Aspartate oxidase mutant, engineering bacterium and application of aspartate oxidase mutant and engineering bacterium in preparation of L-glufosinate-ammonium through oxidation-reduction coupling
A technology of aspartate oxidase and mutants, applied in the direction of oxidoreductase, biochemical equipment and methods, enzymes, etc., can solve the problems of incomplete conversion of raw material PPO, troublesome separation of L-glufosinate, expensive chiral resolution Separate reagents and other issues to achieve the effect of easy separation and purification, shortened reaction time and high conversion rate
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Embodiment 1
[0057] The construction of embodiment 1 expression vector and engineering bacterium
[0058] 1. Recombinant Escherichia coli E.coli BL21(DE3) / pET28b-CeDAAO
[0059]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 of the encoded protein The sequence is shown in SEQ ID NO.2) primers were designed, and NcoI and XhoI restriction enzyme sites were introduced in the primers respectively:
[0060] Upstream primer: 5'-TATACCATGGCGAACATCATCCCGAAAATC-3';
[0061] Downstream primer: 5'-CTCGAGTTACAGACCCCAGCGCGGTTTTAAC-3';
[0062] Using the pET-28b(+) plasmid as an expression vector, construct E.coli BL21(DE3) / pET28b-CeDAAO: Initiated by the above primers, using the D-aspartate oxidase gene sequence as a template, using high-fidelity Pfu DNA polymerase is amplified to obtain the gene sequence of D-aspartate oxidase with restriction sites, and af...
Embodiment 2
[0089] Example 2: Induced expression of glufosinate-ammonium dehydrogenase mutant-glucose dehydrogenase recombinant bacteria and aspartate oxidase recombinant bacteria
[0090](1) Wet bacteria containing D-aspartate oxidase: the engineered bacteria E.coli BL21(DE3) / pET28b-CeDAAO constructed in Example 1 containing the D-aspartate oxidase gene was inoculated into In 50μg / mL kanamycin-resistant LB liquid medium, culture at 37°C and 200rpm for 12h, then inoculate 1% (v / v) inoculum into fresh LB liquid containing 50μg / mL kanamycin resistance culture medium, at 37°C, 150rpm to cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 24μg / mL, induce culture at 28°C for 14h, centrifuge at 4°C, 8000rpm for 20min, discard the supernatant, collect the precipitate, and wash with pH 7.5, 20mM phosphate buffered saline (PBS ) was washed twice to obtain wet thalli.
[0091] (2) Wet cells containing glufosinate-ammonium dehydrogenase mutant-glucose dehydrogenase: the reco...
Embodiment 3
[0092] Example 3: Construction of aspartate oxidase gene mutation library and its high-throughput screening
[0093] Through homology modeling and molecular docking of CeDAAO, the 16th, 34th, 50th, 54th, 57th, 58th, 210th, 219th, 312th, and 313rd positions of the amino acid sequence shown in SEQ ID NO.2 were selected for site-directed saturation mutation, The primer design is shown in Table 3.
[0094] Table 3 Primer design
[0095]
[0096]
[0097] 1. Establishment of high-throughput screening method
[0098] The structure of glufosinate-ammonium shows that it is an amino acid structure, which 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 and The derivatization reaction between N-acetyl-L-cysteine and glufosinate-ammonium produces isoindole, a substance with fluorescence absorption characteristics, which can be detected under a fl...
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