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CN-122012457-A - Candida antarctica lipase B mutant and application thereof

CN122012457ACN 122012457 ACN122012457 ACN 122012457ACN-122012457-A

Abstract

The invention relates to the technical field of genetic engineering and biological engineering, in particular to a candida antarctica lipase B mutant and application thereof. The candida antarctica lipase B mutant provided by the invention is obtained by the combined mutation of the 143 th site and 284 th site on an amino acid sequence shown in a wild type lipase B. Compared with wild type enzyme, the mutant of candida antarctica lipase B constructed by the invention has the advantages that the enzyme activity of the mutant is improved by 12.5 times, the protein expression quantity reaches 1.8-1.9 g/L under the scale of a 5L fermentation tank, the yield can be obviously improved, and the production cost is reduced. In addition, the candida antarctica lipase B mutant can be used for synthesizing high-added-value and non-natural substrates such as vitamin C fatty acid ester, vitamin A oleate and the like, and lays a foundation for the application of the lipase in the field of fine chemical engineering.

Inventors

  • Jiao Yinshan
  • WU WENHUI
  • Cha Yaping
  • QIAO JINFANG
  • Xu Peiyue

Assignees

  • 杭州合碳创物科技有限公司
  • 深圳合碳生物科技有限公司
  • 常德合碳生物科技有限公司
  • 合碳创物科技(连云港)有限公司

Dates

Publication Date
20260512
Application Date
20260415

Claims (9)

  1. 1. The candida antarctica lipase B mutant is characterized by being obtained by combined mutation at the 143 rd and 284 th positions of an amino acid sequence shown in SEQ ID No. 2.
  2. 2. The candida antarctica lipase B mutant of claim 1, which has the amino acid sequence shown in SEQ ID No. 4.
  3. 3. A nucleic acid molecule encoding the candida antarctica lipase B mutant of claim 1 or 2.
  4. 4. The nucleic acid molecule of claim 3, wherein the nucleotide sequence of said nucleic acid molecule is set forth in SEQ ID No. 3.
  5. 5. A recombinant vector comprising the nucleic acid molecule of claim 3 or 4.
  6. 6. A recombinant cell comprising the nucleic acid molecule of claim 3 or 4 or the recombinant vector of claim 5.
  7. 7. The recombinant cell of claim 6, wherein the recombinant cell is pichia pastoris Pichia pastoris GS115,115.
  8. 8. Use of a candida antarctica lipase B mutant according to claim 1 or 2, a nucleic acid molecule according to claim 3 or 4, a recombinant vector according to claim 5, a recombinant cell according to claim 6 or 7 for the preparation of vitamin C fatty acid esters.
  9. 9. Use of a candida antarctica lipase B mutant according to claim 1 or 2, a nucleic acid molecule according to claim 3 or 4, a recombinant vector according to claim 5, a recombinant cell according to claim 6 or 7 for the preparation of vitamin a oleate.

Description

Candida antarctica lipase B mutant and application thereof Technical Field The invention relates to the technical field of genetic engineering and biological engineering, in particular to a candida antarctica lipase B mutant and application thereof. Background Lipase (EC 3.1.1.3) is an important industrial biocatalyst, and is widely applied to the fields of food processing, medical synthesis, biofuel production, fine chemical manufacturing and the like because of the advantages of high substrate selectivity, mild reaction conditions, environmental friendliness and the like. Among them, lipase B (CALB) derived from Candida antarctica (CANDIDA ANTARCTICA) has become one of the most widely used lipases commercially due to its excellent catalytic performance and high stability. However, although CALB has been commercially supplied, its large-scale industrial application still has many problems. First, the wild-type CALB has a low expression level in a natural host, resulting in high downstream isolation and purification costs. Secondly, the wild type CALB in a free state has poor stability in a non-aqueous phase reaction system, has limited catalytic activity and conversion efficiency on a non-natural substrate, and is difficult to meet the synthesis requirement of high added value products. Currently, the prior art has attempted to engineer CALBs to improve their performance by protein engineering means. Patent document CN119709695A discloses a candida antarctica lipase B mutant with high transesterification activity and application thereof. The candida antarctica lipase B mutant with high transesterification activity is obtained by carrying out mutation on 11、13、15、18、24、25、58、74、88、96、98、99、126、139、141、144、163、168、189、206、208、242、243、244、249、250、263、278、281、282、285、286、301、305、307 th site on the basis of wild candida antarctica lipase B gene sequence. The candida antarctica lipase B mutant with high transesterification activity has higher fat hydrolysis activity and transesterification activity than the original enzyme, can be used for preparing biodiesel by an enzyme method, and has potential industrial application value. Patent document CN120442595A discloses a candida antarctica lipase B mutant and application thereof. The mutant is characterized in that based on a wild candida antarctica lipase B gene sequence, 146 th alanine is replaced by glycine, 207 th glycine is replaced by alanine, 223 rd aspartic acid is replaced by glycine, and 278 th leucine is replaced by methionine. Compared with a wild type, the candida antarctica lipase B mutant has higher enzyme activity and thermal stability, can be used for synthesizing chlorogenic acid derivatives, and has good industrial application prospect. However, the mutant design of candida antarctica lipase B at present is mostly aimed at the site of the active pocket cover region to mutate, mainly relies on high-throughput screening, and the promotion amplitude of the catalytic activity is usually not high, so that the requirement cannot be met. Therefore, research and development of a lipase mutant which has significantly higher catalytic activity than the wild type and is compatible with a high-efficiency expression system is still a problem to be solved. Disclosure of Invention In order to solve the defects of the prior art, the invention provides a candida antarctica lipase B mutant with better enzyme activity effect and high expression efficiency, which is used for solving the problems of poor catalytic activity and low catalytic efficiency of the existing wild candida antarctica lipase B during preparation. Meanwhile, the candida antarctica lipase B mutant provided by the invention can be also applied to the preparation of non-natural substrates such as vitamin C derivatives, vitamin A derivatives and the like, and meets the synthesis requirement of high-added-value products. In order to achieve the above purpose, the present invention discloses the following technical solutions: The nucleotide sequence of the wild Candida Antarctica Lipase B (CALB) derived from the lipase B (CANDIDA ANTARCTICA) of candida antarctica is shown as SEQ ID NO.1, and the amino acid sequence coded by the gene is shown as SEQ ID NO. 2. The invention provides a candida antarctica lipase B mutant, which is obtained by combining mutation at the 143 th site and 284 th site of an amino acid sequence shown in SEQ ID NO. 2. Further, the candida antarctica lipase B mutant is obtained by mutating proline at 143 rd position on the amino acid sequence of wild candida antarctica lipase B to leucine and mutating alanine at 284 th position to glutamic acid. Further, the amino acid sequence of the candida antarctica lipase B mutant is shown as SEQ ID NO. 4. The invention also provides a nucleic acid molecule which codes for the candida antarctica lipase B mutant. Further, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 3. In addition, the invention also provides a re