Tryptophan-2,3-dioxygenase mutant and preparation method and application thereof
A technology of dioxygenase and tryptophan, applied in the field of protein engineering, can solve the problems of limited application prospects and low yield of HPIC, and achieve the effect of simple reflection system, excellent diastereoselectivity and mild reaction conditions
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Embodiment 1
[0036] The construction of the gene expression vector of embodiment 1 xcTDO
[0037] The xcTDO fragment synthesized from the whole gene (sequence shown in SEQ ID NO: 3, synthesized by Suzhou Jinweizhi Biological Co., Ltd.) was subjected to restriction endonuclease Nd and wxya (New England Biolabs Company) After performing double enzyme digestion according to the instructions, use T4 ligase (New England Biolabs Company) to connect to the Nd and wxya Double-digested expression vector pET22b (+). The ligation product was used to transform Escherichia coli DH5α competent cells (Tiangen Biochemical Technology Co., Ltd.). Pick the successfully transformed monoclonal colony from the solid LB medium plate containing 100 μg / ml ampicillin, and place it overnight in the LB liquid medium containing the same concentration of ampicillin at 37°C with a shaker speed of 220rpm to cultivate. The recombinant plasmid was extracted from the cultured bacterial liquid with a small plasmid ext...
Embodiment 2
[0038] The establishment of embodiment 2 mutant library
[0039] Three rounds of site-directed saturation mutagenesis were used.
[0040] The xcTDO residues for the first round of site-directed saturation mutagenesis were: Y24, Y27, L28, F51, H55, Y113, R117 and L120. They are located in the αB (F51 and H55) and αD (Y113, R117, and L120) helices and in the amino-terminal residues from other monomers (Y24, Y27, and L28), all on one side of the active center. During the screening process, using the expression plasmid (cloned on the plasmid with the nucleotide sequence encoding SEQ ID NO: 3) synthesized as a template, primers were designed around each mutation site to construct a single mutation library. Screening revealed that most mutants in the F51X library displayed significantly higher HPIC production than wild-type xcTDO, while little or no production of HPIC was detected with mutants from other libraries.
[0041] (2) Construct the second round of saturation mutation lib...
Embodiment 3
[0043] Example 3 Activation and induced expression of wild-type xcTDO and mutant libraries
[0044] Transform the recombinant plasmid into Escherichia coli BL21 (DE3) (Tiangen Biochemical Technology Co., Ltd.), and pick a single clone from the plate cultured overnight to fill 300 μl of LB liquid with a final concentration of 100 μg / ml ampicillin The medium was cultured in a 96-deep-well plate overnight at 37°C with a shaker at 250 rpm. On the next day, draw 15 μl of culture solution from the 96 deep-well plate cultured overnight, and add it to a fresh 1.5 ml 96 deep-well plate containing ampicillin TB liquid medium with a final concentration of 100 μg / ml, at 37°C, Shaker speed 250 rpm, wait for OD 600 At about 0.6, induce by adding the heme precursor δ-aminolevulinic acid and 0.5 mM IPTG at a final concentration of 0.5 mM. After expressing at 22°C for 20 h at a shaker speed of 250 rpm, centrifuge at a speed of 5000 rpm for 10 min, and discard the supernatant.
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