Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of β-glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL-A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in β-glucosidase activity at 25 and 50°C compared to the wild type, without compromising high-temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL-A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard-informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes.

中文翻译：

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基于热点向导的超嗜热 β-葡萄糖苷酶工程，可增强低温下的酶活性


超耐热酶由于其高热稳定性而成为工业酶的重要来源。不幸的是，大多数超嗜热酶在低温（例如环境温度）下活性降低，限制了它们的适用性。此外，进化超嗜热酶以提高低温活性而不损害其他所需特性通常是困难的。在当前的研究中，通过在 HotSpot Wizard 分析指导下构建饱和诱变文库，然后筛选其活性和热稳定性，对来自激烈火球菌(PfBGL) 的 β-葡萄糖苷酶变体进行了改造，以增强低温下的酶活性。通过该文库构建和筛选，一种包含 Q214S/A264S/F344I 突变的 PfBGL 突变体 PfBGL-A4 在 25 和 50°C 下的 β-葡萄糖苷酶活性比野生型增加两倍以上，且不影响高温活性、热稳定性和底物特异性。我们的实验和计算特征表明，PfBGL-A4 的发现可能是由于活性位点周围局部构象灵活性的提高，同时稍微压缩了整体蛋白质结构。这项研究展示了基于 HotSpot Wizard 的超嗜热酶工程的潜力，并强调了这些酶的温度、酶活性和构象灵活性之间的相互作用。