Dataset Information

Cloning and characterization of a new ?-glucosidase from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis.

ABSTRACT: BACKGROUND: The study on the second generation bio-fuel is a hot area of current research of renewable energy. Among series of key points in this area, the role of ?-glucosidase in the degradation of intermediate gluco-oligosaccharides limits the rate of the complete saccharification of lignocellulose. RESULTS: In this study, a new ?-glucosidase gene, unglu135B12, which was isolated from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis by the function-based screening, encodes a 779 amino acid polypeptide that contains a catalytic domain belonging to glycoside hydrolase family 3 (GH3). It was recombinantly expressed, purified and biochemically characterized. The recombinant ?-glucosidase, unglu135B12, displayed optimum enzymatic activity at pH 5.0 at 38°C, and showed the highest specific activity of 2.5 × 10(3) U/mg under this optimal condition to p-nitrophenyl-?-D-glucopyranoside (pNPG), and its Km and Vmax values were 0.309 mmol/L and 7.292 ?mol/min, respectively. In addition, the presence of Ca2+, K+, Na+ slightly improved ?-glucosidase activity of unglu135B12 by about 5%, while about 10~85% loss of ?-glucosidase activity was induced by addition of Mn2+, Fe3+, Zn2+, Cu2+. Interestingly, unglu135B12 was activated by glucose at the concentration lower than 40 mM. CONCLUSIONS: Our findings indicate that unglu135B12 is a new ?-glucosidase derived from rumen of cattle, and it might be a potent candidate for saccharification of lignocellulose in industrial application.

SUBMITTER: Li Y

PROVIDER: S-EPMC4287584 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Cloning and characterization of a new β-glucosidase from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis.

Li Yadan Y Liu Ning N Yang Hui H Zhao Fei F Yu Ye Y Tian Yun Y Lu Xiangyang X

BMC biotechnology 20141002

<h4>Background</h4>The study on the second generation bio-fuel is a hot area of current research of renewable energy. Among series of key points in this area, the role of β-glucosidase in the degradation of intermediate gluco-oligosaccharides limits the rate of the complete saccharification of lignocellulose.<h4>Results</h4>In this study, a new β-glucosidase gene, unglu135B12, which was isolated from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis by the function-based ...[more]

PMID: 25274487

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Project description:BACKGROUND:Enzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance. RESULTS:Here, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Escherichia coli. Four of the eight enzymes had significant activities on either pNP-?-d-galactopyranoside, pNP-?-d-xylopyranoside, pNP-?-l-arabinopyranoside or pNP-?-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the pNP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a ?-galactosidase (74%) and (2) a ?-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The ?-galactosidase- and ?-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the ?-xylosidase-like protein 2 showed activities with both pNP-Xyl and pNP-Ara in the temperature range 40-50 °C and pH range 8.0-10.0. Moreover, proteins 5 and 6 showed thermotolerant ?-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential "GGDEF" catalytic site, encoding ?-glucosidase activity, whereas that of protein 6 showed a "GDSL" site, encoding a 'new family' ?-glucosidase activity. CONCLUSION:Using a rational screening approach, we identified and characterized four thermo-alkaliphilic glycosyl hydrolases that have the potential to serve as constituents of enzyme cocktails that produce sugars from lignocellulosic plant remains.

Dataset Information

Cloning and characterization of a new ?-glucosidase from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis.

Publications

Cloning and characterization of a new β-glucosidase from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis.

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