A metagenomic approach to discover a novel β-glucosidase from bovine rumens

Abstract

Abstractβ-Glucosidases play an important role in biomass degradation as they hydrolyze cellobiose to glucose in a final step of cellulolysis. In particular, ruminant animals rely onβ-glucosidases from rumen microorganisms for conversion of plant cellulosic materials into glucose. In this study, we are interested in characterization of a novelβ-glucosidase from rumen microorganisms. However, most rumen microorganisms are obligate anaerobes, which require special cultivation conditions. Presently, the metagenomic techniques, which enable isolation and characterization of microbial genes directly from environmental samples, have been applied to overcome these problems. In this study, the sequence-based screening approach was successfully applied to identify a novelβ-glucosidase gene,Br2, from a bovine rumen metagenomic sample. A 1338-bp complete coding sequence ofBr2encodes a 51-kDa GH1β-glucosidase of 445 amino acid residues with 59% sequence identity to aβ-glucosidase fromCellulosilyticum ruminicolaJCM 14822. The recombinantly expressed Br2 exhibited an optimal activity at pH 6.5 and 40°C, reflecting its rumen bacterial origin, and relatively higher catalytic efficiencies toward glucoside and fucoside substrates than other glycosides, similar to many previously reported bacterialβ-glucosidases. Our sequence-based screening approach can be applied to identify other genes of interest from environmental samples.

Structural and mutational analysis of glycoside hydrolase family 1 Br2 β-glucosidase derived from bovine rumen metagenome

“…The catalytic residues (Glu163 and Glu350) and those likely involved in substrate binding in Br2 (GenBank accession number APM84071 ) were targeted for site-directed mutagenesis. PCR-based site-directed mutagenesis reactions [ 14 ] were performed using sense and antisense primers ( Supplementary Table S1 ) and the pET15b-Br2 plasmid as a template [ 3 ]. The sequences of Br2 mutants were confirmed by DNA sequencing before transformation into E. coli BL21(DE3) by electroporation.…”

“…The recombinant Br2 showed an apparent subunit molecular weight of 53 kDa, and optimal activity at pH 6.5 and 40 °C. Interestingly, Br2 lacks the signal peptide for extracellular secretion and exhibited very low efficiency toward cellobiose, suggesting that Br2 was probably not involved in cellulolysis as we initially assumed [ 3 ]. Therefore, in this study, we aimed to further characterize Br2 by determining its three-dimensional structure and the amino acid residues responsible for substrate specificities toward various glucooligosaccharides.…”

“…Among these enzymes, β -glucosidases play an essential role in cellulolysis as they hydrolyze cellobiose to glucose; in addition, β -glucosidases participate in the hydrolysis of various glucosides involved in physiological functions, such as plant‒microbe and plant‒insect interactions, hormone activation, defense mechanisms, degradation of glycolipids and exogenous glucosides, cell wall catabolism, and lignification [ 2 ]. β -Glucosidases are present in all organisms, including the residential microbes in the bovine rumen [ 3 ]. Since the structure of the cyanogenic β -glucosidase from Trifolium repens was first elucidated in 1995 (PDB code 1CBG ) [ 4 ], all members of GH1 have been found to exhibit a common ( β / α ) 8 -barrel fold.…”

“…Most reported β -glucosidases from rumen metagenomes belong to the GH3 family [ [9] , [10] , [11] ], but only the structures of a GH3 broad-specificity β -glucosidase/ β -xylosidase from cow rumen are available [ 12 ]. On the other hand, only two GH1 enzymes from the ruminal metagenome have been characterized thus far, which are Br2 and PersiBGL1 from bovine and sheep rumens, respectively [ 3 , 13 ], but their three-dimensional structures have yet to be reported.…”

“…These include endo- β -1,4-glucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91), and β -glucosidases [ 11 ]. We previously employed a metagenomic approach to identify a 1338-bp gene from a bovine rumen sample, which encodes a novel 445-amino acid GH1 Br2 β -glucosidase [ 3 ]. The residues Glu163 and Glu350 were identified as the acid/base catalyst and nucleophile, respectively, by amino acid sequence alignment with other GH1 β -glucosidases ( Supplementary Fig.…”