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Transaminase mutant, enzyme preparation, recombinant vector, recombinant cell, preparation method of recombinant cell and application of transaminase mutant, enzyme preparation, recombinant vector and recombinant cell

A technology of recombinant cells and transaminases, applied in the fields of recombinant cells and their preparation, recombinant vectors, transaminase mutants, and enzyme preparations, can solve the problems of low activity of transaminases

Active Publication Date: 2020-05-08
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

However, the existing transaminases have low enzymatic activity and cannot meet the actual needs

Method used

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  • Transaminase mutant, enzyme preparation, recombinant vector, recombinant cell, preparation method of recombinant cell and application of transaminase mutant, enzyme preparation, recombinant vector and recombinant cell
  • Transaminase mutant, enzyme preparation, recombinant vector, recombinant cell, preparation method of recombinant cell and application of transaminase mutant, enzyme preparation, recombinant vector and recombinant cell
  • Transaminase mutant, enzyme preparation, recombinant vector, recombinant cell, preparation method of recombinant cell and application of transaminase mutant, enzyme preparation, recombinant vector and recombinant cell

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preparation example Construction

[0060] The method for preparing recombinant cells in the above embodiment includes the following steps S110-S130:

[0061] S110. Perform error-prone PCR amplification on the coding sequence of the wild-type transaminase in the above embodiment, digest it and connect it to an empty vector to obtain a transaminase mutant library plasmid.

[0062] Wherein, in the step of performing error-prone PCR amplification on the coding sequence of the wild-type transaminase in the above embodiment, the error-prone PCR amplification is carried out by using a pair of primers whose sequences are shown in SEQ ID No.3-SEQ ID No.4. Specifically, the sequence shown in SEQ ID No.3 is: 5'-TACCAGACGACGA catTTTGACGGCCTGG -3', wherein, the underlined base is the recognition site of restriction endonuclease Nde I. The sequence shown in SEQ ID No.4 is: 5'-CTTCCATAGCCAA ggatcc TTCAAGACTTTTCTTCAACGA-3', wherein the underlined base is the recognition site of restriction endonuclease BamH III.

[0063] ...

Embodiment 1

[0080] Cloning of the coding sequence of wild transaminase WP_0532423951.1

[0081] The coding sequence of wild-type transaminase WP_0532423951.1 was codon-optimized with Escherichia coli as the host, and was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., using sequences such as those shown in SEQ ID No.3 to SEQ ID No.4 The primer pair PCR amplifies the coding sequence of the wild-type aminotransferase, and the PCR amplification uses PrimeSTAR Max polymerase from Takara Company. The reaction conditions of PCR were: 98°C, 3min, 1 cycle; 98°C, 10sec, 55°C, 10sec, 72°C, 15sec, 30 cycles; 72°C, 5min, 1 cycle. Wherein, the amino acid sequence of the wild-type transaminase WP_0532423951.1 is shown in SEQ ID No.1; the nucleotide sequence of the wild-type transaminase is shown in SEQ ID No.2.

[0082] After the reaction, the PCR product was detected by agarose gel electrophoresis with a mass percent content of 1.2%, and a 1.0 kb band was obtained, the length of which was ...

Embodiment 2

[0084] Expression, purification and activity determination of wild transaminase WP_0532423951.1

[0085] The engineering bacteria containing the pET28a-WP_0532423951.1 recombinant plasmid in the glycerol tube were inoculated at 1% by volume into a 4mL LB medium test tube containing 100ug / mL kanamycin, and cultured at 37°C and 220rpm for 11h. Transfer 4 mL of the cultured bacteria solution to 1L LB liquid medium containing 50ug / mL kanamycin, and culture it in a shaker flask at 37°C and 220rpm for about 2.5h, so that the OD 600 When it reached about 0.8, 0.1 mM IPTG inducer was added, and cultured at 25° C. and 200 rpm for 12 hours. The Escherichia coli suspension harvested after induction was separated from solid and liquid, and after centrifugation to collect the bacteria, the bacteria liquid was resuspended and broken up and centrifuged again, and the supernatant was subjected to a step of Ni-NTA affinity chromatography to obtain a purity of 90 % above wild-type transaminase...

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Abstract

The invention relates to a transaminase mutant, an enzyme preparation, a recombinant vector, a recombinant cell, a preparation method of the recombinant cell and application of the transaminase mutant, the enzyme preparation, the recombinant vector and the recombinant cell. An amino acid sequence of the transaminase mutant is obtained through mutation of an amino acid sequence of wild transaminase, the amino acid sequence of the wild transaminase is represented by SEQ ID No.1, and mutated amino acid sites include at least one of an amino acid site 226, an amino acid site 281, an amino acid site 312 and an amino acid site 406. The transaminase mutant is relatively high in enzyme activity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a transaminase mutant, an enzyme preparation, a recombinant vector, a recombinant cell and a preparation method and application thereof. Background technique [0002] Transaminase (Transaminase, TA, EC 2.6.1X), also known as aminotransferase (Aminotransferase), is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that can catalyze the conversion of amino groups between ketoacids and amino acids. reversible transfer reaction. Transaminases are ubiquitous in nature and play a key role in nitrogen metabolism. According to the different substrates and products in the catalytic reaction, transaminases can be divided into α-transaminases that only catalyze α-amino and α-keto acids and ω-transaminases that can catalyze amino acids with remote carboxylic acid groups. Because ω-transaminase can catalyze the amino transfer between ketoacids, aldehydes and ketones, and has the advantages of hig...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/10C12N15/70C12N1/21C12R1/19
CPCC12N9/1096C12N15/1031C12N15/70C12Y206/01C12Q2531/113
Inventor 张丽星张艺凡郭天杰马富强杨广宇
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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