| Summary: | 碩士 === 靜宜大學 === 化粧品科學系 === 104 === Thermobifida fusca BCRC 19214 is a thermophilic actinobacteria with ability to degrade plant biomass or other organic compounds and to catalyze oxidation of phenolic compounds. In this study, the superoxide dismutase gene (Tfu 0957) was cloned into pET Dute-1 expression vector and expressed in Escherichia coli BL21 (DE3) expression host. According to the results of this study, the optimize induction conditions were induced with 0.1mM IPTG at 16oC for 20 hrs that could make the minimal insoluble inclusion bodies. The recombinant superoxide dismutase can be easily purified by Ni2+-NTA chromatography. According to the activity affected by different metal ions, the enzyme belongs to zinc-copper containing oxidase.
Furthermore, the Tfu 0957 and xylanase genes were cloned into pPICαKan and pGAPZαA expression vectors, respectively and then transformed successively into Pichia pastoris X-33 by electroporation. The transformant, P. pastoris-Tfu 0957 Xyl selected by YPDS plates containing 100 μg/mL Zeocin and 4 mg/mL G418, could express two recombinant proteins, one is constitutively expressed xylanase and another is inducibly expressed Tfu 0957. After cultivation in YPD for 5 days and methanol induction at 48 hours, 0.65014 U/mL of Tfu 0957 and 20.9 U/mL of xylanase activities were harvested using 2,6-dimethoxyphenol (2,6-DMP) and xylan as substrates, respectively. This enzyme using sugar cane bagasse as substrate has 12.85 U/mL hydrolytic activity.
|
|---|
