Molecular Engineering Of Oleate Hydratase And Reaction Optimization For The Cascade Synthesis Of 10-oxostearic Acid

Oleate hydratase can catalyze the hydration of renewable oleic acid to produce 10-hydroxystearic acid,which can be further converted to 10-oxostearic acid by a 10-oxostearic acid dehydrogenase(MlADH).Both 10-hydroxystearic acid and 10-oxostearic acid are fine chemicals with high values.MlADH is an NAD+-depended dehydrogenase and NAD+ needs to be recycled during the reaction to reduce the cost of expensive coenzyme.In this study,molecular modification of oleate hydratase PaOH was carried out to improve its activity and thermal stability;NAD+was regenerated by NADH oxidase with O2 as oxidant,which was supplied in-situ by catalase-mediated decomposition of the H2O2 continuouly supplemented into the reaction system.Therefore,the reaction cost of 10-oxostearic acid production was dramatically reduced.The main work of this study are as follows:(1)Construction and evaluation of a high-throughput screening method for oleate hydratase.Accompanied with the bioconversion of 10-hydroxystearic acid into 10-oxostearic acid,NADH is formed which can be chemically converted to a blue-violet product,with an absorption peak at 580 nm.The detection limit of 10-hydroxystearic acid is 0.01 mM,and the linearity is good at the concentration of 10-hydroxystearic acid between 0.01-1.00 mM.Therefore,this method is suitable for the activity screening of oleate hydratases.(2)Molecular engineering of oleate hydratase to improve its activity and thermostability.This work was started from the wild type of oleate hydratase from Paracoccus aminophilus(PaOH),which was previously reported by our group.Semi-rational design and error-prone PCR methods were used for the molecular evolution of PaOH,and three better variants were harvested,whose activities were 1.4-3.4 times that of the wild type.The catalytic efficiency(kcat/KM)of the best mutant PaOH-M4 was 3.5 times that of the wild type.(3)Optimization of the cascade reaction for biocatalytic synthesis of 10-oxostearic acid.To maximize the reaction performance,we adopted the one-pot,two-step cascade reaction system using PaOH-M4 and MlADH.Several parameters of the cascade reaction,such as catalysts dose and the loading of hydrogen peroxide,were optimized.Under the optimized condition(substrate loading 50 g·L-1,PaOH-M4(cell-free extra)loading 2,500 U·L-1),the conversion reached over 98.5%after 2 h.After the first step reaction,the pH of the reaction mixture was adjusted to 7.5,and the second step reaction was initiated by supplementing NAD+,SmNOXV193R/V194H,catalase,and MlADH.After another 4 h,the conversion of 10-hydroxy stearic acid to 10-oxostearic acid reached 98.2%.Finally,25.3 g of final product was isolated with 79.5%yield and 99.0%purity.