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標題: | Enzymatic characterization of Bacillus licheniformis gamma-glutamyltranspeptidase fused with N-terminally truncated forms of Bacillus sp TS-23 alpha-amylase |
作者: | Hu, Hui-Yu Yang, Jia-Ci Chen, Jiau-Hua Chi, Meng-Chun Lin, Long-Liu |
貢獻者: | 食品科技系 |
關鍵字: | Bacillus Licheniformis Gamma-Glutamyltranspeptidase Autocatalytic Processing Alpha-Amylase Starch-Binding Domain |
日期: | 2012-07-15 |
上傳時間: | 2014-03-21 16:15:10 (UTC+8) |
出版者: | Elsevier Science Inc |
摘要: | Bacillus licheniformis gamma-glutamyltranspeptidase (BIGGT) was fused at its C-terminal end with N-terminally truncated forms of Bacillus sp. TS-23 alpha-amylase. BIGGT and six fusion enzymes, BIGGT/SBD, BIGGT/AMY Delta N476, BIGGT/AMY Delta N443, BIGGT/AMY Delta N376, BIGGT/AMY Delta N195, and BIGGT/AMY Delta N34, were over-expressed in Escherichia coli M15 cells and purified to apparent homogeneity by metal-affinity chromatography. The fusion constructions had no significant effect on the autocatalytic processing of BIGGT. Progressive decrease in the GGT activity of fusion proteins was associated with an increasing level of truncation, and only BIGGT/AMY Delta N34 reserved the amylolytic activity. The protein fusions did not alter the optimal temperature and pH of BIGGT. However, as compared with the parental BIGGT, a significant change in circular dichorism and fluorescence spectra was observed in the fusion enzymes. Thermal unfolding of BIGGT, BIGGT/AMY Delta N476, BIGGT/AMY Delta N443, and BIGGT/AMY Delta N376 followed the two-state unfolding process with a transition point (T-m) of 61.3-63.1 degrees C, whereas BIGGT/AMY Delta N195 and BIGGT/AMY Delta N34 displayed two temperature transitions at 40.6 and 46.7 degrees C as well as at 62.8 and 62.9 degrees C, respectively. All of the fusion enzymes exhibited the raw-starch-binding ability, and the adsorbed proteins could be eluted from the adsorbent by 50 mM Tris-HCl (pH 9.0) containing 2% soluble starch. (c) 2012 Elsevier Inc. All rights reserved. |
關聯: | Enzyme And Microbial Technology, 51(2), 86-94 |
顯示於類別: | [ 食品科技系 ] 期刊論文
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