The Expression And Characterization Of A Recombinant Thermostable Short Chain Dehydrogenase/Reductase

Thermostable short-chain dehydrogenases/reductases have great potential application in synthesis of chiral alcohol. A short-chain dehydrogenase/reductase SDR3from the extremely thermophilic bacterium Carboxydothermus hydrogenoformans SP. was heterologously expressed and then explored in its biochemical characterization relation to asymmetric reduction. One substrate in its substrates spectrum was researched for the development of biocatalysis technology.The gene shows an open reading frame of747bp nucleotides, encoding a sequence of248amino acids. The encoded protein has typical residues patterns belonged to the short-chain dehydrogenases/reductases superfamily. With pET28a, a prokaryotic expression system SDR3was heterologusly expressed in E.coli. The best induction condition was as follow:incubation temperature25℃, IPTG concentration0.05mmol/L, induction time14h.The purified SDR3had an optimum temperature at70℃, pH at6and a good activity in α-keto esters. The SDR3was stable in high temperature and retained residual activity more than90%after15min in70℃. However, SDR3was significantly inhibited by Zn2+and Co2+in1mM.A process for asymmetric reduction of Ethyl benzoylformat by SDR3was developed, in which a NADH-regenerating system with glucose dehydrogenase was used. The NADH consumption was greatly reduced by this system. At last R-ethyl mandelate was harvested in6h at yield55.27%, e.e. more than99%.