Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance

Xia et al. Biotechnol Biofuels (2016) 9:147 DOI 10.1186/s13068-016-0560-8 Biotechnology for Biofuels Open Access RESEARCH Engineering a highly active thermophilic β‑glucosidase to enhance its pH stability and saccharification performance Wei Xia1,2†, Xinxin Xu3†, Lichun Qian2, Pengjun Shi1*, Yingguo Bai1, Huiying Luo1, Rui Ma1 and Bin Yao1* 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, 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. Conclusions: In 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. Keywords: β-Glucosidase, Talaromyce leycettanus, Saccharification, pH stability, O-glycosylation, Pichia pastoris Background 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]. For the closest *Correspondence: ; ; † Wei Xia and Xinxin Xu contributed equally to this paper 1 Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People’s Republic of China Full list of author information is available at the end of the article decades, developing efficient technologies to convert biomass materials into fuels has attracted focused attention of researchers [2, 3]. Moreover, the biodegradation of cellulosic materials has been reported to have potential importance in kinds of industrial and agricultural applications [4–7]. Some glycoside hydrolases (GHs) are the most effective enzymes to depolymerize cellulose. As generally known, endo-β-glucanase (EC 3.2.1.4, EG) that catalyzes the breakdown of internal β-1,4-linkages at random position of the glucose polymers, cellobiohydrolase (EC 3.2.1.91, CBH I and CBH II) that cuts off cellobiose © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 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]. In detail, 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. And recent researches reveal that a class of enzymes now known as lytic polysaccharide monooxygenases (LPMOs) are also important for the decomposition of recalcitrant biological macromolecules such as plant cell wall and chitin polymers [9]. 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 now classified as auxiliary activities 9 (formerly GH61), 10 (formerly CBM33) and 11 in the CAZy database [11]. Several cellulolytic GHs have been commercialized for industrial production of biofuels and chemicals [12–15]. For example, Celluclast 1.5L (Novo Nodisk A/S) from Trichoderma reesei ATCC 26921 and newly developed Cellic® CTec2 and Cellic® CTec3 are the most widely used commercial cellulolytic preparation [16, 17]. However, its low β-glucosidase activity makes supplementation of exogenous enzyme necessary for efficient biomass conversion [18, 19]. Since β-glucosidase plays a vital role in cellulose hydrolysis by undertaking the ratelimiting final step of hydrolyzing cellobiose, which is an intermediate product of cellulose hydrolysis and also a strong inhibitor of cellulase activities, into glucose [20, 21], it’s a common practice to supplement exogenous β-glucosidase to enhance the saccharification efficiency of cellulosic materials [22–24]. This challenge remains a major bottleneck in the bioconversion process, and recent research has, therefore, shown increased interest in the search for novel β-glucosidases. Based on the amino acid sequences, β-glucosidases have been classified into GH families 1, 3, 5, 9, 30 and 116. Although enzymes from different families and different organisms vary greatly in properties and functions, co (...truncated)