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Alpha-aminopherase and mutant and application of alpha-aminopherase and mutant to asymmetric synthesis of L- glufosinate-ammonium

The technology of transaminase and mutant is applied in the application field of asymmetric synthesis of L-glufosinate-ammonium, which can solve the problems of less enzyme source, low enzyme activity, low conversion rate and the like, and achieves the effect of good application prospect.

Active Publication Date: 2019-04-12
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problems of the existing transaminase process are that there are few enzyme sources, low enzyme activity, low conversion rate, and expensive amino donors.

Method used

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  • Alpha-aminopherase and mutant and application of alpha-aminopherase and mutant to asymmetric synthesis of L- glufosinate-ammonium
  • Alpha-aminopherase and mutant and application of alpha-aminopherase and mutant to asymmetric synthesis of L- glufosinate-ammonium
  • Alpha-aminopherase and mutant and application of alpha-aminopherase and mutant to asymmetric synthesis of L- glufosinate-ammonium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Screening of novel transaminase

[0032] 1. Enzyme screening and synthesis

[0033] Through the analysis of the catalytic pocket and key residues of the reported transaminases, three enzymes were mined from the enzyme database, and their sources were Ashbya gossypii (GenBank number NP_987025.1), Azoarcus sp. (GenBank number WP_011766092.1) and Acinetobacter sp. (GenBank number WP_104500271.1), and named AgTA, AsTA and AcTA. Codon optimization was carried out according to the codon preference of Escherichia coli, and three selected nucleotide sequences were synthesized by the method of total synthesis through the routine operation of genetic engineering, such as SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO. 6; the amino acid sequence encoding the enzyme is shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5. Add 6×his-tag tags at the end of the nucleic acid sequence, add restriction sites Xba I and Xho I at both ends, clone the gene into the Xba I and Xho I sites...

Embodiment 2

[0041] Example 2: Construction and screening of AcTA single point mutants

[0042] 1. Mutant construction

[0043] Select the recombinant bacterium with the highest enzyme activity, according to the AcTA parent (the parent AcTA comes from Acinetobacter sp., GenBank numbering is WP_104500271.1) sequence (the amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6 Shown) to design mutation primers for site-directed mutations, using rapid PCR technology, using the recombinant vector pET28b / AcTA as a template, and introducing a single mutation at position 87, the primers are:

[0044] Forward primer GGGTAAAATGGCA NNK GCCATGATG (the underline is the mutated base)

[0045] reverse primer CGTACATCATGGC NNK TGCCATTTTAC (the underline is the mutant base)

[0046] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase ...

Embodiment 3

[0061] Example 3: Construction and screening of AcTA two-site mutants

[0062] According to the single mutant AcTA1 sequence constructed in Example 2, the mutation primers for site-directed mutation were designed, using the rapid PCR technology, using the recombinant vector pET28b / AcTA1 as a template, and introducing a single mutation at the 144th position, the primers were:

[0063] Forward primer CGATGGTATG NNK CTGGTGAAAG (the underline is the mutated base)

[0064] reverse primer CTTCTTTCACCAG NNK CATACCATC (the underline is the mutated base)

[0065] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.

[0066] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 50°C for 15s, 59°C for 6.5min) for 30 cycles; 72°C for 5min.

[0067] The PCR product was transformed into E.coli BL21 (DE3) competent cells, ...

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Abstract

The invention discloses an application of novel aminopherase and a high-vitality mutant thereof to preparation of L-glufosinate-ammonium. The amino acid sequence of the aminopherase is as shown in SEQID:5, and the mutant of the aminopherase is prepared through performing single-site mutation or multi-site mutation on one or a plurality of 124th, 144th, 237th, 250th and 328th in the amino acid sequence as shown in the SEQ ID:5. The high-efficiency expression of vitality mutant genes of high-conversion rate aminopherase can be realized, and the highest enzyme activity is 840U / mg. The highest optimum reaction temperature of the aminopherase mutant can reach 67 DEG C, and during asymmetric synthesis of the L-glufosinate-ammonium by catalyzing 800mM of glufosinate-ammonium precursor ketone atthe temperature through inorganic amine and n-butylamine, the conversion rate is as high as 100%. The AcTA mutant solves the technical difficult problems that few enzyme sources exist, the enzyme activity is low, substrate tolerance is bad and amino donors are expensive in a current L-glufosinate-ammonium preparation technology from the aminopherase, and has better application prospects.

Description

(1) Technical field [0001] The invention relates to a novel transaminase and its high-activity mutant, and its application in the asymmetric synthesis of L-glufosinate-ammonium. (2) Background technology [0002] Glufosinate-ammonium, 4-[hydroxy(methyl)phosphono]-DL-homoalanine (phosphinothricin, PPT), is a broad-spectrum, contact-killing, destructive, non-residual herbicide with high efficiency, low toxicity, Easy to degrade and so on. PPT is a racemic mixture containing two optical isomers, but only the L-form is phytotoxic. Since the herbicide glyphosate has withdrawn from the pesticide market, the preparation of optically pure L-PPT has gained unprecedented market opportunities. [0003] The preparation of optically pure L-PPT mainly includes chemical synthesis, chiral resolution and asymmetric synthesis. The chemical synthesis method has many process steps, and the required synthetic reagents are expensive, resulting in high cost investment, and some reagents have ce...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P13/04
CPCC12N9/1096C12P13/04C12Y206/01
Inventor 薛亚平贾东旭郑裕国刘子健徐海鹏李军良金利群柳志强程峰
Owner ZHEJIANG UNIV OF TECH
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