A Mutant of Phaseolin Epoxide Hydrolase with Improved Enantioselectivity
A technology of epoxides and mutants, applied in the fields of genetic engineering and protein expression, can solve problems such as low enantioselectivity and limited application potential
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Embodiment 1
[0018] Embodiment 1: Construction of mutant enzyme gene and its expression plasmid
[0019] 1. Acquisition of plasmid pET-28a(+)-pveh1
[0020] The medium composition of recombinant E.coli BL21(DE3) / pET-28a(+)-pveh1 is: peptone 1%, yeast extract 0.5%, NaCl 1%.
[0021] The recombinant bacteria were inoculated in a test tube with a medium filling volume of 5 mL and cultured at 37° C. and 215 rpm for 12 h with shaking. After the cultivation, the cells were centrifuged at 12,000 rpm for 1 min and the cells were collected, and the plasmid pET-28a(+)-pveh1 was extracted using the plasmid extraction kit Pure PlasmidMiniKit (Kangwei Century Biotechnology Co., Ltd.); wherein the plasmid pET-28a( The amino acid sequence of the epoxide hydrolase PvEH1 in +)-pveh1 is shown in SEQ ID NO.2 (the nucleotide sequence is shown in SEQ ID NO.4).
[0022] 2. Construction of recombinant E. coli BL21(DE3) / pET-28a(+)-pveh1(W102L)
[0023] Design and synthesize specific site-directed mutagenesis p...
Embodiment 2
[0028] Example 2: Mutant enzyme PvEH1 W102L the acquisition of
[0029] E.coli BL21-pveh1 W102L Inoculate a single colony in 2 mL of LB medium containing 100 μg / mL kanamycin, and culture overnight at 37°C and 215 r / min; transfer 1 mL of the culture solution to 50 mL of the same medium, and culture to OD 600 When it is 0.6-0.8, add IPTG (final concentration 0.5mmol / L) and induce at 20°C for 10h. Collect the thalli, 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
[0030] Example 3: Mutant enzyme PvEH1 W102L Determination of enantioselectivity
[0031] Add 40 μL of bacterial suspension and 910 μL of phosphate buffer to a 2 mL EP tube, incubate at 25°C for 5 min, then add 50 μL of racemic o-methylphenyl glycidyl ether (rac-o-GMPE, final concentration 10 mmol / L) and immediately The reaction was timed, and 100 μL of the reaction solution was regularly added to 1 mL of ethyl acetate to terminate the reaction. After microfiltration, samples were analyzed by normal phase HPLC, OD-H column and UV detector. The mobile phase is n-hexane / isopropanol (80:20, v / v), the flow rate is 0.8mL / min, the detection wavelength is 220nm, the peak time of (R)-o-GMPE and (S)-o-GMPE They are 6.52min and 8.02min respectively. Substrate e.e. s =[(S-R) / (R+S)]×100%; E=ln[(1-c)×(1-e.e. s )] / ln[(1-c)×(1+e.e. s )]. Where: R and S represent (R)- and (S)-o-GMPE concentration, c represents rac-o-GMPE conversion rate. The wild-type enzyme can retain the (R)-o-GMPE s...
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