Identification and Characterization of Bacterial Alliinase: Resource and Substrate Stereospecificity.

Limited alliinase resources cause difficulties in the biosynthesis of thiosulfinates (e.g., allicin), restricting their applications in the agricultural and food industries. To effectively biosynthesize thiosulfinates, this study aimed to excavate bacterial alliinase resources and elucidate their catalytic properties. Two bacterial cystathionine β-lyases (MetCs) possessing high alliinase activity (>60 U mg ) toward L-(-)-alliin were identified from rhizosphere isolates. Metagenomic exploration revealed that cystathionine β-lyase from (BcPatB) possessed high activity toward both L-(±)-alliin and L-(+)-alliin (208.6 and 225.1 U mg ), respectively. Although these enzymes all preferred l-cysteine S-conjugate sulfoxides as substrates, BcPatB had a closer phylogenetic relationship with alliinases and shared several similar features with alliinase. Interestingly, the TrpIleAlaAsp Met motif in a cuspate loop of BcPatB, especially sites 31 and 32 at the top of the motif, was modeled to locate near the sulfoxide of L-(+)-alliin and is important for substrate stereospecificity. Moreover, the stereoselectivity and activity of mutants I31V and A32G were higher toward L-(+)-alliin than those of mutant I31L/D33E toward L-(-)-alliin. Using bacterial alliinases and chemically synthesized substrates, we obtained thiosulfinates with high antimicrobial and antinematode activities that could provide insights into the protection of crops and food.