Ogonda Lydia A, Saumonneau Amélie, Dion Michel, Muge Edward K, Wamalwa Benson M, Mulaa Francis J, Tellier Charles
Université de Nantes, CNRS, UFIP, UMR6286, 2, rue de la Houssinière, 44322, Nantes, France.
Department of Biochemistry, School of Medicine, College of Health Sciences, University of Nairobi, P.O BOX 30197-00100, Nairobi, Kenya.
Biotechnol Lett. 2021 Mar;43(3):691-700. doi: 10.1007/s10529-020-03056-z. Epub 2021 Jan 1.
To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering RESULTS: Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 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. BpGH48 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 BpGH9 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 BpGH9 on crystalline cellulose.
A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.
寻找新型嗜碱纤维素酶，并通过分子工程提高其对结晶纤维素的作用效率。结果：从一株短小芽孢杆菌中鉴定、克隆并对两种新型纤维素酶BpGH9和BpGH48进行了生化特性分析。BpGH9是一种模块化内切纤维素酶，属于糖苷水解酶9家族（GH9），包含一个催化模块（GH）和一个属于3类c亚类的碳水化合物结合模块（CBM3c）。该酶对高碱性pH具有极强的耐受性，在pH 10时仍具有显著活性。BpGH48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BpGH9的截短形式以及与额外CBM3a模块的嵌合融合体。CBM3c模块的缺失导致催化活性显著下降。然而，CBM3a的融合，尽管处于非天然位置，却增强了BpGH9对结晶纤维素的活性。结论：克隆了一种新型嗜碱内切纤维素酶BpGH9，并将其工程化为融合蛋白（CBM3a-BpGH9），从而提高了其对结晶纤维素的活性。
