Division of Applied Life Sciences (BK21), PMBBRC, Gyeongsang National University, 900 Gajwa-dong, Jinju, Gyeongnam 660-701, Republic of Korea.
Bioresour Technol. 2012 May;112:10-7. doi: 10.1016/j.biortech.2012.02.066. Epub 2012 Feb 22.
The chimeric proteins viz. CBM3-Cel9A, CBM4-Cel9A and CBM30-Cel9A, are constructed by fusion of family 3, 4, and 30 cellulose binding modules (CBMs) to N-terminus of family 9 endoglucanase (Cel9A) from Alicyclobacillus acidocaldrious. The chimeric enzymes were successfully expressed in Escherichia coli and purified to homogeneity. The chimeric enzymes showed significant increase in Avicel (8-12 folds) and filter paper (7-10 folds) degradation activities compared to Cel9A endoglucanase. Computational protein modeling and simulation on the chimeric enzymes were applied to analyze the fused CBMs effect on the increased insoluble cellulosic substrates degradation activity. Thin layer chromatography analysis of the enzymatic hydrolysis products and distribution of reducing sugars between soluble and insoluble fractions indicated processive cleavage of insoluble cellulosic substrates by the chimeras. The fused CBMs played a critical accessory role for the Cel9A catalytic domain and changed its character to facilitate the processive cleavage of insoluble cellulosic substrates.
构建了融合了家族 3、4 和 30 纤维素结合模块（CBMs）的杂合蛋白 CBM3-Cel9A、CBM4-Cel9A 和 CBM30-Cel9A，这些杂合蛋白的 N 端融合了来自极端耐热嗜酸热杆菌的家族 9 内切葡聚糖酶（Cel9A）。这些杂合酶在大肠杆菌中成功表达并纯化至均一性。与 Cel9A 内切葡聚糖酶相比，杂合酶对滤纸（7-10 倍）和 Avicel（8-12 倍）的降解活性显著提高。对杂合酶进行计算蛋白质建模和模拟，以分析融合的 CBM 对增加的不溶性纤维素底物降解活性的影响。酶解产物的薄层层析分析以及可溶性和不溶性部分之间还原糖的分布表明，杂合酶对不溶性纤维素底物进行了连续切割。融合的 CBMs 对 Cel9A 催化结构域起着至关重要的辅助作用，并改变了其性质，以促进不溶性纤维素底物的连续切割。
