抄録
β-glucosidases (BGLs) plays an important role in enzymatic saccharification of cellulosic biomass by converting cellooligosaccharides to glucose. This final step is critical for efficient production of glucose but most BGLs known to date are commonly inhibited by low (〜mM) concentration of glucose. Therefore, BGLs that are insensitive to glucose have a great biotechnological merit. Here we applied a metagenomic approach to screen for such glucose-tolerant BGLs. A metagenomic library was created in Esherichia coli (approximately 10,000 colonies) and grown on LB-agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-glucoside to yield 828 positive colonies. They were then arrayed in 96-well plates, grown in LB broth and secondary screened for activities in the presence of 10% (w/v) glucose. Seven glucose-tolerant clones were identified in which two types of bgl genes were identified whose deduced amino acid sequences (452 aa, glycosyl hydrolase family 1) were identical each other. The recombinant protein (Ks5A7) was overproduced to be 54 kDa by SDS-PAGE and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45℃ and pH 5.0-6.5, and retained full or slightly enhanced activity in the presence of 0.1-0.5 M glucose. It had low KM (78 μM with p-nitrophenyl β-D-glucoside; 0.36 mM with cellobiose) and high k_<cat> (1045 s^<-1> mM^<-1> with p-nitrophenyl β-D-glucoside; 386 s^<-1> mM^<-1> with cellobiose) and was free from substrate (0.1 M cellobiose) inhibition. The efficient use of Ks5A7 for enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose when added to Trichoderma reesei cellulases.
