Studies on heat tolerance and substrate-recognition mechanism of cellulase

本研究旨在通过基因分析和蛋白鉴定，了解热胞梭菌和粪孢梭菌F-9纤维素酶的耐热性和底物识别特性。所得结果如下：AC.stercorarium基因编码纤维素酶XynB，全长1101 bp，共367个氨基酸，不含半胱氨酸，属于纤维素酶家族f。第6保守区ITELD的谷氨酸被认为是该酶的活性中心。其他保守区域的谷氨酸和天冬氨酸被评估为对底物的识别作出贡献。纯化后的XynB除显示木聚糖酶活性外，还显示纤维素酶活性。该酶在pH6.1、80C时具有最大活性，在pH6.1、100C加热10 min后，在60C时恢复了约60%的初始活性。由于蛋白质聚集体的形成，这种活性恢复在pH5.8时可以忽略不计。8 M尿素对团聚体增溶后活性有部分恢复，表明活性损失除可逆变性外，还可能由不可逆变性引起。差示扫描量热法（DSC）分析表明，该蛋白具有倒绕肽形成天然蛋白结构的能力。为了研究底物识别机制，在XynB中加入了底物类似物1-脱氧吉莉明，它与酶水解糖苷键的活性中心结合，但没有被抑制。这表明底物识别机制与淀粉酶不同。用硝酸钠修饰C.thermocellum cellulase（CelC）基因形成随机突变。该突变基因编码纤维素酶，其中107Glu和209Lys两个氨基酸分别被替换为107Gly和209 Glu。这种突变降低了温度和最佳pH值。添加

The present research aimed at getting some properties of heat tolerance and substrate recognition of cellulases from Clostridium thermocellum and Clostridium stercorarium F-9 by gene analysis and protein characterization. The results obtained here were as follows ; AC.stercorarium gene consisting of 1101 bp, i.c., 367 amino acids with no cysteine was encoded a cellulase XynB, which belongs to cellulase family F.In the amino acid sequence of the protein, 8 conserved regions were found outo by homology analysis. The glutamic acid in the 6th conserved region, ITELD, was estimated to be the active center of the enzyme. Glutamic and aspartic acids in the other conserved regions were evaluated to contribute to the recognition of substrates. Purified XynB revealed cellulase activity in addition to the xylanase activity. This enzyme had maximum activity at pH6.1, 80C and recovered about 60% of initial activity at 60C, even after heating at 100C for 10 min at pH 6.1. This activity recovery was negligible at pH5.8 due to the formation of protein aggregates. Solubilization of the aggregates with 8 M urea showed a partial activity recovery, suggested that activity loss might be caused by the irreversible denaturation in addition to the reversible denaturation. Differential scanning calorimetry (DSC) analysis showed that this protein had a power to rewind the peptide for forming a native protein structure. For studying on the substrate-recognition mechanism, a substrate analog, 1-deoxynogirimicine which binds to active center of the enzyme hydrolyzing glucoside bond, was added to XynB but it was not inhibited. This indicate that the substrate recognition mechanism is different from amylase. The C.thermocellum cellulase(CelC) gene was modified by Sodium nitrate to form random mutation. The mutated gene was encoded the cellulase, in which two amino acids, 107Glu and 209Lys, were replaced to 107Gly and 209 Glu, respectively. This mutation lowered temperature and pH optima. Adding