The Screening,Cloning And Expression Of The Hydroxy Acid Dehydrogenase

Optically pure aliphatic hydroxy acids and derivatives have been extensively studied as versatile chiral building blocks for preparation of many valuable bioactive compounds or natural products.It is well established that the most efficient and convenient route to chiral aliphatic hydroxy acid is the asymmetric reduction of the related ketoacids.Significant advancements have been made in the asymmetric reduction of ketoacids.However,these methods are mainly limited to aromatic ketoesters and short chain aliphatic ketoesters.Unfortunately,little progress has been achieved for asymmetric reduction of long-chain aliphatic ketoacids,especially for ?-,?-or higher ketoacids.Therefore,it is still highly challenging to develop an efficient catalytic system for asymmetric reduction of long chain aliphatic ketoacids with high enantioselectivity and broad range of substrates.A new ideal biocatalyst,Pseudomonas panipatensis with high enantioselectivity towards 4-oxodecanoic acid,was successfully isolated from soil samples through the strategies of target reaction-oriented screening and the rapid analysis of TLC and GC.The complete genome of Pseudomonas panipatensis was then sequenced.An NADPH-dependent reductase(PpCR)from Pseudomonas panipatensis was discovered by genome hunting method.With this enzyme as the template,SmCR from Serratia marcescens was discovered by data mining.SmCR was purified to homogeneity and its catalytic properties were studied.The maximum activity was observed at pH 6.0 and the optimum temperature was found to be 40?.SmCR was relatively stable at 30? with half-lives of 90 h,compared to 50 h and 15 h at 40? and 50? respectively.The Km value of SmCR for 4-oxodecanoic acid was 1.26 mM and the kcat was 0.128 min-1.The preparation experiment of y-decalactone was performed in 100 mL system of sodium phosphate buffer(100 mM,pH 6.0)with 2 mM 4-oxodecanoic acid.The product yield reached to 72%with 99%ee of(R)-y-decalactone.The reduction was then extended to 5-oxodecanoic acid,which provided 70%yield of the corresponding(R)-?-decalactone in 95%ee.