Abstract
Backgroundβ-Glucosidase is an important member of the biomass-degrading enzyme system, and plays vital roles in enzymatic saccharification for biofuels production. Candidates with high activity and great stability over high temperature and varied pHs are always preferred in industrial practice. To achieve cost-effective biomass conversion, exploring natural enzymes, developing high level expression systems and engineering superior mutants are effective approaches commonly used.ResultsA newly identified β-glucosidase of GH3, Bgl3A, from Talaromyces leycettanus JCM12802, was overexpressed in yeast strain Pichia pastoris GS115, yielding a crude enzyme activity of 6000 U/ml in a 3 L fermentation tank. The purified enzyme exhibited outstanding enzymatic properties, including favorable temperature and pH optima (75 °C and pH 4.5), good thermostability (maintaining stable at 60 °C), and high catalytic performance (with a specific activity and catalytic efficiency of 905 U/mg and 9096/s/mM on pNPG, respectively). However, the narrow stability of Bgl3A at pH 4.0–5.0 would limit its industrial applications. Further site-directed mutagenesis indicated the role of excessive O-glycosylation in pH liability. By removing the potential O-glycosylation sites, two mutants showed improved pH stability over a broader pH range (3.0–10.0). Besides, with better stability under pH 5.0 and 50 °C compared with wild type Bgl3A, saccharification efficiency of mutant M1 was improved substantially cooperating with cellulase Celluclast 1.5L. And mutant M1 reached approximately equivalent saccharification performance to commercial β-glucosidase Novozyme 188 with identical β-glucosidase activity, suggesting its great prospect in biofuels production.ConclusionsIn this study, we overexpressed a novel β-glucosidase Bgl3A with high specific activity and high catalytic efficiency in P. pastoris. We further proved the negative effect of excessive O-glycosylation on the pH stability of Bgl3A, and enhanced the pH stability by reducing the O-glycosylation. And the enhanced mutants showed much better application prospect with substantially improved saccharification efficiency on cellulosic materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0560-8) contains supplementary material, which is available to authorized users.
Highlights
As one of the most abundant renewable energy sources on Earth, plant biomass mainly consists of lignocellulose, which is a complicated heterogeneous complex made up of hemicellulose, lignin and cellulose [1]
EGs catalyze the breakdown of internal β-1, 4-linkages at random position of the glucose polymer chain, while CBHs cut-off cellobiose residues from the ends (CBH I and CBH II cuts from the reducing and nonreducing ends, respectively)
At the last step, generated cellobiose or cello-oligosaccharides are hydrolyzed into single units of glucose from the nonreducing end by β-glucosidases
Summary
As one of the most abundant renewable energy sources on Earth, plant biomass mainly consists of lignocellulose, which is a complicated heterogeneous complex made up of hemicellulose, lignin and cellulose [1]. Xia et al Biotechnol Biofuels (2016) 9:147 residues from the reducing or nonreducing ends, and β-glucosidase (EC 3.2.1.21) that hydrolyzes single units from the nonreducing end into glucose [3, 8]. LPMOs cleave the chains at the surface of the crystalline polymer by oxidation of the polysaccharide chain to contribute to further enzymatic action and eventual degradation [10]. These enzymes were originally designated glycoside hydrolase family 61 and carbohydrate-binding module family 33, but are classified as auxiliary activities 9 (formerly GH61), (formerly CBM33) and in the CAZy database [11]
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