In previous studies, we isolated a novel β-glucosidase from Penicillium oxalicum 16 (16BGL), which is useful for producing cellulosic ethanol. However, 16BGL has a relatively low enzyme activity and product tolerance; besides, a huge gap exists between the optimum temperature of 16BGL (70 °C) and the fermentation temperature for producing cellulosic ethanol (40 °C). Here, we present a directed evolution-based study, which combines one-round error-prone PCR with three rounds of high-throughput screening, for coevolving multiple enzymatic characteristics of 16BGL. We identified an improved variant Y-1-B1 with a triple mutant (G414S/D421V/T441S). Y-1-B1 had an optimum temperature of 50 °C, much closer to the fermentation temperature. The catalytic efficiency of Y-1-B1 for hydrolyzing pNPG was 1355 mM-1 s-1 at 50 °C and pH 5, significantly higher than that of 16BGL (807 mM-1 s-1). Y-1-B1 also achieved a slightly reduced strength of product inhibition of 1.1 at a glucose concentration of 20 mM, compared with the ratio of 1.3 for 16BGL. A maximum titer of 6.9 g/L for ethanol production was achieved in the reaction with Y-1-B1, which was 22% higher than that achieved with 16BGL. Structure modeling revealed that the mutations are distant from the active-site pocket. Therefore, we performed molecular dynamics (MD) simulations to understand why these mutations can improve catalytic efficiency. MD simulation revealed that the nucleophilic residue Asp261 had a much closer contact with the glucosidic center of pNPG in the simulation with Y-1-B1 than that with 16BGL, suggesting that the mutant is more favorable for catalysis. KEY POINTS: • Multiple enzymatic properties of Penicillium oxalicum 16 BGL were coevolved. • A catalytically efficient triple mutant G414S/D421V/T441S was reported. • Molecular dynamics simulation supported the enhanced catalytic activity.

中文翻译：

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通过草酸青霉 16 中 β-葡萄糖苷酶的多种酶学特性的协同进化来提高纤维素乙醇的生产。

在之前的研究中，我们从草酸青霉 16 (16BGL) 中分离出一种新型 β-葡萄糖苷酶，可用于生产纤维素乙醇。然而，16BGL 的酶活性和产品耐受性相对较低；此外，16BGL的最适温度（70°C）与生产纤维素乙醇的发酵温度（40°C）之间存在巨大差距。在这里，我们提出了一项基于定向进化的研究，该研究将一轮易错 PCR 与三轮高通量筛选相结合，共同进化 16BGL 的多种酶学特征。我们鉴定了具有三重突变体 (G414S/D421V/T441S) 的改良变体 Y-1-B1。Y-1-B1 的最适温度为 50 °C，更接近发酵温度。Y-1-B1 在 50 °C 和 pH 5 条件下水解 pNPG 的催化效率为 1355 mM-1 s-1，显着高于 16BGL (807 mM-1 s-1)。在葡萄糖浓度为 20 mM 时，Y-1-B1 的产物抑制强度也略有降低，为 1.1，而 16BGL 的比率为 1.3。在与 Y-1-B1 的反应中，乙醇生产的最大效价为 6.9 g/L，比使用 16BGL 所达到的效价高 22%。结构模型显示突变远离活性位点口袋。因此，我们进行了分子动力学 (MD) 模拟以了解为什么这些突变可以提高催化效率。MD 模拟显示亲核残基 Asp261 在与 Y-1-B1 的模拟中与 pNPG 的糖苷中心的接触比与 16BGL 的更接近，表明该突变体更利于催化。关键点：• 草酸青霉 16 BGL 的多种酶学特性是共同进化的。• 报道了一种催化高效的三重突变体 G414S/D421V/T441S。• 分子动力学模拟支持增强的催化活性。