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Omega transaminase as well as mutant, recombinant plasmid, genetically engineered bacterium and application thereof

A technology of genetically engineered bacteria and recombinant plasmids, applied in the fields of genetic engineering, application, transferase, etc., can solve the problems of low conversion efficiency, low catalytic activity of 1-propiophenone, low conversion rate, etc., and achieve excellent catalytic activity, reaction Mild conditions and high stereoselectivity

Active Publication Date: 2021-03-12
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently reported natural S-type transaminases with catalytic activity for this substrate, such as OATA and PDTA, have very low catalytic activity for 1-propiophenone
Although the catalytic activity of the modified mutant strain to the substrate has been improved, the conversion efficiency is not high. For example, when the modified PDTA catalyzes 4.2 mM 1-propiophenone, the reaction time is 3 days, and the conversion rate is only 3 days. 79%, although its e.e. value is greater than 99%

Method used

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  • Omega transaminase as well as mutant, recombinant plasmid, genetically engineered bacterium and application thereof
  • Omega transaminase as well as mutant, recombinant plasmid, genetically engineered bacterium and application thereof
  • Omega transaminase as well as mutant, recombinant plasmid, genetically engineered bacterium and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: Amplification of the omega transaminase gene bpta derived from Burkholderia tumoriformis

[0046] The ω transaminase gene bpta of the present invention is derived from Burkholderia phymatum STM815, the ω transaminase BPTA of the present invention belongs to type I PLP-dependent enzyme, and the nucleotide of the ω transaminase gene bpta encoding the ω transaminase BPTA The sequence is shown as SEQ ID No: 1, and the size is 1437bp.

[0047] Using the Shanghai Sangon Biotech Gram-negative Bacteria Genomic DNA Mini-Prep Kit, and in strict accordance with the instructions on the operation of Burkholderia tumoriform DNA samples were purified to obtain Burkholderia tumoriformum (Burkholderiaphymatum STM815 ) whole genome.

[0048] According to the sequence analysis results, the primers for amplifying the omega transaminase gene bpta were designed:

[0049] bptaF:5'-ggaattccatatgagtttcaggacagaagaaatcgc-3'

[0050] bptaR:5'-cccaagcttacgcaagcccgagttgttgt-3'

[0051...

Embodiment 2

[0053] Example 2: BPTA W82A , BPTA W82A / M78F , BPTA W82A / I284F , BPTA W82A / T440Q , BPTA M78F / W82A / I284F / T440Q Mutant strain construction

[0054] The recombinant plasmid containing the mutant ω transaminase gene bpta-M78F / W82A / I284F / T440Q used in the present invention is constructed in an iterative manner: first, the recombinant plasmid bpta-pET-28a is used as a template, and the following primer pair ① As a primer, the recombinant plasmid bpta-W82A-pET-28a was obtained; then, the recombinant plasmid bpta-W82A-pET-28a was used as a template, and the following primer pair ② was used as a primer to obtain the recombinant plasmid bpta-W82A / M78F-pET-28a; Then use the recombinant plasmid bpta-W82A / M78F-pET-28a as a template, and the following primer pair ③ as primers to obtain the recombinant plasmid bpta-M78F / W82A / I284F-pET-28a; finally use the recombinant plasmid bpta-M78F / W82A / I284F -pET-28a was used as a template, and the following primer pair ④ was used as a primer to obt...

Embodiment 3

[0066] Embodiment 3: Contain the construction of the recombinant genetic engineering strain of transaminase gene bpta and mutant gene thereof

[0067] Escherichia coli BL21 (DE3) was inoculated in LB medium, and BL21 competent cells were prepared according to conventional methods in the art.

[0068] Transfer the recombinant plasmid obtained in the above-mentioned Example 2 into E. coli BL21 competent cells, evenly spread it on a kanamycin-resistant plate, culture it upside down at 37°C for 16 hours, pick a single colony and verify that it is a positive clone, and pass through LB medium Cultivate and obtain recombinant genetically engineered strains.

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Abstract

The invention provides omega transaminase as well as a mutant, a recombinant plasmid, a genetically engineered bacterium and application thereof. The amino acid sequence of the omega transaminase is shown as SEQ ID No: 2, wherein the omega transaminase mutant is obtained by mutation of the amino acid sequence of the omega transaminase and has at least 90% of homology with the SEQ ID No: 2. According to the invention, the omega transaminase gene is extracted from Burkholderia phymatum, the heterologous expression of the omega transaminase gene is realized, and the generated omega transaminase strain shows excellent catalytic activity on a series of aryl alkyl substrates. The omega transaminase is mutated to obtain a series of omega transaminase mutants, and the omega transaminase mutants are coupled with different enzymes to be applied to the production of asymmetric synthesis of (S)-1-amphetamine and other chiral amine products by a biological enzyme method, so that the method has thecharacteristics of economy, environmental protection and high chiral selectivity; thus, the invention provides a potential choice for industrial application of transaminase.

Description

technical field [0001] The invention relates to the technical field of bioengineering, and more specifically relates to an omega transaminase and its mutant, a recombinant plasmid, a genetically engineered bacterium and applications thereof. Background technique [0002] At present, 95% of the drugs in the world are chiral drugs, and 70% of the chiral drugs need to use chiral amines as chiral precursors to synthesize the next target product. The mainstream methods for synthesizing chiral amines are still chemical methods or a combination of biochemical methods. However, in recent years, the country's supervision of chemical synthesis has become more stringent. Correspondingly, the bio-manufacturing industry is being vigorously supported. This is due to the danger of the harsh synthesis conditions of the chemical method itself and the serious environmental pollution caused by the subsequent sewage discharge. consistent situation. Especially for the synthesis of chiral drugs...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/1096C12Y206/01C12N15/70C12P13/001
Inventor 王华磊魏东芝谢有余
Owner EAST CHINA UNIV OF SCI & TECH
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