微紫青霉外切葡聚糖纤维二糖水解酶(CBHI)的纤维素结合结构域及其链结区非水解性破坏纤维素结晶区结构(英文)  

Non-hydrolytic Disruption of Crystalline Structure of Cellulose by Cellulose Binding Domain and Linker Sequence of Cellobiohydrolase I from Penicillium janthinellum

作　　者：高培基[1] 陈冠军[1] 汪天虹[1] 张颖舒[1] 刘洁[1] 

机构地区：[1]山东大学微生物技术国家重点实验室,济南250100

出　　处：《生物化学与生物物理学报》2001年第1期13-18,共6页

基　　金：国家自然科学基金资助项目 !No .39430 0 20&&

摘　　要：天然纤维素的结晶区必需在内、外切纤维素酶的协同作用下 ,始可被降解 ,这是纤维素降解的限速步骤。内、外切纤维素酶均为β 1,4 糖苷键的水解酶 ,但单一的内、外切纤维素酶却都不能水解天然纤维素的结晶区。内、外切纤维素酶怎样协同降解纤维素的机理一直未得阐明 ,是天然纤维素降解机制研究中的难点。纤维素酶分子是由具有催化功能的催化结构域(catalyticdomain ,CD)和具有结合纤维素功能的纤维素结合 (吸附 )结构域 (cellulosebindingdomain ,CBD)及连结它们的链结区 (linker)序列组成。已知一细菌的CBD在吸附纤维素后 ,导致纤维素聚合物断裂形成短小纤维 ,但这一现象还未在真菌中有类似发现。通过对插入质粒 pUC 18上的微紫青霉外切葡聚糖纤维二糖水解酶CBHI的cDNA基因 ,进行系列序列定向缺失等体外操作 ,得到了催化结构域序列缺失的重组质粒 ,转化大肠杆菌JM10 9后 ,利用纤维素结合结构域CBD可吸附纤维素的特性 ,筛选到含CBD编码区的转化子PUC18G ,生产出了LacZ CBD融合蛋白。经木瓜蛋白酶有限酶切后 ,分离纯化得到了CBD结构域及其链结区称为 :CBDCBHI。经X光衍射、红外光谱分析、热活力测定和扫描电镜观察表明 ,CBDCBHI吸附纤维素后 ,能够导致纤维素聚合物氢键断裂 ,结晶度减低和形成短纤维 。The cooperation between cellobiohydrolase (CBHI) and endoglucanase (EG) is necessary for biodegradation of native cellulose, but its mechanism is still poorly understood. The present paper report at the first time that an isolated component, the cellulose binding domain with its linker sequence of cellobiohydrolase I from Penicillium janthinellum (CBD CBHI ), plays an important role in the synergism between CBHI and EGI during cellulose biodegradation. A recombinant plasmid (pUC18C), containing the gene fragment encoding CBD CBHI from P. janthinellum was derived from pUC18 181. In pUC 18C, the catalytic domain region of cbh I gene was deleted by in vitro DNA manipulations and then E.coli JM 109 was transformed for the production of LacZ CBD fusion protein. The active LacZ CBD fusion protein was digested by papain and then purified by re exclusion chromatography. The purified peptide sequence of CBD CBHI had the ability of binding crystalline cellulose. The detailed morphological and structural changes of cotton fibers after binding CBD CBHI were investigated by using scanning electron microscopy, calorimetric activity and X ray diffraction. The results demonstrated that the CBD CBHI not only has a high binding capacity to cellulose, but also causes non hydrolytic disruption of crystalline cellulose, which leads to the release of short fibers. IR spectroscopy and X ray diffraction show that destabilization is caused by the non hydrolytic disruption of cellulose and the disruption of hydrogen bonds in crystalline cellulose. The efficiency of crystalline cellulose degradation was enhanced by synergistic action of CBD CBHI with EGI. These results suggest that the cellulose binding domain with its linker plays an important role in crystalline cellulose degradation.

关 键 词：纤维素降解 外切纤维素酶I 纤维素 结合结构域 

分 类 号：Q946[生物学—植物学]