| Chiral synthesis that has got a lot of attention in recent years is always a hot topic in the world.With the development of pharmaceutical,agrochemical and fine chemical sectors,an ever-increasing demand for optically pure intermediates or synthons has been observed.Chiral epoxides and vicinal diols are considered as the crucial and highly value-added building blocks.As a supplement to chemical synthesis,the low-cost and eco-friendly biocatalysis conforms to the social ideas of“green industry”and“sustainable development”.Epoxide hydrolase（EH）,a promising biocatalyst,can stereoselectively catalyze the ring-opening of racemic（rac-）epoxides,retaining single epoxide enantiomers and/or generating corresponding enantiopure vicinal diols.Therefore,researches related to the excavation and application of EH are still of great significance.In this study,the cDNA and genomic DNA sequences of a novel EH（PvEH2）were cloned from Phaseolus vulgaris.Then,the recombinant PvEH2 was successfully expressed in Escherichia coli,and its catalytic properties towards 13 monosubstituted epoxides were assayed.Based on the substrate spectrum of PvEH2,new reaction medium（system）was constructed to improve the maximum allowable substrate concentration.According to the result of computer-aided analysis,cap loop substitution of PvEH2 was performed to investigate the effects of this region on enzymatic catalytic properties.The kinetic resolution or enantioconvergent hydrolysis of an epoxide was achieved,respectively,using a fusion mutant independently or by chemoenzymatic strategy.（1）A 951-bp cDNA complete fragment coding for PvEH2（a total of 316 amino acids）was amplified by RT-PCR.The multiple sequence alignment result indicated that PvEH2 belongs toα/β-hydrolase fold superfamily.Then,recombinant PvEH2 was heterogeneously expressed in E.coli Rosetta（DE3）.Using rac-styrene oxide（1a）as substrate,the induction conditions were optimized through single-factor and orthogonal tests.As a result,the EH activity of fermentation broth reached up to 34.3 U·L-1 from 17.4 U·L-1 under the optimal induction conditions(OD600 value of 0.8,IPTG concentration of 0.2 mmol·L-1,induction period of 9 h and induction temperature of 20℃).The temperature and pH optima of purified PvEH2 were determined to be 30℃and 7.0.The half-life period of PvEH2’s activity at 40 and 45℃were about 82 and 40 min.After incubated at 25℃for 2 h at a pH range of 6.58.0,PvEH2displayed high stability（i.e.,relative activities were over 80%）.The kinetic parameter assay showed that the Km and kcat values of purified PvEH2 for（S）-1a were 2.12 mmol·L-1 and 18.86s-1,while those for（R）-1a were 19.65 mmol·L-1 and 1.02 s-1.Consequently,the affinity and catalytic efficiency of PvEH2 for（S）-1a was obviously higher than those for（R）-1a.（2）Using PvEH2-expressing transformant,E.coli/Pveh2,as catalyst,substrate spectrum assay exhibited that PvEH2 had the highest catalytic activities for rac-1a and rac-1,2-epoxyhexane（13a）(8.0 and 7.8 U·g-1 wet cell weight).Except for 13a,PvEH2enantiopreferentially hydrolyzed（S）-enantiomers of all tested epoxides.In enantioselectivity,PvEH2 exhibited an enantiomeric ratio（E）value of over 200 towards rac-1a,which,to our knowledge,was the highest among those of the known wild-type EH.PvEH2 showed moderate E values（6.223.0）towards rac-aryl glycidyl ethers（6a10a）.In regioselectivity,PvEH2 possessed high and complementary regioselectivities towards m-nitrostyrene oxide（3a）and m-chlorostyrene oxide（5a）,that is,the regioselectivity coefficientsαS andβR for 3a were 90.3%and 96.4%,while for 5a were 93.0%and 95.8%.The above results indicated that PvEH2 had potentials to mediate the kinetic resolution of rac-1a and the enantioconvergent hydrolysis of rac-3a and rac-5a.（3）A new transformant E.coli/pveh2 was constructed by transferring PvEH2-encoding gene from pET28a（+）into a cold-shock plasmid pCold II,after which the EH activity of fermentation broth was improved from 34.3 U·L-1 to 82.3 U·L-1,when using rac-1a as substrate.The poor solubility of substrate as well as the substrate and product inhibitions were proved to be the main reasons for the low substrate concentrations in PvEH2-mediated asymmetric hydrolysis.Using the lyophilized E.coli/pveh2 cell as biocatalyst,n-octane and glycerol were selected for constructing buffer/organic solvent reaction systems from five hydrophilic organic solvents and eleven hydrophobic ones.In the optimized n-octane/phosphate buffer biphsic system（volume ratio of two phases and weight ratio of rac-1a to E.coli/pveh2 were 4:6 and 0.8）,the maximum allowable rac-1a concentration was elevated to 200 mmol·L-1,which was 10 folds that in only buffer phase.The hydrolysis of 200mmol·L-1 rac-1a was conducted,affording（R）-1a with>99.5%ees and 47.8%yield at 25℃for 12 h,and the volumetric productivity(0.96 g·L-1·h-1)was the highest ever reported.Simultaneously,（R）-phenyl-1,2-ethanediol（1b）with 93.6%eep,50.4%yield and 1.16g·L-1·h-1 productivity was also formed.In case of rac-3a,the optimized reaction system was phosphate buffer containing 5%(v·v-1)glycerol,in which its maximum concentration increased from 10 mmol·L-1 to 40 mmol·L-1.（R）-1-（3-nitrophenyl）-1,2-ethanediol（3b）with84.9%eep and 90.2%yield was obtained in 12 h with a substrate conversion ratio（c）of97.6%.（4）To enhance the enantioselectivity of PvEH2 towards rac-13a and investigate the influence of cap loop on PvEH2’s catalytic properties for other epoxides,its partial cap loop was separately replaced with other four plant-derived EH（StEH,PvEH1,Vr EH1 and VrEH2）,creating four corresponding fusion mutants（Pv2St,Pv2Pv1,Pv2Vr1 and Pv2Vr2）.The investigation of substrate spectrum indicated that loop replacement decreased the activities in various degrees,where Pv2St and Pv2Pv1 still retained over 50%relative activities.Although the enantiopreference of PvEH2 did not switch after the substitution,the E values changed obviously.In detail,the E value of Pv2Pv1 towards rac-1a was reduced from over 200 to 30.9,while that of Pv2St for rac-13a was increased from 2.1 to 24.2.However,there was no obvious effect of loop substitution on the regioselectivity.By means of the Pv2St-expressing transformant（E.coli/pv2st）,up to 280 mmol·L-1 rac-13a can be efficiently resolved,producing 103.3 mmol·L-1（S）-13a with>99.5%ees,36.9%yield and 2.30 g·L-1·h-1productivity.The chemoenzymatic preparation of（R）-1,2-dihydroxyhexane（13b）was performed,affording（R）-13b with 73.0%eep and 86.5% yield. |
