Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Xia, Wei; Xu, Xinxin; Qian, Lichun; Shi, Pengjun; Bai, Yingguo; Luo, Huiying; Ma, Rui; Yao, Bin
• Is Part Of: Biotechnology for biofuels. Volume 9:Issue 1(2016)
• Page Start: 1
• Page End: 12
• Publication Date: 2016-12
• Subjects: β-Glucosidase -- Talaromyce leycettanus -- Saccharification -- pH stability -- O-glycosylation -- Pichia pastoris; Biotechnology -- Periodicals; Biomass energy -- Periodicals; Energy-Generating Resources -- Periodicals; 662.88
• DOI: 10.1186/s13068-016-0560-8
• Languages: English
• ISSNs: 1754-6834

Abstract

Abstract Background β-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. Results A newly identified β-glucosidase of GH3, Bgl3A, fromTalaromyces leycettanus JCM12802, was overexpressed in yeast strainPichia 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 onp NPG, 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 excessiveO -glycosylation in pH liability. By removing the potentialO -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. Conclusions In this study, we overexpressed a novel β-glucosidase Bgl3A with high specific activity and high catalytic efficiency inP. pastoris. We further proved the negative effect of excessiveO -glycosylation on the pH stability of Bgl3A, and enhanced the pH stability by reducing theO -glycosylation. And the enhanced mutants showed much better application prospect with substantially improved saccharification efficiency on cellulosic materials.