sleh1 mutants and their use in enantionormalized hydrolysis of epoxides
A technology of epoxides and mutants, applied in the fields of application, hydrolytic enzymes, and microbial-based methods, can solve problems such as difficult normalized hydrolysis of EHs
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Embodiment 1
[0040] Example 1 Construction of mutant enzyme gene and its corresponding recombinant bacteria
[0041] For the construction method of the genetically engineered bacteria E.coli / sleh1, please refer to the patent application with the patent publication number CN109652354A. The recombinant bacteria E.coli / sleh1 were inoculated into 5 mL of LB medium, and cultured with shaking at 37° C. and 220 rpm for 12 h. After the cultivation, the cells were centrifuged at 13,000 rpm for 1 min and the cells were collected. The recombinant plasmid pET28a (+ )-sleh1.
[0042] Design specific primers for single-point iterative mutagenesis. Design and synthesize specific site-directed mutagenesis primers as follows:
[0043] W106T-F:5-TTTGTTGTTGCGCATGAT GGCGCGTTTATTGCTTGG-3', with mutation site;
[0044] W106L-F:5-TTTGTTGTTGCGCATGAT GGCGCGTTTATTGCTTGG-3', with mutation site;
[0045] F189I-F:5-TACCGCGATCCTGCACCA TGTTTTCCTAAAGGCAAA-3', containing the mutation site;
[0046] F189L-F:5-T...
Embodiment 2
[0049] Example 2 Induction, expression and purification of mutant enzymes
[0050] Inducible expression of mutant enzymes: four recombinant strains E.coli / sleh1 W106T / F189L , E.coli / sleh1 W106T / F189I , E.coli / sleh1 W106L / F189L and E.coli / sleh1 W106L / F189I A single colony was inoculated in 2 mL of LB medium containing 100 μg / mL kanamycin, and cultured overnight at 37 °C and 220 r / min; 2 mL of the culture medium was transferred to 100 mL of LB culture that also contained 100 μg / mL kanamycin. medium, cultured to OD 600 When the temperature is 0.6 to 0.8, add 10 μL IPTG (final concentration 0.05 mmol / L), and after induction at 25 °C for 8 h, the cells are collected by centrifugation, and 5 mL of sodium phosphate buffer (Na 2 HPO 4 -NaH 2 PO 4 , 50mmol / L, pH 7.5) to obtain four wet bacterial suspensions with a final concentration of 200mg / mL.
[0051] Purification of mutant enzymes: The recombinant cells of the four mutant enzymes collected by centrifugation after inducti...
Embodiment 3
[0052] Example 3 Determination of normality and regioselectivity of mutant enzymes
[0053] The bacterial suspension was prepared according to the method of Example 2, and the normalization and yield of the mutant enzyme were determined, and 250 μL of recombinant bacteria E.coli / sleh1 were taken respectively. W106T / F189L , E.coli / sleh1 W106T / F189I , E.coli / sleh1 W106L / F189L and E.coli / sleh1 W106L / F189I The bacterial suspension was respectively suspended in 200 μL sodium phosphate buffer (50 mmol / L, pH 7.5), and then 50 μL rac-1,2-epoxyoctane solution (200 mmol / L, solvent was methanol) was added to the final concentration. At 20 mmol / L, the reaction was carried out at 20 °C and 120 r / min in a constant temperature shaking reactor for 3 h. 200 μL of the reacted sample was extracted with 1 mL of ethyl acetate, and the organic phase was dried over anhydrous magnesium sulfate. Detected by gas chromatography, the results showed that the mutant enzyme SlEH1 W106T / F189L , SlEH1 W1...
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