Cloning, expression, and characterization of two β-glucosidases from isoflavone glycoside-hydrolyzing Bacillus subtilis natto

On the basis of the genomic sequence of Bacillus subtilis 168, two beta-glucosidase genes (bgIH and yckE) from B. subtilis natto, which has been reported to have high isoflavone glucoside-hydrolyzing activity, were cloned and overexpressed in E coli M15. The temperature for the optimal p-nitrophenyl-beta-D-glucoside hydrolyzing activity of both enzymes was between 37 and 45 degrees C, but BgIH had a higher thermal stability than YckE. Both showed high activity at pH 6.0, but YckE was stable over a wider pH range than BgIH. Recombinant BgIH was inhibited 73%, 63%, and 43% by 1.0 mM Cd2+, Fe2+, or Cu2+, respectively, while other divalent metal ions resulted in 0-23% inhibition, whereas YckE was inhibited by less than 20% by any of the divalent metal ions we tested. Among the substrate we used, BgIH showed the highest affinity for genistin and YckE showed the highest affinity for p-nitrophenyl-beta-8-D-fructopyranoside. Both BgIH and YckE hydrolyzed genistin and daidzin into their isoflavone aglycones, genistein and daidzein, but BgIH was more efficient than YckE in isoflavone glucoside hydrolysis (20-fold higher k(cat)). Our results suggest that recombinant BgIH may be applicable in the process of isoflavones deglycosylation.