Objectives To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering Results Two novel cellulases, Bp GH9 and Bp GH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. Bp GH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. Bp GH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-β1,4 glucanes. A truncated form of Bp GH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of Bp GH9 on crystalline cellulose. Conclusions A new alkaliphilic endocellulase Bp GH9, was cloned and engineered as a fusion protein (CBM3a- Bp GH9), which led to an improved activity on crystalline cellulose.

中文翻译：

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两种新的 GH9 和 GH48 纤维素酶的表征和改造，它们来自从博戈里亚湖分离的短小芽孢杆菌

目的通过分子工程寻找新的嗜碱性纤维素酶并提高它们对结晶纤维素的效率 结果鉴定、克隆和生化表征了来自短小芽孢杆菌菌株的两种新型纤维素酶 Bp GH9 和 Bp GH48。Bp GH9 是属于糖苷水解酶 9 家族 (GH9) 的模块化内切纤维素酶，其中包含催化模块 (GH) 和属于 3 类和 c 亚类 (CBM3c) 的碳水化合物结合模块。该酶对高碱性 pH 具有极强的耐受性，并在 pH 10 时保持显着活性。Bp GH48 是一种外切纤维素酶，属于糖苷水解酶 48 家族 (GH48)，作用于低聚 β1,4 葡聚糖的还原端。构建了截短形式的 Bp GH9 和具有额外 CBM3a 模块的嵌合融合体。CBM3c 模块的删除导致催化活性显着下降。然而，CBM​​3a 的融合虽然处于非天然位置，但增强了 Bp GH9 对结晶纤维素的活性。结论 一种新的嗜碱性内切纤维素酶 Bp GH9 被克隆并设计为融合蛋白 (CBM3a-Bp GH9)，从而提高了对结晶纤维素的活性。