99A-9 |
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D.-S. LIAN1, C.-Y. Chang1, J.-R. Too1, and S.-T. Yu2. (1) Department of Bioindustry Technology, Da-Yeh University, 112 Shan-Jiao Rd., Dah-Tsuen, Chang-Hua, 515, Taiwan, (2) Department of Environmental Engineering, Da-Yeh University, 112 Shan-Jiao Rd., Dah-Tsuen, Chang-Hua, 515, Taiwan Chitin, a beta-1, 4-linked homopolymer of N-acetyl-D-glucosamine, is the second most abundant polysaccharides in nature. It can be hydrolyzed to oligosaccharides by microbial chitinases. The physiological functions of N-acetylchitooligosaccharides are important for immune system, including infection preventive, anti-tumor, anti-fungi and anti-bacterial capabilities. The purpose of this study is to produce N-acetylchitooligosaccharides and characterization the chinases by Aeromonas sp. DYU-Too 7, isolated from indigenous soil in Taiwan, using chitin as the major carbon substrate. The activity of chitinase was assayed by colorimetric method to measure the amount of the reducing-end-groups in N-acetylglucosamine (NAG). One unit of the enzyme activity is defined as the amount of enzyme that liberates 1 £gmole of NAG per min at 40 °C and pH 6.5. The hydrates of N-acetylchitooligosaccharides were analyzed by HPLC. The microbes of Aeromonas sp. DYU-Too 7 were cultivated in a 3-L fermenter. There was a maximum production of N-acetylglucosamine, 0.7 g/L after 36 h of cultivation in chitin broth. The concentrations of N-acetylchitobiose (GlcNAc)2 were 0.16 g/L in chitin broth and 0.28 g/L in phosphorus-limiting broth, and the highest concentration of N-acetylchitohexaose (GlcNAc)6 was about 0.06 g/L in both media. It is indicated that limiting a nutrient would affect the hydrolysis of N-acetylchitoologosaccharides. A feasible approach to increase the production of higher-degree-polymer of N-acetylchitooligosaccharides is by controlling the microbial growth and the production of chitinase under limiting a certain nutrient. The characteristics of chitinases from Aeromonas sp. DYU-Too7 were analyzed by concentrated with 20-80% ammonium sulfate and purified using DEAE-sepharose CL-6B and Sephadex G-100. Two extracellular chitinases were separated with the molecular mass of 35 and 60 kDa, respectively determined by SDS-PAGE. The optimum pH and temperature for the both separated chitinases were 4-5 and 60-70°C, respectively.
Session 99A, Biotechnology: General
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