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Method for producing candida antarctica lipase B and specific DNA molecule used by method

A Candida Antarctica and DNA molecule technology, applied in the biological field, can solve the problems of high cost of eukaryotic expression, difficult to popularize industrial application, etc., and achieve the effects of increasing yield and simple production process

Active Publication Date: 2021-02-26
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The cost of eukaryotic expression is high, and industrial application is difficult to promote, so low-cost prokaryotic expression has become a research trend

Method used

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  • Method for producing candida antarctica lipase B and specific DNA molecule used by method
  • Method for producing candida antarctica lipase B and specific DNA molecule used by method
  • Method for producing candida antarctica lipase B and specific DNA molecule used by method

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Embodiment 1

[0067] Embodiment 1, adopt CALB fusion gene to produce Candida antarctica lipase B

[0068] 1. Construction of recombinant plasmids

[0069] 1. Optimize the codon according to the amino acid sequence of the CALB protein, and artificially synthesize the CALB fusion gene with 6 His tags at the N-terminal.

[0070] The nucleotide sequence of the CALB fusion gene is shown in SEQ ID No:3.

[0071] The CALB fusion gene encodes the CALB fusion protein shown in SEQ ID No:4.

[0072] 2. Construction of recombinant plasmid pET30a-CALB

[0073] The small DNA fragment between the restriction endonucleases NdeI and HindIII of the plasmid pET30a was replaced with the DNA molecule shown in the 4th to 978th ​​positions from the 5' end of SEQ ID NO: 3, and the other sequences were unchanged to obtain the recombinant plasmid pET30a -CALB.

[0074] The nucleotide sequence of the recombinant plasmid pET30a-CALB is shown in SEQ ID NO:8.

[0075] The recombinant plasmid pET30a-CALB expresses t...

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Abstract

The invention discloses a method for producing candida antarctica lipase B and a specific DNA molecule used by the method. The nucleotide sequence of the specific DNA molecule is shown as SEQ ID NO: 3. Experiments prove that the recombinant plasmid pET30a-CALB is used for prokaryotic expression of the CALB inclusion body, and then renaturation is performed to obtain the CALB protein with high purity (more than 95%) and high activity; the enzyme activity of CALB protein obtained from 1L culture solution is about 21252U, and the specific activity is about 253U / mg, which is far higher than that of the CALB protein prepared by the existing literature. Therefore, the method provided by the invention can improve the yield of the CALB remarkably, the production process is simple, and the method can be applied to actual production The invention has important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing Candida antarctica lipase B and the specific DNA molecule used therein. Background technique [0002] Candida antarctica Lipase B (CALB) is a lipase extracted from Candida antarctica samples, which can catalyze the synthesis of biological organic esters (such as surfactants, pharmaceutical intermediates, etc.) , hydrolysis of esters, transesterification and other types of reactions, it has strong catalytic activity, high stereoselectivity and high stability for water-soluble and water-insoluble substances, and has become one of the most widely used lipases in the world. The low-cost mass production will make it have a broader application prospect in the field of green biochemical medicine. [0003] In 1994, Uppenberg J. determined the amino acid sequence and its three-dimensional structure of CALB (UppenbergJ., Patkar S., Bergfors T. and Jones T.A....

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/70C12N1/21C07K19/00C12N9/20C12R1/19
CPCC12N9/20C12Y301/01003C12N15/70C07K2319/21
Inventor 江红邹振
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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