| Co-Investigator(Kenkyū-buntansha) |
TANAKA Akiyoshi Mie Univ., Fac.Education, Assoc.Professor, 生物資源学部, 助教授 (10155111)
SAKKA Kazuo Mie Univ., Fac.Bioresources, Assoc.Professor, 生物資源学部, 助教授 (20154031)
SHIMADA Kyo Mie Univ., Fac.Bioresources, Professor, 生物資源学部, 教授 (20024549)
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| Research Abstract |
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
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