Fungus-derived GH-7 family cellobiohydrolase I (CBHI, EC 3.2.1.91) is one of the most important industrial enzymes for cellulosic biomass saccharification. Talaromyces cellulolyticus is well known as a mesophilic fungus producing a high amount of CBHI. Thermostability enhances the economic value of enzymes by making them more robust. However, CBHI has proven difficult to engineer, a fact that stems in part from its low expression in heterozygous hosts and its complex structure. Here, we report the successful improvement of the thermostability of CBHI from T. cellulolyticus using our homologous expression system and protein engineering method. We examined the key structures that seem to contribute to its thermostability using the 3D structural information of CBHI. Some parts of the structure of the Talaromyces emersonii CBHI were grafted into T. cellulolyticus CBHI and thermostable mutant CBHIs were constructed. The thermostability was primarily because of the improvement in the loop structures, and the positive effects of the mutations for thermostability were additive. By combing the mutations, the constructed thermophilic CBHI exhibits high hydrolytic activity toward crystalline cellulose with an optimum temperature at over 70°C. In addition, the strategy can be applied to the construction of the other thermostable CBHIs.

中文翻译：

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通过蛋白质工程从真菌Talaromycescellulolyticus构建热稳定的纤维二糖水解酶I。

真菌衍生的GH-7家族纤维二糖水解酶I（CBHI，EC 3.2.1.91）是纤维素生物质糖化最重要的工业酶之一。纤维素分解Talaromyces cellulolyticus是产生大量CBHI的中温真菌。通过使酶更稳定，热稳定性提高了酶的经济价值。然而，事实证明CBHI难以工程改造，这一事实部分源于其在杂合宿主中的低表达及其复杂的结构。在这里，我们报道了使用我们的同源表达系统和蛋白质工程方法成功改善了溶纤支球菌CBHI的热稳定性。我们使用CBHI的3D结构信息检查了似乎有助于其热稳定性的关键结构。艾美氏菌（Talaromyces emersonii）CBHI的某些结构被嫁接到T中。构建了纤维素分解酶CBHI和热稳定突变体CBHI。热稳定性主要是由于环结构的改善，而突变对热稳定性的积极影响是可加的。通过组合突变，所构建的嗜热CBHI在高于70°C的最佳温度下对结晶纤维素表现出高水解活性。另外，该策略可以应用于其他热稳定的CBHI的构造。所构建的嗜热CBHI在超过70°C的最佳温度下，对结晶纤维素表现出高水解活性。另外，该策略可以应用于其他热稳定的CBHI的构造。所构建的嗜热CBHI在超过70°C的最佳温度下，对结晶纤维素表现出高水解活性。另外，该策略可以应用于其他热稳定的CBHI的构造。