Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7 alpha-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids

Bakonyi D, Hummel W (2017)
ENZYME AND MICROBIAL TECHNOLOGY 99: 16-24.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Abstract / Bemerkung
A gene encoding a novel 7 alpha-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 degrees C, but the enzyme is inactivated at this temperature with a half-life time of 5 min. Contrary to other known 7 alpha-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 7 alpha-HSDH from Clostridium difficile was altered to accept NAD(H). This mutant was biochemically characterized and compared to the wild-type.(C) 2016 Published by Elsevier Inc.
Stichworte
7 alpha-hydroxysteroid dehydrogenase; Clostridium difficile; Bile acids; 7-ketolithocholic acid; Cofactor specificity; NAD(P)H oxidase
Erscheinungsjahr
2017
Zeitschriftentitel
ENZYME AND MICROBIAL TECHNOLOGY
Band
99
Seite(n)
16-24
ISSN
0141-0229
eISSN
1879-0909
Page URI
https://pub.uni-bielefeld.de/record/2916563

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Bakonyi D, Hummel W. Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7 alpha-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids. ENZYME AND MICROBIAL TECHNOLOGY. 2017;99:16-24.
Bakonyi, D., & Hummel, W. (2017). Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7 alpha-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids. ENZYME AND MICROBIAL TECHNOLOGY, 99, 16-24. doi:10.1016/j.enzmictec.2016.12.006
Bakonyi, D., and Hummel, W. (2017). Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7 alpha-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids. ENZYME AND MICROBIAL TECHNOLOGY 99, 16-24.
Bakonyi, D., & Hummel, W., 2017. Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7 alpha-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids. ENZYME AND MICROBIAL TECHNOLOGY, 99, p 16-24.
D. Bakonyi and W. Hummel, “Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7 alpha-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids”, ENZYME AND MICROBIAL TECHNOLOGY, vol. 99, 2017, pp. 16-24.
Bakonyi, D., Hummel, W.: Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7 alpha-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids. ENZYME AND MICROBIAL TECHNOLOGY. 99, 16-24 (2017).
Bakonyi, Daniel, and Hummel, Werner. “Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7 alpha-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids”. ENZYME AND MICROBIAL TECHNOLOGY 99 (2017): 16-24.

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