| Summary: | 碩士 === 國立臺灣大學 === 農業化學研究所 === 90 === In this study, various agricultural wastes were used as carbon source for thermophilic actinomycete Thermobifida fusca NTU22 to produce extracellular xylanase. The maximum xylanase activity (14.0 U/mL ) could be obtained by using 2% bagasse as carbon source. After the culture filtrate was treated at 70℃ for 30 min, 90% of b-xylosidase activity could be removed. However 100% of xylanase activity was still maintained. After 2% of oat-spelt xylan was hydrolyzed by 10 U/mL of heat-treated xylanase at 60℃ and pH 7.0 for 10 h, the yield of xylooligosaccharides was about 40.1% containing 61% xylobiose, 13% xylotriose and 26% xylotetraose.
Xylooligosaccharides were recovered by active charcoal chromatography. One gram of active charcoal (Norit) could adsorb 35.1 mg of xylooligosaccharides. The xylose and xylooligosaccharides adsorbed on the active charcoal could be eluted by water and 30% ethanol, respectively. The recovery of xylooligosaccharides was about 95% and the purity was about 71.4%.
On the other hand, the optimum medium compositions for production of extracellular peroxidase by T. fusca NTU22 was 2% bagasse, 0.4% NH4NO3, and 0.68% KH2PO4 (pH 9.0). The maximum extracellular peroxidase activity (96.2 mU/mL) could be obtained after cultured at 50℃for 48 h in a 5-L fermentor under the following fermentation conditions : working volume, 3 L； agitation speed, 300 rpm and aeration rate, 1 vvm. The peroxidase was purified through purification steps involving ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. It was found that four different extracellular peroxidases were produced from T. fusca NTU22. The main peroxidase P1 purified 119-fold as to specific activity could be obtained after the successive purification step of preparative electrophoresis. The overall yield of the peroxidase P1 was about 0.37%.
The peroxidase P1 was conformed to be a glycoprotein and lack of heme component in the tertiary structure. The molecular weight was 63 kDa, and the pI value was 4.6. The Km and Vmax for the peroxidase P1 activity were determined to be 2.47 mM and 4.32 U/mg protein, respectively, using 2,4-dicholorophenol (2,4-DCP) as a substrate. The optimum temperature and pH of the enzyme were 70℃ and 7.0. The enzyme exhibited broad pH stability (pH 5.0~9.0) and maintained about 50% activity after treated at 70℃ for 3h.
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