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Transaminase mutant, construction method and application thereof

A technology of mutants and transaminases, applied in the field of transaminase mutants and their construction, can solve problems such as poor thermal stability of transaminases, and achieve excellent stereoselectivity, simple culture conditions, and good thermal stability

Active Publication Date: 2020-04-10
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Therefore, the technical problem to be solved in the present invention is to overcome the defects of poor thermostability of existing transaminases, thereby providing a transaminase mutant with good thermostability, a method for constructing a transaminase mutant, and a transaminase mutant in catalyzing the synthesis of chiral amines. application in

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  • Transaminase mutant, construction method and application thereof
  • Transaminase mutant, construction method and application thereof

Examples

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Embodiment 1

[0037]This embodiment provides a transaminase mutant, which is a molecularly engineered derivative protein based on the amino acid sequence of the wild-type transaminase (WP_0532423951.1), wherein the wild-type transaminase (WP_0532423951.1) is derived from Aspartate aminotransferase (aspartate aminotransferase is a member of the omega transaminase family) gene of the Chromobacter sp.DMS1 strain is expressed after codon optimization, and the nucleic acid sequence encoding the protein is SEQ ID NO .1, the amino acid sequence is SEQ ID NO.2.

[0038] The transaminase mutants provided in this embodiment include:

[0039] (a1) a derivative protein having the same function as the protein shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; or,

[0040] (a2) A derivative protein having at least 92% homology with the protein shown in SEQ ID NO.2 by replacing one or more positions of the amino acid sequence ...

Embodiment 2

[0049] The present embodiment provides a gene encoding the transaminase mutant as described in Example 1:

[0050] (1) The nucleic acid sequence encoding the transaminase mutant whose mutation site is A259F is SEQ ID NO.3;

[0051] (2) The nucleic acid sequence encoding the transaminase mutant whose mutation site is G431T is SEQ ID NO.4;

[0052] (3) The nucleic acid sequence encoding the transaminase mutant whose mutation site is E264A is SEQ ID NO.5;

[0053] (4) The nucleic acid sequence encoding the transaminase mutant whose mutation site is A269E is SEQ ID NO.6;

[0054] (5) The nucleic acid sequence encoding the transaminase mutant whose mutation site is S381E is SEQ ID NO.7;

[0055] (6) The nucleic acid sequence encoding the transaminase mutant whose mutation site is G431T / E264A is SEQ ID NO.8;

[0056] (7) The nucleic acid sequence encoding the transaminase mutant whose mutation site is G431T / A269E is SEQ ID NO.9;

[0057] (8) The nucleic acid sequence encoding th...

Embodiment 3

[0059] This example provides a recombinant plasmid comprising the gene provided in Example 2, which is a plasmid vector that can be used to integrate the transaminase gene into a host cell for stable expression, such as pET28a, pUC18, pUC19, pET15b, etc.; the host cell can be selected Gram-invisible bacteria (such as Escherichia coli), Gram-positive bacteria (such as Bacillus subtilis), fungi (such as Aspergillus), yeasts (such as Saccharomyces cerevisiae), actinomycetes (such as Streptomyces), etc., can be used to express transaminase mutant proteins.

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Abstract

Belonging to the technical field of biology, the invention particularly relates to a transaminase mutant, a construction method and application thereof. The transaminase mutant provided by the invention comprises a variety of mutants subjected to single-point mutation and combined mutation at the 259th site, 431st site, 264th site, 269th site and 381st site in an amino acid sequence of wild transaminase (WP_0532423951.1), and compared with the wild type transaminase (WP_0532423951.1), the mutant has a longer half-life period at 55DEG C, the half-life period of an optimal combined mutant is about 5 times that of the wild type transaminase (WP_0532423951.1), the thermal stability is better, and the mutant is suitable for catalytic synthesis of chiral amine at high temperature. The transaminase mutant obtained by the construction method provided by the invention shows excellent stereoselectivity and catalytic activity when used for catalytic synthesis of chiral amine at high temperature,and has good application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a transaminase mutant and its construction method and application. Background technique [0002] Chiral amines widely exist in nature and are important intermediates for the synthesis of natural products and chiral drugs. In-depth research on the asymmetric synthesis of chiral amines has great economic benefits and application value. Taking diabetes as an example, the main component of sitagliptin, an anti-diabetic oral drug Januvia, is R-type amine. In the process of synthesizing sitagliptin, the process of using transaminase to catalyze the transamination reaction to generate the key intermediate of sitagliptin is Reversible, by changing the catalytic reaction conditions to promote the reaction balance between the amino group and the ketone group to shift to the chiral amine generation direction, the synthesis efficiency of the chiral amine can be improved, and the theo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P13/00
CPCC12N9/1096C12P13/001C12Y206/01
Inventor 马富强张艺凡郭天杰杨广宇
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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