期刊
CHEMBIOCHEM
卷 11, 期 4, 页码 533-538出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200900711
关键词
acid/base catalysts; enzyme catalysis; hydrolases; synthesis; thioglycoligases
资金
- Natural Sciences and Engineering Research Council of Canada
- Austrian Science Fund
- Erwin Schrodinger Fellowship
Thioglycoligases are engineered enzymes for the synthesis of thioglycosides that are derived from retaining glycosidases by replacing the acid/base catalyst. The optimal choice of substitution for the acid/base mutant is currently unknown, so to investigate this question a complete acid/base library of the model glycosidase Bacillus circulans xylanase (Bcx) was generated by using site-saturation mutagenesis. A novel screening approach combining active site titration with semiquantitative product analysis by thin layer chromatography was established and used to evaluate specific activities of each mutant enzyme within crude cell lysates. The six most active Bcx variants were analyzed in more detail, a pH optimum of 8.5 was established and the identity of reaction products was confirmed. Optimal choices for substitution were small, preferably polar amino acids such as threonine, cysteine, and serine. We discuss the resultant data in the context of previously published studies on thioglycoligases.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据