Ketopantolactone Reductase:Cloning,Expression And Application For Efficiently Asymmetric Synthesis Of D-pantolactone

D-pantothenic acid,which is well known as vitamin B5,can maintain hair,blood and skin health,and it can be used as a precursor of Coenzyme A(CoA).By now,D-pantothenic acid was synthesised using D-pantolactone and ?-alanine as raw materials through the chemical strategy.The precursor,D-pantolactone,is obtained by chemical synthesis and kinetic resolution using the hydrolysis enzyme.But the chemical synthesis process is complicated and polluted,while the hydrolysis kinetic resolution also requires chemical racemization and lactonization using acid which certainly increase the additional cost.Therefore,it is very significant for the development of new synthesis process of D-pantolactone.In this paper,Sce CPR1 gene was successfully cloned from the Saccharomyces cerevisiae using its genome as template,and the enzyme regeneration system of SceCPR1 and glucose dehydrogenase(EsGDH),enzyme-coupled system,was successfully constructed to realize the efficient asymmetric reduction of ketopantolactone to D-pantolactone.SceCPR1 and EsGDH protein molecular weight were 35 kDa and 27 kDa,respectively.Through the optimization of the reduction conditions,the enzyme activity of unit mass of wet cells containing of SceCPR1 and Es GDH finally reached 1179.2 U/g and 442.8 U/g,respectively.The enzymatic properties showed that the optimum pH was 5.5,the temperature was 45 ?.And then it is easy to denature at 45 ?,but5 mM NADPH,which is added simultaneously,can ensure its temperature stability;Then,SceCPR1 does not belong to the metal ion-dependent reductase,but 5mM Fe3+ and most of organic solvents can inhibit the enzyme activity;The substrate spectrum showed that the enzyme had the highest activity for ketopantolactone,although,it had no activity for keto-pantoic acid and D-or L-pantolactone.Under the low substrate concentration,the Km values of SceCPR1 for ketoprenalactone and NADPH were 0.164 mM and 0.029 mM,respectively,and the Vmax of the reaction was 131.03 U/mg and 137.81 U/mg,respectively.When lyophilized cells BL21(DE3)/p ACYCDuet 1-SceCPR1/EsGDH was used as biocatalyst,the reaction conditions were optimized as follows: pH 5.5,35 ?,the biocatalyst dosage of 0.03 g/mL,the molar ratio of glucose and substrate of 1.5:1.0 as well as the agitation rate of400 rpm.Through the study of substrate hydrolysis,it was found that thespontaneous hydrolysis of substrate was the most important factor affecting product yield.Thus,under the optimum catalytic conditions,the ketopantolacone and glucose were dissolved in pH 2.5,and the final product concentration reached 475 mM,95% yield,e.e.p ? 99.9%.