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α-Transaminases and mutants and their application in asymmetric synthesis of l-glufosinate

A mutant and transaminase technology, which is applied in the application field of asymmetric synthesis of L-glufosinate, can solve the problems of low product conversion rate, low enzyme activity, few enzyme sources, etc., and achieves the effect of good application prospect.

Active Publication Date: 2020-06-19
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problems existing in the existing transaminase process are few enzyme sources, low enzyme activity, poor substrate tolerance, and low product conversion rate

Method used

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  • α-Transaminases and mutants and their application in asymmetric synthesis of l-glufosinate
  • α-Transaminases and mutants and their application in asymmetric synthesis of l-glufosinate
  • α-Transaminases and mutants and their application in asymmetric synthesis of l-glufosinate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Screening of novel transaminase

[0037] 1. Enzyme screening and synthesis

[0038] Through the analysis of the catalytic pocket and key residues of reported α-transaminases, three enzymes were obtained from the enzyme database, and their sources were Neisseria meningitidis serogroup B (GenBank number WP_014582472. ) and Rhizopus microsporus (GenBank accession number XP_023463024.1), and named NsTA, BiTA and RmTA. 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 shown; 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 corr...

Embodiment 2

[0047] Example 2: Construction and screening of NsTA single point mutants

[0048] 1. Mutant construction

[0049] Select the recombinant bacterium with the highest enzyme activity, according to the NsTA parent (the parent NsTA comes from Neisseriameningitidis serogroup B, GenBank numbering is WP_014582472.1) sequence (amino acid sequence is shown in SEQ ID NO.1, nucleotide sequence is shown in SEQ ID NO.2 Shown) to design mutation primers for site-directed mutations, using rapid PCR technology, using the recombinant vector pET28b / NsTA as a template, and introducing a single mutation at position 87, the primers are:

[0050] Forward primer GGGCGAGGCG NNK ATTGTTG (the underline is the mutant base)

[0051] reverse primer CAACAACAAT NNK CGCCTCGC (the underline is the mutated base)

[0052] 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, ad...

Embodiment 3

[0066] Example 3: Construction and screening of NsTA two-site mutants

[0067] According to the single mutant NsTA1 sequence constructed in Example 2, the mutation primers for site-directed mutation were designed, using the rapid PCR technology, using the recombinant vector pET28b / NsTA1 as a template, and introducing a single mutation at the 108th position, the primers were:

[0068] Forward primer CGAAACCGCCGAC NNK TATACGCC (the underline is the mutated base)

[0069] reverse primer CTCAAAGGCGTATA NNK GTCGGCGG (the underline is the mutated base)

[0070] 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 DNAPolymerase 50U, add ddH 2 0 to 50 μL.

[0071] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 50°C for 15s, 62°C for 6.5min) for 30 cycles; 72°C for 5min.

[0072] 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 asymmetric synthesis of L-glufosinate-ammonium. The amino acid sequence of the aminopherase is as shown in SEQ ID:1, and the mutant of the aminopherase is prepared through performing single-site mutation or multi-site mutation on one or a plurality of 87th, 108th, 167th, 304th and 357th in the aminoacid sequence as shown in the SEQ ID:1. The high-efficiency expression of vitality mutant genes of high-conversion rate aminopherase can be realized, and the highest enzyme activity is 818.4U / mg. Thehighest optimum reaction temperature of the aminopherase vitality mutant can reach 67 DEG C, and during asymmetric synthesis of the L-glufosinate-ammonium through catalyzing 800mM of glufosinate-ammonium precursor ketone at the temperature, the conversion rate is as high as 100%. The NsTA mutant solves the technical difficult problems that few enzyme sources exist, the enzyme activity is low, substrate tolerance is bad and the conversion rate is low in a current L-glufosinate-ammonium preparation technology from the aminopherase, and has better application prospects.

Description

[0001] (1) Technical field [0002] The invention relates to a new α-transaminase and its mutant, and the application of the α-transaminase and the mutant in asymmetric synthesis of L-glufosinate-ammonium. [0003] (2) Background technology [0004] 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. [0005] 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 inv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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