Characterization of a Novel β-Glucosidase from a Compost Microbial Metagenome with Strong Transglycosylation Activity

Taku Uchiyama,Kentaro Miyazaki,Katsuro Yaoi

doi:10.1074/jbc.m113.471342

Abstract

The β-glucosidase encoded by the td2f2 gene was isolated from a compost microbial metagenomic library by functional screening. The protein was identified to be a member of the glycoside hydrolase family 1 and was overexpressed in Escherichia coli, purified, and biochemically characterized. The recombinant β-glucosidase, Td2F2, exhibited enzymatic activity with β-glycosidic substrates, with preferences for glucose, fucose, and galactose. Hydrolysis occurred at the nonreducing end and in an exo manner. The order of catalytic efficiency for glucodisaccharides and cellooligosaccharides was sophorose > cellotetraose > cellotriose > laminaribiose > cellobiose > cellopentaose > gentiobiose, respectively. Intriguingly, the p-nitrophenyl-β-D-glucopyranoside hydrolysis activity of Td2F2 was activated by various monosaccharides and sugar alcohols. At a D-glucose concentration of 1000 mM, enzyme activity was 6.7-fold higher than that observed in the absence of D-glucose. With 31.3 mM D-glucose, Td2F2 catalyzed transglycosylation to generate sophorose, laminaribiose, cellobiose, and gentiobiose. Transglycosylation products were detected under all activated conditions, suggesting that the activity enhancement induced by monosaccharides and sugar alcohols may be due to the transglycosylation activity of the enzyme. These results show that Td2F2 obtained from a compost microbial metagenome may be a potent candidate for industrial applications.

Highlights

There is an ongoing search for a ␤-glucosidase that has insensitivity to product inhibition
Screening of Clones with ␤-Glucosidase Activity from the Metagenomic Library—Many positive clones exhibiting ␤-glucosidase activity were screened from the metagenomic library
Corona௡ CAD analyses revealed that the reaction of Td2F2 with pNPGlc plus 31.3 mM D-glucose resulted in the transglycosylation products laminaribiose, cellobiose, sophorose, and gentiobiose (Fig. 7)

Summary

Background

There is an ongoing search for a ␤-glucosidase that has insensitivity to product inhibition. The search for a ␤-glucosidase that is insensitive to product inhibition by glucose has been newly reinvigorated Due to their ability to activate glycosidic bonds, ␤-glucosidases have been considered as promising biocatalysts for the synthesis of stereo- and regiospecific glycosides or oligosaccharides, which are in turn are potentially useful as functional materials, nutraceuticals, or pharmaceuticals because of their biological recognition, signaling mechanisms, and antibiotic properties [1]. Td2F2 exhibits the highest potential for use in cellulosic ethanol production and in the synthesis of stereo- and regiospecific glycosides, which is afforded by insensitivity to glucose inhibition and a relatively high transglycosylation activity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION