Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance.
Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance.
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设计高活性嗜热 β-葡萄糖苷酶以增强其 pH 稳定性和糖化性能
DOI：
10.1186/s13068-016-0560-8
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发表时间：
2016
影响因子：
6.3
通讯作者：
Yao B
中科院分区：
文献类型：
--
作者：
Xia W;Xu X;Qian L;Shi P;Bai Y;Luo H;Ma R;Yao B
β-葡萄糖苷酶是生物质降解酶系统的重要成员，在生物燃料生产的酶促糖化中发挥着至关重要的作用。在工业实践中，在高温和不同pH值下具有高活性和高稳定性的候选物始终是首选。为了实现具有成本效益的生物质转化，探索天然酶、开发高水平表达系统和工程优良突变体是常用的有效方法。新鉴定的 GH3 β-葡萄糖苷酶 Bgl3A（来自 Talaromyces leycettanus JCM12802）在酵母菌株毕赤酵母 GS115 中过表达，在 3 L 发酵罐中产生了 6000 U/ml 的粗酶活性。纯化的酶表现出出色的酶学特性，包括适宜的温度和 pH 值（75 °C 和 pH 4.5）、良好的热稳定性（在 60 °C 下保持稳定）和高催化性能（对 pNPG 的比活性和催化效率分别为 905 U/mg 和 9096/s/mM）。然而，Bgl3A 在 pH 4.0-5.0 范围内的稳定性较窄，限制了其工业应用。进一步的定点诱变表明过度的 O-糖基化在 pH 敏感性中的作用。通过去除潜在的 O-糖基化位点，两个突变体在更宽的 pH 范围 (3.0–10.0) 内表现出更高的 pH 稳定性。此外，与野生型Bgl3A相比，突变体M1在pH 5.0和50℃下具有更好的稳定性，与纤维素酶Celluclast 1.5L配合，糖化效率大幅提高。突变体M1的糖化性能与商业β-葡萄糖苷酶Novozyme 188大致相当，且具有相同的β-葡萄糖苷酶活性，表明其在生物燃料生产中的巨大前景。在本研究中，我们在毕赤酵母中过表达了一种具有高比活性和高催化效率的新型β-葡萄糖苷酶Bgl3A。我们进一步证明了过度的O-糖基化对Bgl3A pH稳定性的负面影响，并通过减少O-糖基化来增强pH稳定性。增强的突变体显示出更好的应用前景，显着提高了纤维素材料的糖化效率。本文的在线版本 (doi:10.1186/s13068-016-0560-8) 包含补充材料，可供授权用户使用。
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. 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. 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. The online version of this article (doi:10.1186/s13068-016-0560-8) contains supplementary material, which is available to authorized users.