Exploring the fatty acid double bond hydration activities of Lacticaseibacillus rhamnosus strains

The fatty acid double bond hydratases (FADBH) are involved in the detoxification of unsaturated fatty acids and the production of multi-functional hydroxy fatty acids. Thereby, we have investigated the fatty acid hydration activities of Lacticaseibacillus rhamnosus strains including the ones isolated from the oral microbiome. Interestingly, the fatty acid hydration activities were different among L. rhamnosus strains; one of the isolated oral strains (DM065) exhibited 0.14 mu mol/g dry cells/min (U/g dry cells) for hydration of linoleic acid, whereas the wellknown probiotics, L. rhamnosus GG (LGG) showed 1.66 U/g dry cells. With an aim to examine the critical factors to determine microbial hydration activities, the FADBH of LGG (OhyLr_LGG) was cloned, engineered to have maltose-binding protein as a soluble fusion partner, and subjected to kinetic and mutation studies. The catalytic efficiencies of OhyLr_LGG for oleic and linoleic acid were 0.102 and 0.066 s- 1 mu M-1, respectively. These values are comparable to those of OhyA from Stenotrophomonas maltophilia (Sm_OhyA2), which was one of the most active enzymes. In addition, the T545K mutation of OhyLr_LGG led to a 76 % reduction in the linoleic acid hydration activity (0.016 s- 1 mu M-1). Since the OhyLr_DM065 possesses K545, the T545K substitution could be one of the major reasons for the lower hydration activities of the DM065 strain. Thereby, it was concluded that the amino acid sequence of FADBH would be one of the key factors to determine the production of hydroxy fatty acids of Lactobacillus probiotics.