کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
4752827 1416372 2017 40 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Cloning, expression, and biochemical characterization of a novel NADP+-dependent 7α-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
Cloning, expression, and biochemical characterization of a novel NADP+-dependent 7α-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids
چکیده انگلیسی
A gene encoding a novel 7α-specific NADP+-dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60 °C, but the enzyme is inactivated at this temperature with a half-life time of 5 min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition. In order to demonstrate the applicability of this enzyme, a small-scale biotransformation of the bile acid chenodeoxycholic acid (CDCA) into 7-ketolithocholic acid (7-KLCA) was carried out with simultaneous regeneration of NADP+ using an NADPH oxidase that resulted in a complete conversion (<99%). Furthermore, by a structure-based site-directed mutagenesis, cofactor specificity of the 7α-HSDH from Clostridium difficile was altered to accept NAD(H). This mutant was biochemically characterized and compared to the wild-type.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Enzyme and Microbial Technology - Volume 99, April 2017, Pages 16-24
نویسندگان
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