The Study Of Stability Of 1,3-propanediol Oxidoreductase (DhaT) Of Klebsiella Pneumoniae J2B

1,3-propanediol oxidoreductase?DhaT?,which can catalyze the conversion of3-hydroxypropionaldehyde?3-HPA?to 1,3-propanediol?1,3-PD?with the oxidation of NADH to NAD⁺,is a key enzyme in the production of 1,3-PD from glycerol.However,DhaT is known to be severely inactivated by its physiological substrate,3-HPA,due to the reaction of 3-HPA with the thiol group of the cysteine residues,which is a serious constraints on industrial application.In this study,using site-directed mutagenesis,four cysteine residues?Cys28,Cys93,Cys107 and Cys154?in Klebsiella pneumoniae J2B Dha T were substituted to alanine,the amino acid commonly found in cysteine's positions in other DhaT,individually and in combination in order to improve the stability of DhaT against3-HPA.The results showed that,after inculated 60 min with 10 mM 3-HPA,all single mutants have no signification improvement on DhaT stability,the double mutants DhaT₂-1?C28A_C93A?,DhaT₂-2?C28A_C107A?,DhaT₂-5?C93A_C107A?and triple mutants DhaT₃-1?C28A_C93A_C107A?retained 28%,24%,24%and 30%of their corresponding initial activities while wild-type DhaT retained only 19%of its initial activity,and the corresponding enzyme half-lives were 1.65,1.41,1.41 and1.62 times greater than that of wild-type enzyme.Furthermore,the observed enhancement of DhaT₃-1 were pronounced at lower3-HPA concentration,DhaT₃-1 retained 54%of its initial activity while wild-type DhaT retained only 30%of its initial activity after 60 min incubation with 5 mM3-HPA,and more than 2 times greater enzyme half-life than wild-type enzyme.DhaT₂-2 and DhaT₃-1 exhibited approximately 50%-and 16%-higher activity than the wild-type enzyme,respectively,after 60 min incubation with 10 mM 3-HPA in both forward and reverse reaction.At the same time we performed the effects of pH and temperature of DhaT,DhaT₂-2 and DhaT₃-1,the results should that the optimum pH and temperature are same between them,are pH 9.0 and 55°C.According to detailed kinetic studies,the double mutant DhaT₂-2 and triple mutant DhaT₃-1 had similar kinetic properties(V_max,K_cat and K_m for both 3-HPA and NADH)with wild-type DhaT.This study shows that DhaT stability against 3-HPA can be increased by cysteine-residue removal,albeit to a limited extent,the stable mutants can use for the green production of 1,3-propanediol.