Background:To produce second-generation biofuels, enzymatic catalysis is required to convert cellulose from lignocellulosic biomass into fermentable sugars. β-Glucosidases finalize the process by hydrolyzing cellobiose into glucose, so the efficiency of cellulose hydrolysis largely depends on the quantity and quality of these enzymes used during saccharification. Accordingly, to reduce biofuel production costs, new microbial strains are needed that can produce highly efficient enzymes on a large scale. Results:We heterologously expressed the fungal β-glucosidase D2-BGL from a Taiwanese indigenous fungus Chaetomella raphigera in Pichia pastoris for constitutive production by fermentation. Recombinant D2-BGL presented significantly higher substrate affinity than the commercial β-glucosidase Novozyme 188 (N188; Km = 0.2 vs 2.14 mM for p-nitrophenyl β-d-glucopyranoside and 0.96 vs 2.38 mM for cellobiose). When combined with RUT-C30 cellulases, it hydrolyzed acid-pretreated lignocellulosic biomasses more efficiently than the commercial cellulase mixture CTec3. The extent of conversion from cellulose to glucose was 83% for sugarcane bagasse and 63% for rice straws. Compared to N188, use of D2-BGL halved the time necessary to produce maximal levels of ethanol by a semi-simultaneous saccharification and fermentation process. We upscaled production of recombinant D2-BGL to 33.6 U/mL within 15 days using a 1-ton bioreactor. Crystal structure analysis revealed that D2-BGL belongs to glycoside hydrolase (GH) family 3. Removing the N-glycosylation N68 or O-glycosylation T431 residues by site-directed mutagenesis negatively affected enzyme production in P. pastoris. The F256 substrate-binding residue in D2-BGL is located in a shorter loop surrounding the active site pocket relative to that of Aspergillus β-glucosidases, and this short loop is responsible for its high substrate affinity toward cellobiose. Conclusions:D2-BGL is an efficient supplement for lignocellulosic biomass saccharification, and we upscaled production of this enzyme using a 1-ton bioreactor. Enzyme production could be further improved using optimized fermentation, which could reduce biofuel production costs. Our structure analysis of D2-BGL offers new insights into GH3 β-glucosidases, which will be useful for strain improvements via a structure-based mutagenesis approach.

译文

背景:为生产第二代生物燃料,需要酶催化才能将纤维素从木质纤维素生物质转化为可发酵糖。 β-葡糖苷酶通过将纤维二糖水解为葡萄糖来完成这一过程,因此纤维素水解的效率很大程度上取决于糖化过程中所用这些酶的数量和质量。因此,为了降低生物燃料的生产成本,需要可以大规模生产高效酶的新微生物菌株。
结果:我们在巴斯德毕赤酵母中异源表达了台湾原产的一种真菌,即南美白对虾中的真菌β-葡萄糖苷酶D2-BGL,用于发酵生产。重组D2-BGL的底物亲和力明显高于商品β-葡萄糖苷酶Novozyme 188(N188;对硝基苯基β-d-吡喃葡萄糖苷Km = 0.2 vs 2.14mM,纤维二糖Km = 0.2 vs 2.14mM)。当与RUT-C30纤维素酶结合时,它比商业纤维素酶混合物CTec3更有效地水解酸预处理的木质纤维素生物质。对于甘蔗渣,从纤维素到葡萄糖的转化程度为83%,对于稻草为63%。与N188相比,D2-BGL的使用通过半同时糖化和发酵过程生产最大量乙醇所需的时间减少了一半。我们使用1吨生物反应器在15天内将重组D2-BGL的生产规模提升至33.6 U / mL。晶体结构分析表明D2-BGL属于糖苷水解酶(GH)家族3。通过定点诱变去除N-糖基化N68或O-糖基化T431残基会对巴斯德毕赤酵母中的酶产生产生负面影响。相对于曲霉β-葡萄糖苷酶,D2-BGL中的F256底物结合残基位于围绕活性位点袋的较短环中,并且该短环是其对纤维二糖的高底物亲和力的原因。
结论:D2-BGL是木质纤维素生物质糖化的有效补充剂,我们使用1吨生物反应器提高了该酶的生产规模。通过优化发酵可以进一步改善酶的生产,这可以降低生物燃料的生产成本。我们对D2-BGL的结构分析为GH3β-葡萄糖苷酶提供了新的见解,这将有助于通过基于结构的诱变方法改善菌株。

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