The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form
The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form
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DOI：
10.1128/jb.179.1.46-52.1997
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发表时间：
1997-01
影响因子：
3.2
通讯作者：
C. Reverbel‐Leroy;S. Pagès;A. Bélaich;J. Belaich;C. Tardif
中科院分区：
文献类型：
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作者：
C. Reverbel‐Leroy;S. Pagès;A. Bélaich;J. Belaich;C. Tardif
由C-末端组氨酸尾标记的溶纤维梭菌的纤维素酶CelF的重组形式在大肠杆菌中过量产生。融合蛋白经Ni-次氮基三乙酸柱亲和层析纯化。完整形式的CelF（Mr，79，000）在C末端迅速降解，产生较短的稳定形式，称为截短的CelF（Mr，71，000）。完整和截短的纯化形式均降解无定形纤维素（kcat分别为42和30 min（-1））和微晶纤维素（kcat分别为13和10 min（-1））。从无定形纤维素中释放的可溶性还原末端与不溶性还原末端的高比率表明CelF是一种加工酶。然而，在反应开始时由截短的CelF从磷酸溶胀的纤维素中释放的纤维糊精的多样性表明该酶可能随机水解β-1，4键。粘度测量和CelF和内切葡聚糖酶CelA能够降解一些相同的纤维素位点的发现支持了这一假设。因此，CelF被称为加工性内切纤维素酶。免疫印迹分析结果表明，CelF与C.纤维素分解菌它被鉴定为纤维素体的三个主要成分之一。通过相互作用蛋白质印迹（免疫印迹）和使用基于BIAcore生物传感器的分析系统的结合测定来监测CelF的整个形式与CipC（多纤维素酶体整合蛋白）或mini-CipC 1（CipC的重组截短形式）相互作用的能力。
The recombinant form of the cellulase CelF of Clostridium cellulolyticum, tagged by a C-terminal histine tail, was overproduced in Escherichia coli. The fusion protein was purified by affinity chromatography on a Ni-nitrilotriacetic acid column. The intact form of CelF (Mr, 79,000) was rapidly degraded at the C terminus, giving a shorter stable form, called truncated CelF (Mr, 71,000). Both the entire and the truncated purified forms degraded amorphous cellulose (kcat = 42 and 30 min(-1), respectively) and microcrystalline cellulose (kcat = 13 and 10 min(-1), respectively). The high ratio of soluble reducing ends to insoluble reducing ends released by truncated CelF from amorphous cellulose showed that CelF is a processive enzyme. Nevertheless, the diversity of the cellodextrins released by truncated CelF from phosphoric acid-swollen cellulose at the beginning of the reaction indicated that the enzyme might randomly hydrolyze beta-1,4 bonds. This hypothesis was supported by viscosimetric measurements and by the finding that CelF and the endoglucanase CelA are able to degrade some of the same cellulose sites. CelF was therefore called a processive endocellulase. The results of immunoblotting analysis showed that CelF was associated with the cellulosome of C. cellulolyticum. It was identified as one of the three major components of cellulosomes. The ability of the entire form of CelF to interact with CipC, the cellulosome integrating protein, or mini-CipC1, a recombinant truncated form of CipC, was monitored by interaction Western blotting (immunoblotting) and by binding assays using a BIAcore biosensor-based analytical system.