Study On Producing Enantiopure(2R,3S)-3-Phenylglycidate By Biotransformation

(2R,3S)-3-phenylglycidate,containing three-membered ring with a O atom,is an efficient intermediate for taxol.This subject is intended to use microbial epoxide hydrolases for the preparation of enantiopure 3-pheny lglyci date.By using 3-phenylglcidol as the sole carbon,Up to 30 strains showing enantioselectivity toward the epoxide 3-phenylglycidate were screened from more than two hundred bacteria and fungus strains,which were isolated from oil-contaminated soil samples collected from many places.A fungi strain with best enantioselectivity in catalyzing the hydrolysis of tranps-3-phenylglycidate with retaining(2R,3S)-3-Phenylglycidate was further studied and identified as Galactomyces geotrichum based on morphology,bology and ITS sequence,which was named as G.geotrichum ZJUTZQ 200.Then,the fermentation conditions for G.geotrichum ZJUTZQ 200 were investigated based on single factor experiments,including carbon sources,nitrogen sources,metal ions,time for fermentation,initial inoculum and the initial pH of medium.They influenced the biomass,enzyme activity and e.e.s value.Simultaneously,the additive amounts of carbon source,nitrogen source and metal ions were got through orthogonal test.The best result were obtained under the following condition:Glycerol 1.0%,soybean flour 1.5%,NaNO3 0.3%,K2HPO4·3H2O0.1%,KCl 0.05%,MgSO4·7H2O 0.05%,30?,pH 5.0,48 h.In this contidion,the e.e.s value of the substrate increased from the initial 85.5%to 93.2%,the conversion rate of the substrate from the initial 78.3%to 87.6%,the biomass from the initial 8.42 g/L to 34.3 g/L,nearly four times.Afterward,the conditions for kinetic resolution of racemic 3-phenylglycidate by G.geotrichum ZJUTZQ 200 were studied including the initial pH,co-solvents,initial substrate concentration,bioconversion time and so on.It showed the best resolution conditions as follows:substrate/cell 5%in DMSO(4%)was hydrolyzed in 10 mL KPB(0.1 M,pH7.2)using wet myceliam of G.geotrichum ZJUTZQ 200(wet cell 1.0 g)at 30? for 8 h.It gave the best e.e.S value 99.9%and E value 49 under the best resolution.Finally,following above bioresolution condition,0.51 g(2R,3S)-3-phenylglycidate were obtained within 8 hours in 200 mL buffer.Then 0.102 g(2R,,3S)-3-amino-3-phenyl-2-hy-droxypropionic acid was then successfully prepared using above-mentioned enantiopure compound as starting material by three steps.The research on substrate specificity of this enzyme concluded that this strain showed high enantioselectivity towards phenylglycidates expoxide analogs.