the art to develop a low temperature lipase with high enzymatic activity. Disclosure of Invention In view of the deficiencies in the prior art, the present disclosure provides efficient lipase mutants to achieve efficient hydrolysis of lipid compounds at low temperatures. According to one aspect of the present disclosure, there is provided a lipase mutant, corresponding to the amino acid sequence of a wild-type lipase, having a mutation at one or more of amino acids 10, 40, 45, 47, 156, 159, wherein the wild-type lipase is a wild-type lipase a derived from bacillus subtilis (Bacillus subtilis), preferably the amino acid sequence of the wild-type lipase is as shown in SEQ ID No. 1. In some embodiments, the lipase mutant has one or more mutations in I10M, W40I, T45N, Y47R, G S and Y159A. In some embodiments, the lipase mutant has a mutation of one of I10M, W40I, T45N, Y47R, G S and Y159A. In some embodiments, the lipase mutant has I10M and W40I mutations. In some embodiments, the lipase mutant has I10M and Y47R mutations. In some embodiments, the lipase mutant has I10M and G156S mutations. In some embodiments, the lipase mutant has I10M and G159A mutations. In some embodiments, the lipase mutant has the I10M, W I and Y47R mutations. In some embodiments, the lipase mutant has the I10M, W I and G156S mutations. In some embodiments, the lipase mutant has the I10M, W I and Y159A mutations. In some embodiments, the lipase mutant has the I10M, W40I, Y R and G156S mutations. In some embodiments, the lipase mutant has the I10M, W40I, Y R and Y159A mutations. In some embodiments, the lipase mutant has the I10M, W40I, T45N, Y47R and G156S mutations. In some embodiments, the lipase mutant has the I10M, W40I, Y47R, G S and Y159A mutations. In some embodiments, the lipase mutant has the I10M, W40I, T45N, Y47R, G S and Y159A mutations. In some embodiments, the lipase mutants have the amino acid sequence shown in one or more of SEQ ID NOS: 12-28, or an amino acid sequence in which one or more of the amino acid sequences shown in SEQ ID NOS: 12-28 has been added, deleted, substituted or modified with one or more amino acids. In some embodiments, the lipase mutants have an optimal reaction temperature of 10-35 ℃,20-30 ℃, e.g., 10 ℃, 15 ℃,20 ℃,25 ℃, 30 ℃,35 ℃, or any value therebetween. In some preferred embodiments, the lipase mutants have an optimal reaction temperature of 20-30 ℃. In some embodiments, the relative activity of the lipase mutant compared to the wild-type lipase is greater than 1.0, preferably greater than 1.1, greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 2, greater than 4, greater than 6, greater than 8, greater than 10, and the like. In some embodiments, the lipase mutant has a relative activity compared to the wild-type lipase of 1.3-8.2, e.g., 1.3, 1.5, 2, 2.5, 3, 3.5, 4, 4