Abstract
BackgroundCellulose, an abundant and renewable polysaccharides, constitutes the largest resource for bioconversion of biofuels. Plant polysaccharides hydrolysis is catalyzed by cellulases, which include endoglucanases, exoglucanases, and β-glucosidases. Converting cellulose and hemicellulose to short chains of oligosaccharides by endo-/exoglucanases is the key step for biofuel transformation. Intriguingly, β-glucanases with transglycosylation activity not only can relieve product inhibition of glucan hydrolysis but also has potential application as biocatalysts for functional materials.ResultsHere, a metagenomic fosmid library was constructed from a paddy soil for cellulase screening. One purified clone showing carboxymethylcellulase activity was isolated, and the complete β-glucanase gene (umcel9y-1) was cloned and overexpressed in Escherichia coli. Phylogenetic analysis indicated that β-glucanase Umcel9y-1 belonged to the theme C of glycoside hydrolase family 9. Amino acids sequence showed 58.4 % similarity between Umcel9y-1 and its closest characterized reference, cellulase Cel01. Biological characterization showed that Umcel9y-1 was an efficient endoglucanase and also exhibited high activities of exoglucanase and transglycosylation. The transglycosylation products of Umcel9y-1 including sophorose, laminaribiose, and gentiobiose, and transglycosylation was detected under all activated conditions. The order of catalytic efficiency for polysaccharides, cellooligosaccharides, and aryl-β-glycosides was p-nitrophenol-D-cellobioside, barley glucan, cellopentaose, cellotetraose, cellotriose, hydroxyethylcellulose, cellohexose, laminarin, and carboxymethylcellulose, respectively. The barley glucan was the optimal polysaccharides for Umcel9y-1 with Km and Kcat/Km values of 13.700 mM and 239.152 s−1 mM−1, respectively.ConclusionBiological characterizations of recombinant Umcel9y-1 showed that the versatile β-glucanase had efficient endoglucanase activity to barley glucan and also exhibited high activities of exoglucanase and transglycosylation. The optimum conditions of recombinant Umcel9y-1 was pH 6.5–7.0 at 37 °C with predominant halotolerance and high-thermal stability. These results indicate that the novel metagenomic-derived β-glucanase may be a potent candidate for industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0449-6) contains supplementary material, which is available to authorized users.
Highlights
Cellulose, an abundant and renewable polysaccharides, constitutes the largest resource for bioconversion of biofuels
Cellulases with transglycosylation activity are attracted as substitute of glycosyltransferases for the synthesis of stereo- and regiospecific glycosides which are considered as functional materials, nutraceuticals, or pharmaceuticals [9, 10]
The CMCase gene umcel9y-1 was expected to encode a protein of 680 amino acid residues (Additional file 1: Figure S1) and was identified as endoglucanase Umcel9y-1 consisting of an immunoglobulin-like domain (Cel-N) at the N-terminal (Amino acids 76–161th: pfam 02927) and an endoglucanase catalytic domain (Amino acids 166–621th: pfam 00759) (Additional file 2: Figure S2)
Summary
An abundant and renewable polysaccharides, constitutes the largest resource for bioconversion of biofuels. Plant polysaccharides hydrolysis is catalyzed by cellulases, which include endoglucanases, exoglucanases, and β-glucosidases. Cellulose is the most abundant and renewable polysaccharide and constitutes one-third of all existing plant cell-wall material, which is the largest potential resource for bioconversion of ethanol and other biofuels, feedstock chemicals, and pharmaceuticals [2,3,4]. Cellulases catalyze cellulolysis through hydrolyzing β-1, 4-glycosidic bonds in cellulose, and mainly consisted of three types of enzymes: endoglucanases (EC3.2.1.4), exoglucanase. Cellulases (including β-glucanase) with transglycosylation activity are attracted as substitute of glycosyltransferases for the synthesis of stereo- and regiospecific glycosides (or oligosaccharides) which are considered as functional materials, nutraceuticals, or pharmaceuticals [9, 10]. Biochemical characterizations of cellulases require intensive efforts but are likely to generate new opportunities for the use of renewable resource as biofuels, food and drug additives, and other agricultural products [6,7,8,9,10,11,12]
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