| 论文题目: | Site-saturation mutagenesis of central tyrosine 195 leading to diverse product specificities of an alpha-cyclodextrin glycosyltransferase from Paenibacillus sp 602-1 |
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| 作者: | Xie Ting, Song Binghong, Yue Yang, Chao Yapeng*, Qian Shijun |
| 联系作者: | Chao Yapeng* |
| 刊物名称: | Journal of Biotechnology |
| 期: | 170 |
| 卷: | |
| 页: | 10-16 |
| 年份: | 2014 |
| 影响因子: | 3.34 |
| 论文下载: | |
| 摘要: | Central tyrosine 195 plays an important role in the active site of cyclodextrin glycosyltransferase (CGTase) that is highly conservative among various CGTases. However, a detailed functional understanding of this subsite is lacking. In this study, we applied site-directed saturation mutagenesis to investigate the effect of tyrosine 195 on the hydrolytic activity and cyclization specificity of an alpha-CGTase. A total of 17 mutant CGTases were obtained and heterologously expressed in E. coli. The mutant Y195F alpha-CGTase showed similar characteristics with wild-type a-CGTase. The other mutant a-CGTases showed considerably lower activity for starch-degradation and cyclodextrin (CD) formation. Interestingly, we found that the main product of mutant Y195R a-CGTase was gamma-CDs (50%), not alpha-CDs (35%). The mutant Y195I alpha-CGTase drastically altered the CD specificity of alpha-CGTase, which showed a switch toward the synthesis of both beta- and gamma-CDs with percentages of 34% and 38%, respectively. Other mutant CGTases retained the alpha-CD as the main product but with lower percentages than wild-type alpha-CGTase. Mutant Y195F, Y195I, and Y195R CGTases showed an optimal temperature of 50 degrees C and pH 6.5. The mutants Y195I and Y195R also showed better thermostability. These findings suggested that aromatic amino acids Tyr or Phe at the 195 position were important for the amylolytic activity and cyclization specificity of a-CGTase. The mutants Y195I CGTase and Y195R CGTase have potential applications for gamma-CD production in the future. (C) 2013 Elsevier B.V. All rights reserved. |
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