Double-coenzyme dependent glufosinate-ammonium dehydrogenase mutant and application thereof to catalytic synthesis of L-glufosinate-ammonium
A technology of glufosinate-ammonium and mutants, applied in double coenzyme-dependent glufosinate-ammonium dehydrogenase mutants, the application field of catalyzing the synthesis of L-glufosinate-ammonium, can solve the problem of incomplete conversion of raw material PPO, L-glufosinate-ammonium Troublesome phosphine separation, expensive chiral resolution reagents, etc., to achieve the effect of easy separation and purification, high conversion rate, and improved activity of mutants
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Embodiment 1
[0052] The construction of embodiment 1 expression vector and engineering bacterium
[0053] (1) Recombinant Escherichia coli E.coli BL21(DE3) / / pETDuet-1-VgPPTDH
[0054] A glufosinate-ammonium dehydrogenase derived from Ventosimonas gracilis was selected in the NCBI database, and the NCBI accession number was WP_068388615.1, and gene synthesis was carried out. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is Shown in SEQ ID NO.2.
[0055] Primers were designed according to the nucleotide sequence shown in SEQ ID NO.1, and Sac I and NotI restriction enzyme sites were introduced in the primers respectively:
[0056] Upstream primer: 5'-GAGCTCATGACTGTATCTGTTGACTCCTT-3';
[0057] Downstream primer: 5'-GCGGCCGCTTAAACCACGCCTTGCTCCA-3';
[0058] Using the pETDuet-1 plasmid as an expression vector, construct recombinant E. coli E. coli BL21(DE3) / pETDuet-1-VgPPTDH: under the priming of the above primers, use the nucleic acid sequence of the target gen...
Embodiment 2
[0067] Example 2 Recombinant construction of glucose dehydrogenase and glufosinate-ammonium dehydrogenase to obtain recombinant Escherichia coli E. coli BL21(DE3) / pETDuet-1-VgPPTDH-EsGDH.
[0068]In order to further reduce the cost of industrial production, a glucose-glucose dehydrogenase coenzyme cycle system was constructed, and glucose dehydrogenase (NCBI accession number: KM817194.1, shown in amino acid sequence SEQ ID NO.7) was cloned into the expression On the second multiple cloning site of vector pETDuet-1, the glucose dehydrogenase is NADPH / NADH double coenzyme-specific dehydrogenase.
[0069] Acquisition of the glucose dehydrogenase gene with homologous sequences: the respective 20bp sequences in front of and behind the NdeI and PacI restriction sites of the expression vector pETDuet-1 are used as homologous sequences, and the E.coli BL21(DE3) / pET28b-EsGDH (according to NCBI accession number, gene synthesis) was used as a template, primer 1 and primer 2 were designe...
Embodiment 3
[0083] Example 3 Induced expression of glufosinate-ammonium dehydrogenase-glucose dehydrogenase recombinant bacteria and amino acid oxidase
[0084] Wet cells containing glufosinate-ammonium dehydrogenase-glucose dehydrogenase: Inoculate the recombinant Escherichia coli E.coli BL21(DE3) / pETDuet-1-VgPPTDH-EsGDH obtained in Example 2 into 50 μg / mL ampicillin LB liquid medium with resistance to ampicillin was cultured at 37°C for 12 h at 200 rpm, and then inoculated into fresh LB liquid medium containing 50 μg / mL ampicillin resistance at 37 °C at 37 Cultivate at 150rpm to cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 24 μg / mL, induce culture at 22°C for 16 hours, centrifuge at 8000 rpm for 20 minutes at 4°C, discard the supernatant, collect the precipitate, and wash with pH 7.5, 20mM phosphate buffer ( PB) wash twice to obtain the wet cells of recombinant bacterial strain E.coli BL21(DE3) / pETDuet-1-VgPPTDH-EsGDH containing glufosinate-ammonium dehydr...
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