Double coenzyme-dependent glufosinate-ammonium dehydrogenase mutant and its application in catalytic synthesis of l-glufosinate-ammonium
A technology of glufosinate-ammonium and mutants, which is applied in the application field of double coenzyme-dependent glufosinate-ammonium dehydrogenase mutants to catalyze the synthesis of L-glufosinate-ammonium, and can solve complex processes, low single resolution rate, Eliminate the problems of expensive chiral separation reagents, etc., to achieve high conversion rate of raw materials, easy separation and purification, and simple process
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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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