Purification And Characterization Of Carbonyl Reductase With Stereoselectivity From Candida Tropicalis

(R)-ethyl 3-hydroxy-3-phenylpropionate is a key chiral intermediate for synthesis of Atomoxetine that can treat the attention deficit hyperactivity disorder?ADHD?.Atomoxetine hydrochloride?ATX?,listed in 2003,is currently the only non-nervous system stimulant drugs approved by the FDA for use in children,adolescents and adults patients.A strain was screened from the soil,which is capable of transf orming ethyl benzoylacetate to?R?-ethyl 3-hydroxy-3-phenylpropionat e efficiently.The enantiometric excess of the?R?-ethyl 3-hydroxy-3-phenylpropionate was up to 100%.The strain was identified as Can dida by 26S rDNA sequence analysis,and named C.tropicalis zjut412.The fermentation culture conditions of C.tropicalis zjut 412 were optimized by single factor experiment,Plackett-Burman experiment and Box-Benhnken experiment.The optimum conditions were as follows:the glucose concentration 43 g/L,the liquid loading amount 27 mL,43 h.The specific activity of C.tropicalis zjut was increased from the initial 894.4U/g to 1388.66 U/g.The activity was increased by 55.3%.The optimum conditions were as follows:8%glucose as substrate,pH 6.0,substrate concentration 4.8 g/L,biomass 1.92 g/L?dry weight/reaction volume?,35?,24 h.The conversion and enantiomeric excess of?R?-ethyl 3-hydroxy-3-phenylpropionate were all up to 100%.The carbonyl reductase in the C.tropicalis zjut 412 was purified by sonication,ammonium sulfate precipitation,DEAE sepharose FF anion exchange chromatography and Blue agarose 6FF affinity chromatography.The specific activity of carbonyl reductase increased from the initial0.049 U/mg to 6.77 U/mg.The purification multiple was 139 and the recovery rate was 12%.The enzymatic properties of carbonyl reductase in the C.tropicalis zjut 412 were studied.It has the highest catalytic activity at 35?.The enzyme protein is rapidly denatured at 40?.The optimum pH of the catalytic reduction was 6.0.The enzyme activity was only stable between pH 7.0 and 8.5.The effect of EDTA on enzyme activity was not obvious.It was suggested that the enzyme did not need the participation of metal ions in the catalytic reaction.Ca²⁺,Mg²⁺and Mn²⁺ions play an important role in promoting enzyme activity.