Studies on heat tolerance and substrate-recognition mechanism of cellulase

本研究旨在通过对热纤梭菌和粪梭菌F-9纤维素酶的基因分析和蛋白质鉴定，获得其耐热性和底物识别特性。结果表明，粪生AC. stercorarium基因由1101 bp组成，XynB是纤维素酶家族F的一个成员，编码367个氨基酸，不含半胱氨酸。第6保守区ITELD中的谷氨酸可能是该酶的活性中心。评估了其他保守区域中的谷氨酸和天冬氨酸对底物识别的贡献。纯化的XynB除了具有木聚糖酶活性外，还具有纤维素酶活性。该酶在pH6.1，80 ℃时活性最高，在60 ℃时，即使在pH6.1，100 ℃加热10分钟后，仍能恢复约60%的初始活性。由于蛋白质聚集体的形成，在pH 5.8下该活性恢复可忽略不计。用8M尿素溶解聚集体后，其活性部分恢复，表明除了可逆变性外，不可逆变性也可能是导致活性丧失的原因。差示扫描量热法（DSC）分析表明，该蛋白具有能力，以形成一个天然的蛋白质结构的肽倒带。为了研究底物识别机制，将与水解糖苷键的酶的活性中心结合的底物类似物1-deoxynogirimicine添加到XynB中，但其不被抑制。这表明底物识别机制与淀粉酶不同。采用硝酸钠对热纤梭菌纤维素酶基因进行随机突变。突变基因编码纤维素酶，其中107 Glu和209 Lys分别被替换为107 Gly和209 Glu。这种突变降低了最适温度和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