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Amine dehydrogenase mutant with improved thermal stability, construction method and application of genetic engineering bacteria

An amine dehydrogenase, thermal stability technology, applied in the direction of genetic engineering, application, plant genetic improvement, etc., can solve problems such as poor thermal stability of amine dehydrogenase, achieve good thermal stability, good application prospects, excellent stereo selective effect

Active Publication Date: 2020-02-28
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 amine dehydrogenases, thereby providing a genetically engineered strain of amine dehydrogenase mutants and amine dehydrogenase mutants with improved thermostability The construction method of and the application of amine dehydrogenase mutants in the preparation of chiral amines

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  • Amine dehydrogenase mutant with improved thermal stability, construction method and application of genetic engineering bacteria

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Embodiment 1

[0048] This embodiment provides an amine dehydrogenase mutant with improved thermostability, wherein the amine dehydrogenase is a wild-type amine dehydrogenase derived from Thermosediminibacter oceani, named ANDD-TDO protein, and encodes the ANDD-TDO protein The nucleic acid sequence is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2.

[0049] The amine dehydrogenase mutant with improved thermostability provided in this example comprises: the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or more amino acids to form the amino acid sequence shown in SEQ ID NO.2 The amino acid sequence (i.e. ANDD-TDO protein) has the same function as a derivative protein, or the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or more amino acids to form the amino acid sequence shown in the SEQ ID NO.2 Derivative proteins with at least 90% homology in amino acid sequence (ie ANDD-TDO protein).

[0050] Specifically, s...

Embodiment 2

[0055] This embodiment provides a method for constructing an amine dehydrogenase mutant with improved thermostability, comprising the following steps:

[0056] 1. Cloning of wild-type amine dehydrogenase ANDD-TDO gene

[0057] The wild-type amine dehydrogenase gene is codon-optimized with Escherichia coli as the host cell, and the optimized ANDD-TDO gene is obtained. Its nucleic acid sequence is SEQ ID NO.1, and the expressed amino acid sequence is SEQ ID NO.2; ID NO.1 is used as the target gene, using the upstream amplification primer SEQ ID NO.18 and the downstream amplification primer SEQ ID NO.19 to amplify the target gene;

[0058] The nucleic acid sequence of SEQ ID NO.18 is:

[0059] 5'-ACTGCT CATATG GAAAAAATCCGTGTTATCATC-3' (the underline is the recognition site of restriction endonuclease NdeI);

[0060] The nucleic acid sequence of SEQ ID NO.19 is:

[0061] 5'-TCAGCT CTCGAG TTAAGCGTTGTTAACACCG-3' (the underline is the recognition site of restriction endonuclea...

Embodiment 3

[0127] This example provides a gene encoding the amine dehydrogenase mutant with improved thermostability as described in Example 1:

[0128] (1) The nucleic acid sequence encoding the amine dehydrogenase mutant whose mutation site is A35D is SEQ ID NO.28;

[0129] (2) The nucleic acid sequence encoding the amine dehydrogenase mutant whose mutation site is L53R is SEQ ID NO.29;

[0130] (3) The nucleic acid sequence encoding the amine dehydrogenase mutant whose mutation site is S210A is SEQ ID NO.30;

[0131] (4) The nucleic acid sequence encoding the amine dehydrogenase mutant whose mutation site is T321P is SEQ ID NO.31;

[0132] (5) The nucleic acid sequence encoding the amine dehydrogenase mutant whose mutation site is A35D / L53R is SEQ ID NO.32;

[0133] (6) The nucleic acid sequence encoding the amine dehydrogenase mutant whose mutation site is A35D / S210A is SEQ ID NO.33;

[0134] (7) The nucleic acid sequence encoding the amine dehydrogenase mutant whose mutation site...

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Abstract

The invention belongs to the technical field of biology, in particular to an amine dehydrogenase mutant with improved thermal stability, a construction method and an application of genetic engineeringbacteria. The amine dehydrogenase mutant comprises 4 single-site mutants and 11 combined mutants, and compared with wild-type amine dehydrogenase, the single-site mutants and the combined mutants have longer half-life period at 42 DEG C; in particular, the combined mutants show a superposition effect of thermal stability of the single-site mutants, and the half-life period is about 5 times that of the wild-type amine dehydrogenase. The amine dehydrogenase mutant obtained by the construction method has better thermal stability, shows excellent stereoselectivity, regioselectivity and catalyticactivity when being catalyzed for synthesis of chiral amine at higher temperature, and has a better application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of an amine dehydrogenase mutant with improved thermal stability and genetically engineered bacteria. Background technique [0002] Chiral amines are a very important class of amine compounds, which are widely found in medicine, pesticides, synthetic intermediates, natural products and biologically active compounds, especially in the field of chiral drugs. Currently, about 40% of optical The active drug contains a chiral amine structure. The different enantiomers of chiral amines have very similar physical properties, but the difference in their stereostructure will make different chiral enantiomers have different biological activities, and thus the pathways of metabolism, transformation or activation in vivo Different, the physiological activity and toxic effect shown when acting on living organisms are different. Therefore, it is of gr...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12P13/00
CPCC12N9/0014C12P13/001C12Y104/99003
Inventor 马富强郭天杰张艺凡杨广宇
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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