Amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell, preparation methods therefor and application of amine dehydrogenase mutant

A technology of amine dehydrogenase and recombinant cells, which is applied in the biological field and can solve the problems of inability to meet the needs of practical applications and low catalytic efficiency

Active Publication Date: 2020-04-03
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the catalytic efficiency of existing amine dehydrogenases is low, which cannot meet the needs of practical applications.

Method used

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  • Amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell, preparation methods therefor and application of amine dehydrogenase mutant
  • Amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell, preparation methods therefor and application of amine dehydrogenase mutant
  • Amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell, preparation methods therefor and application of amine dehydrogenase mutant

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preparation example Construction

[0055] The method for preparing recombinant cells in the above embodiment includes the following steps S110-S130:

[0056] S110: Perform error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase in the above embodiment, and ligate it into an empty vector after digestion to obtain a plasmid of an amine dehydrogenase mutant library.

[0057] Wherein, in the step of performing error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase in the above embodiment, the error-prone PCR amplification is carried out by using the primer pair whose sequence is as shown in SEQ ID No.3-SEQ ID No.4. increase. Specifically, the sequence shown in SEQ ID No.3 is: 5'-ACTGCTCATATGGAAAAAATCCGTGTTATCATC-3'; the sequence shown in SEQ ID No.4 is: 5'-TCA GCTCTCGAGTTAAGCGTTGTTAACACCG-3'.

[0058] Among them, the error-prone PCR reaction system is: 0.05U / μL DreamTaq TM , 250 μM of dATP, 250 μM of dGTP, 1050 μM of dCTP, 1050 μM of dTTP, 0.4 μ...

Embodiment 1

[0073] Cloning of the coding sequence of wild-type amine dehydrogenase

[0074] The coding sequence of wild-type amine dehydrogenase (GenBank: CP002131.1) was codon-optimized using Escherichia coli as the host, and was synthesized by Suzhou Jinweizhi Co., Ltd. The sequence used is shown in SEQ ID No.5~SEQ ID No.6 The primer pair used for PCR amplification of the coding sequence of wild-type amine dehydrogenase, PCR amplification using Toyobo (Shanghai) Biotechnology Co., Ltd. KOD high-fidelity polymerase, the amplification conditions are: 95 ° C, 2 min; then 56 ° C, 20sec, 72°C, 90sec, a total of 30 cycles; the last 72°C, 10min. Wherein, the amino acid sequence of the wild-type amine dehydrogenase is shown in SEQ ID No.1; the nucleotide sequence of the wild-type amine dehydrogenase is shown in SEQ ID No.2.

[0075] After the reaction, the PCR product was detected by agarose gel electrophoresis with a mass percent content of 1.5%, and a 1.0 kb band was obtained, the length of ...

Embodiment 2

[0077] Expression, purification and activity determination of amine dehydrogenase

[0078] The engineered bacteria containing the pET28a-ANDD-TDO recombinant plasmid in the glycerol tube were inoculated at 1% by volume into a 4 mL LB medium test tube containing 100 μg / mL kanamycin, and cultured at 37°C and 220rpm for 12h. Transfer all the cultured bacterial liquid to 1L LB medium shake flask containing 50 μg / mL kanamycin, culture at 37°C and 220rpm for about 2.5h, and make the OD 600 When it reached about 0.9, 0.1 mM IPTG inducer was added, and cultured at 25° C. and 200 rpm for 16 hours. The Escherichia coli suspension harvested after induction is ultrasonically disrupted, and then subjected to one-step Ni-NTA affinity chromatography to obtain wild-type amine dehydrogenase with a purity of more than 95%. The activity of wild-type amine dehydrogenase was determined, and the result was: K of wild-type amine dehydrogenase M A value of 19.22 μM, the K of wild-type amine dehydro...

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Abstract

The invention relates to an amine dehydrogenase mutant, an enzyme preparation, a recombinant vector, a recombinant cell, preparation methods therefor and an application of the amine dehydrogenase mutant. The amine dehydrogenase mutant is obtained after a 104 glutamic acid of wild type amine dehydrogenase is mutated into alanine, or obtained after a 137 aspartic acid of the wild type amine dehydrogenase is mutated into proline, or obtained after a 174 valine of the wild type amine dehydrogenase is mutated into cysteine, or obtained after a 197 valine of the wild type amine dehydrogenase is mutated into aspartic acid. The amine dehydrogenase is relatively high in catalysis efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an amine dehydrogenase mutant, an enzyme preparation, a recombinant vector, a recombinant cell and a preparation method and application thereof. Background technique [0002] Chiral amines are widely used in organic synthesis, spices, medicine, etc., and are important intermediates. The difference in the three-dimensional structure of different enantiomers of chiral amines makes different chiral enantiomers have different activities and exhibit different physiological activities and toxic effects when they act on living organisms. Therefore, chiral amines have gradually become important indicators for evaluating drug safety and efficacy. [0003] At present, the methods for preparing chiral amines are mainly chemical catalysts and biological enzyme catalysis. Enzyme-catalyzed selective synthesis of chiral drugs has significant technical advantages. Among them, amine dehydrogenase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P13/00
CPCC12N9/0014C12N15/70C12P13/001C12Y104/99003
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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