| Cellulose, one of the most important renewable resources on the earth, and its relevant hydrolysis enzymes, has become the current research hotspots around the world. The alkaline cellulases, which were first found in70s year last century, with the feature of the maximum activity in the alkaline pH range, have been widely utilized in a variety of industries. Among the alkaline cellulases distributed in the nature, the alkaline cellulase produced by bacterial are the currently most important and have been extensively utilized in a variety of industrial sectors, such as the production of detergent, textile and pulp, and the environmental protection, et al. Therefore, it is very significance to explore the novel alkaline cellulases, investigate and improve their enzymatic features.In this dissertation, a Gram-positive bacterial strain that produced high activity CMCase was isolated from the samples collected at Wuming of Nanning, southern of China, and nominated as C73. The strain had rod shaped cell, and was identified as a strain of Jonesia sp by sequence blast of16rDNA. When cultivating the strain in the medium containing1%CMC,1%tryptone,0.5%yeast extract and0.5%NaCl, under372℃,200rpm for40h, the CMCase activity in broth was1.34U/ml. By optimizing the medium formula using the orthogonal analysis, the activity in broth was enhanced36.6%, reached to1.83U/ml under the same condition. The optimum formula of medium for culturing the strain C73for producing the CMCase was1%CMC,1%tryptone and0.5%NaCl (eliminating the yeast extract). The Box-Behnken Design (BBD) response surface analysis was used for further optimizing the cultivating condition for CMCase production. The CMCase activity in broth was further enhanced to2.24U/ml,67.2%higher than that before condition was optimized. The optimized culture condition was that the inoculation was2%, the initial pH value was9.4, the culture temperature was37℃, the rotation of shake flask culture was250rpm and the culture time was50h.The characteristics of CMCase produced by C73in broth were further primarily studied. The results showed that the optimum pH for CMCase activity was pH10, the optimum temperature was50℃, the activity of enzyme was maintaining stable under pH8.0-10.0and temperature of40-55℃, Ca2+, Na+, Li+and Mn2+, Co2+,(NH4)2SO4were activators and inhibitors respectively to CMCase activity, and Zn2+, Pb2+, Ba2+, Cu2+, Fe2+, Mg2+, Ni2+, SDS, EDTA did not exhibit significantly effect on the enzyme activity. The results indicated that the CMCase produced by strain C73was an alkaline cellulase.Based on the activity characteristics, the alkaline cellulase was purified from the broth of C73cultivation by Superdex75chromatography and ion exchange chromatography. A purified CMCase was obtained by the verification of a single protein band in the map of PAGE electrophoresis. After purification the relative activity of the alkaline CMCaes was enhanced5.53-fold comparing the activity in broth, reaching to370.95U/mg protein. The apparent Km and Vmax of the purified CMCase were2.418mM/L and3.484mM/L/min. Comparing with the activity in broth the purified CMCase was more sensitive to the changes of pH and temperature, and the enzyme stability was decreased to the change of reaction condition.Referring to the conserved domains (sugar binding motif), the genes full sequences, and the conserved sequences of the alkaline celluloses previously investigated, and the relevant genes sequences of alkaline cellulase from homologous strains, a variety of degenerate primers were designed using CODEHOP software for PCR amplification of the alkaline cellulase gene from the genome DNA of the strain C73. However, no specific sequence was amplified. Since no Jonesia sp that produced alkaline cellulase was reported previously, we argue that the Strain C73is a novel bacterial strain producing alkaline cellulase and the CMCase produced by C73is much possible as a novel alkaline cellulase. The further effort to explore the gene of the alkaline cellulase should be addressed. |
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