β-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) β-glucosidase isolated from Chaetomella raphigera with high substrate affinity, and is an efficient β-glucosidase supplement to Trichoderma reesei cellulase mixtures for the saccharification of lignocellulosic biomass. We have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in Saccharomyces cerevisiae. Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in Pichia pastoris. Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in Pichia pastoris resulted in 2.7-fold higher production of recombinant protein, higher Vmax and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in P. pastoris. When combined with the T. reesei cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2. D2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 β-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher Vmax than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in P. pastoris. Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.

中文翻译：

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通过定向进化提高毕赤酵母中纤维素分解 β-葡萄糖苷酶 D2-BGL 的生产力和糖化效率

β-葡萄糖苷酶通过催化纤维寡聚体和纤维二糖最终降解为葡萄糖而对纤维素水解至关重要。D2-BGL 是一种真菌糖苷水解酶家族 3 (GH3) β-葡萄糖苷酶，从毛毛虫中分离，具有高底物亲和力，是里氏木霉纤维素酶混合物的有效 ​​β-葡萄糖苷酶补充剂，用于木质纤维素生物质的糖化。我们进行了容易出错的 PCR，以进一步提高野生型 (WT) D2-BGL 的催化效率。三个突变体，每个在 D2-BGL 上替换了两个氨基酸，在酿酒酵母中的初步突变体筛选中表现出增加的活性。通过随后在毕赤酵母中的表达研究了单个氨基酸置换对催化效率和酶产生的影响。替代F256M导致增强对底物抑制的耐受性和比活性，替代D224G导致增加重组酶的产生。产生的最佳 D2-BGL 突变体 Mut M 是通过组合有益突变 D224G、F256M 和 Y260D 构建的。与野生型酶相比，Mut M 在毕赤酵母中的表达导致重组蛋白产量高 2.7 倍，Vmax 更高，对纤维二糖的底物抑制耐受性更高。令人惊讶的是，Mut M 过表达诱导 ER 未折叠蛋白反应的水平低于 P. pastoris 中 WT D2 过表达的水平。当与里氏木霉纤维素酶制剂 Celluclast 1.5L 结合使用时，Mut M 比 WT D2 更有效地水解酸预处理的甘蔗渣。D2-BGL 突变体 Mut M 通过以下定向进化方法成功生成。Mut M 携带三个突变，这些突变在 GH3 β-葡萄糖苷酶的其他定向进化研究中没有报道，与野生型酶相比，该突变体对底物抑制表现出更大的耐受性和更高的 Vmax。除了增强的比活性外，Mut M 在 P. pastoris 中过表达时还表现出比 WT D2 更高的蛋白质效价。我们的研究表明，可以通过蛋白质工程提高纤维素分解酶的催化效率和生产力。除了增强的比活性外，Mut M 在 P. pastoris 中过表达时还表现出比 WT D2 更高的蛋白质效价。我们的研究表明，可以通过蛋白质工程提高纤维素分解酶的催化效率和生产力。除了增强的比活性外，Mut M 在 P. pastoris 中过表达时还表现出比 WT D2 更高的蛋白质效价。我们的研究表明，可以通过蛋白质工程提高纤维素分解酶的催化效率和生产力。