Aspartate Oxidase Mutants, Engineering Bacteria and Their Application in Oxidation-Reduction Coupled Preparation of Refined Glufosinate-ammonium
An aspartate oxidase and mutant technology, applied in the directions of oxidoreductase, biochemical equipment and methods, enzymes, etc., can solve the problem that the raw material PPO cannot be completely converted, the separation of L-glufosinate is troublesome, and the expensive chiral separation. Reagents and other problems, to achieve the effect of easy separation and purification, shortened reaction time, and large conversion rate
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Embodiment 1
[0056] Example 1 Construction of expression vector and engineering bacteria
[0057] 1. Recombinant E.coli BL21(DE3) / pET28b-CeDAAO
[0058]According to the nucleotide sequence (SEQ ID NO.1) of the D-aspartate oxidase gene (NCBI accession number: NP_001370668.1) derived from Caenorhabditis elegans in the gene bank, the amino acid encoding the protein The sequence is shown in SEQ ID NO.2) to design primers, and NcoI and XhoI restriction sites were introduced into the primers respectively:
[0059] Upstream primer: 5'-TATACCATGGCGAACATCATCCCGAAAATC-3';
[0060] Downstream primer: 5'-CTCGAGTTACAGACCCAGCGCGGTTTAAC-3';
[0061] Using the pET-28b(+) plasmid as the expression vector, construct E.coli BL21(DE3) / pET28b-CeDAAO: under the priming of the above primers, using the D-aspartate oxidase gene sequence as the template, the use of high-fidelity Pfu DNA polymerase was amplified to obtain the gene sequence of D-aspartate oxidase with restriction sites. After sequencing, the ampli...
Embodiment 2
[0088] Example 2: Induction and expression of glufosinate-ammonium dehydrogenase mutants-glucose dehydrogenase recombinant bacteria and aspartate oxidase recombinant bacteria
[0089](1) Wet cells containing D-aspartate oxidase: The engineered bacteria E.coli BL21(DE3) / pET28b-CeDAAO containing D-aspartate oxidase gene constructed in Example 1 were inoculated into cells containing D-aspartate oxidase. In LB liquid medium containing 50 μg / mL kanamycin, culture at 37°C for 12 h at 200 rpm, and then inoculate with 1% (v / v) inoculum to fresh LB liquid containing 50 μg / mL kanamycin resistance culture medium, at 37 ° C, 150 rpm to cultivate to bacterial OD 600 After reaching 0.6-0.8, IPTG with a final concentration of 24 μg / mL was added, and after induction and culture at 28 °C for 14 h, centrifuged at 4 °C and 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected. ) was washed twice to obtain wet cells.
[0090] (2) Wet cells containing glufosinate-...
Embodiment 3
[0091] Example 3: Construction of aspartate oxidase gene mutation library and its high-throughput screening
[0092] Through homology modeling and molecular docking of CeDAAO, the 16th, 34th, 50th, 54th, 57th, 58th, 210th, 219th, 312th and 313th positions of the amino acid sequence shown in SEQ ID NO.2 were selected for site-directed saturation mutation. Primer designs are shown in Table 3.
[0093] Table 3 Primer design
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[0095]
[0096] 1. Establishment of high-throughput screening methods
[0097] The structure of glufosinate-ammonium shows that it is an amino acid structure that lacks UV-absorbing groups and is difficult to detect under UV detectors. In order to detect the concentration and optical purity of L-PPT, the derivatization reagent o-phthalaldehyde is used with N-acetyl-L-cysteine is derivatized with glufosinate-ammonium to generate isoindole, a substance with fluorescence absorption properties, which can be detected under a fluorescence detect...
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