Identification Of Crystalline Cellulose-degrading Thermophilic Bacterium, Purification And Characterization Of Avicelase

Cellulose is the major polysaccharidic compound in plants and the most abundant renewable resource on earth. This bioconversion plays an important role in global carbon cycle. The cellulolytic enzymes from thermophil, show high thermostability and strong catalytic efficiency, have attracted much interest because of their enormous potential to convert cellulose. The potential has been reported in various industrial processes and agriculture in the expanding research and development.In our Lab, a thermopholic cellulolytic bacteria was isolated from Techong hot springs in Yunnan province, China. In this paper, we identified its classification, studied on its conditions for avicelase produced, reported the purification and characterization of the avicelase.1. TC-1 strain was a gram-negative facultative aerobe with an optimum growth temperature of about 60℃, able to grow on a completely defined medium with a wide range of carbohydrates, including Avicel, Carboxymethylcellulose, xylan and glucose. On the basis of morphological, physiologic and biochemical characteristics, and 16S rDNA phenotypic analysis, it was ascribed to the genus Meiothermus. The growth of Meiothermus occurred between 40～70℃, many strains had been isolated from land springs and thermo-tun, they had been scarcely reported the degradation of Avicel. So the avicelase from TC-1 strain needs further research.2. TC-1 strain was capable of utilizing Avicel as sole carbon and energy source at 60℃, and could produce strong cellulolytic activity induced by Avicel. The activity on Avicel could reach to 0.17 Units/mg. The result of avicelase activities about different cell compostions showed that TC-1 had an extacellular avicelase. 3. For achieving the production of avicelase secreted by TC-1, "two culture was been taken on the process of the fermentation. The protein purification was performed by DEAE-Sephrose Fast Flow ion exchange and gel filtration chromatography. The avicelase was purified about 9.15-fold to a specific activity of 0.0368 Units/mg with Avicel substrate. According to gel diffusion assay, we obtained a molecular mass of 142 kDa protein with some subunits.4. The maximal activity of the enzyme with the substrate of Avicel was at pH6.0 and 70°C, respectively. The studies on pH and temperature stability showed that it was stable enough between pH5.5～6.5 at 70℃for 1 h, and more than 80 % of the activity still remained when incubation was prolonged to 1 h at 70℃. Among the tested polysaccharides, the avicelase could catalyzed hydrolysis of CMC, Xylan and Avicel.