A Mutant of Phaseolin Epoxide Hydrolase with Improved Stereoselectivity
An epoxide and hydrolase technology, applied in the fields of genetic engineering and protein expression, can solve the problem of not being able to produce high enantiopurity epoxides and vicinal diols, etc.
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Embodiment 1
[0023] Construction of embodiment 1 mutant plasmid
[0024] 1. Plasmid pET28a(+)-pveh1 L105I / M160A / M175I the acquisition
[0025] The recombinant strain E.coli BL21(DE3) / pET28a(+)-pveh1 L105I / M160A / M175I (For the construction method, please refer to the paper Hui H Y, Die H, Xiao L S, et al Directed modification of a novel epoxyhydrolase from Phaseolus vulgaris to improve its enantioconvergence towards styrene epoxy[J]. Catalysis Communications, 2016: 32-35) The liquid volume was 5mL test tubes and cultured with shaking at 37°C and 220rpm for 12h. After the cultivation, the cells were centrifuged at 13,000rpm for 1min and the cells were collected. The cells were extracted from E.coli BL21(DE3) / pET28a(+) -pveh1 L105I / M160A / M175I Plasmids were extracted as templates for iterative mutations for plasmid pET28a(+)-pveh1 L105I / M160A / M175I build.
[0026] 2. Construction of recombinant Escherichia coli E.coli BL21(DE3) / pET-28a(+)-pveh1(PvEH1Z4X4-59)
[0027] Design specific pr...
Embodiment 2
[0036] Induced expression of embodiment 2 mutant enzyme PvEH1Z4X4-59
[0037] Inoculate a single colony of E.coli BL21-PvEH1Z4X4-59 in 2 mL of LB medium containing 100 μg / mL kanamycin, and culture overnight at 37°C and 220 r / min; transfer 2 mL of the culture medium to 100 mL of kanamycin In LB medium, cultured to OD 600 When the temperature is 0.6-0.8, add 100ul IPTG (final concentration 0.5mmol / L), centrifuge to collect the bacteria after induction at 20°C for 10 hours, and use 10mL sodium phosphate buffer (Na 2 HPO 4 -NaH 2 PO 4 , 100mmol / L, pH 7.0) to obtain a bacterial suspension.
Embodiment 3
[0038] Embodiment 3 enantioselectivity and enantionormality determination
[0039] Weigh 50 mg of the recombinant bacteria and suspend them in 1 mL of sodium phosphate buffer (100 mmol L -1 , pH=7.0), draw 950μL of bacterial suspension (50mg·mL -1 ) into a 2mL EP tube, incubated at 25°C for 5min, then added 50μL rac-pCSO or rac-mCSO (200mmol·L -1 , the solvent is methanol) to a final concentration of 10mmol L -1 , 25℃, 800r·min -1 React in a constant temperature shaking reactor for 10 min, take 100 μL of sample and extract with 1 mL of ethyl acetate, and dry the organic phase over anhydrous magnesium sulfate. The samples were analyzed by high performance liquid chromatography Waters e2695, chiral liquid chromatography column and ultraviolet detector. The conditions of rac-pCSO high performance liquid chromatography are: the mobile phase is n-hexane:isopropanol=80:20, the column temperature is 30°C, and the flow rate is 0.8mL min -1 , detection wavelength is 220nm, chiral ...
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