Enzymatic synthesis of 10-oxostearic acid in high space-time yield via cascade reaction of a new oleate hydratase and an alcohol dehydrogenase

Hydroxy fatty acids and carbonyl fatty acids are important multi-functional materials for the manufacture of fragrances and other fine chemicals. In this study, a novel oleate hydratase (PaOH) was cloned from Paracoccus aminophilus DSM 8538 and solubly expressed in Escherichia coli. The recombinant PaOH efficiently catalyzed the hydration of oleic acid, with a specific activity of 5.21 U mg^-1 protein. Enzymatic hydration of oleic acid was optimized for the production of 10-hydroxystearic acid. Under the optimal conditions, a pilot reaction was performed on a 1-L scale, where 90 g L^-1 of oleic acid was converted into 10-hydroxystearic acid by 10 g L^-1 of lyophilized enzyme within 4 h, achieving a conversion of 96.1% and a space-time yield of up to 552 g L^-1 d^-1. The resultant 10-hydroxystearic acid was further converted into 10-oxostearic acid, via enzymatic cascade with a secondary alcohol dehydrogenase (MlADH) from Micrococcus luteus WIUJH20, using a lactate dehydrogenase (LdLDH) from Lactobacillus delbrueckii to drive the co-enzyme regeneration. The cascade reaction was carried out stepwise in one pot. Total conversion reached 95.0% after 10 h reaction at a scale of 1 L, with a space time yield of 217 g L^-1 d^-1. The final yield of 10-oxostearic acid isolated was 52.2% (mol mol^-1), with a purity of >99.0%.