Kao Mu-Rong, Yu Su-May, Ho Tuan-H Ua David
Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and National Defense Medical Center, Taipei, 115, Taiwan.
Institute of Molecular Biology, Academia Sinica, Taipei, 115, Taiwan.
Biotechnol Biofuels. 2021 May 31;14(1):126. doi: 10.1186/s13068-021-01973-3.
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 V 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 V 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.
β-葡萄糖苷酶通过催化纤维寡糖和纤维二糖最终降解为葡萄糖，对纤维素水解至关重要。D2-BGL是一种从嗜热毁丝霉中分离得到的真菌糖苷水解酶家族3（GH3）β-葡萄糖苷酶，具有高底物亲和力，是里氏木霉纤维素酶混合物用于木质纤维素生物质糖化的一种高效β-葡萄糖苷酶补充剂。
我们进行了易错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上都有两个氨基酸的替换，在酿酒酵母的初步突变体筛选中表现出活性增加。随后在毕赤酵母中表达研究了单个氨基酸替换对催化效率和酶产量的影响。F256M替换导致对底物抑制的耐受性和比活性增强，D224G替换导致重组酶产量增加。通过组合有益突变D224G、F256M和Y260D构建了产生的最佳D2-BGL突变体Mut M。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提高2.7倍、更高的Vmax以及对纤维二糖更高的底物抑制耐受性。令人惊讶的是，Mut M的过表达诱导内质网未折叠蛋白反应的水平低于毕赤酵母中WT D2过表达时的水平。当与里氏木霉纤维素酶制剂Celluclast 1.5L组合时，Mut M比WT D2更有效地水解酸预处理的甘蔗渣。
通过定向进化方法成功产生了D2-BGL突变体Mut M。Mut M携带三个在GH3β-葡萄糖苷酶的其他定向进化研究中未报道的突变，并且该突变体比野生型酶表现出对底物抑制更高的耐受性和更高的Vmax。除了比活性增强外，Mut M在毕赤酵母中过表达时还表现出比WT D2更高的蛋白滴度。我们的研究表明，通过蛋白质工程可以提高纤维素分解酶的催化效率和生产力。
