| Summary: | 碩士 === 國立臺東大學 === 生命科學系碩士班 === 106 === α-Galactosidase can be applied to food, sugur, fodder, paper and medicine. The thermostable α-galactosidase of Anoxybacillus flavithermus HY-TTH-D23 (rAFGAL) was cloned into E. coli BL21 (DE3) for heteroexpression.and characterization (rAFGAL-GFP). The rAFGAL-GFP is about 112 kDa, and is the first α-galactosisase cloned from Anoxybacillus flavithermus. This study demonstrates that rAFGAL-GFP produced from E. coli BL21 (DE3) pET GFP rAFGAL, both in the form of inclusion body (IB) and soluble protein. Over 95% of total rAFGAL-GFP was in IB form. There is not obvious effect for reducing the ratio of IB by lowering the culturing temperature, but it was still beneficial to inhence the yeild and the specific activity of soluble rAFGAL-GFP. However, IB could be refolded with urea and Ni-NTA column by dilution or on-column refolding, but the producitivity too low to scale up. As the result shows, the condition of culturing at 37°Ϲ, without glucose and IPTG induction, for 12 hours following by sonication with 1% Triton X-100 and heating at 50°Ϲ for 30 min can achieve the highest producitivity (53.13 ± 8.39 U/L, 11.18 ± 1.33 U/mg, and 2.62 NTD/U). Compared to privious study, the cost has been reduced by 50%. As the product, rAFGAL-GFP keeps activity at pH 7-10 with optimal temperature range from 45-55°Ϲ, and can catalysis melibiose, raffinose and stachyose, espacially melibiose (kcat/Km = 245.20/ mM•s). The results of this study could contribute to the knowledge of Anoxybacillus ssp. α-galactosidase and its application for industrial processes in food, fodder and paper.
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