期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 139, 期 -, 页码 1-7出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2017.07.008
关键词
Cholesterol oxidase; Dehydrogenase; Refolding; Enzymatic activity; Cholest-5-en-3-one; Biocatalyst
类别
资金
- Key Technologies Research and Development Program of Tianjin [14ZCZDSY00012]
- Natural Science Foundation of Tianjin [16JCQNJC09200]
- National Natural Science Foundation of China [21306140]
- Overseas High-level Talents Program of Tianjin University of Science and Technology, China
Cholesterol oxidases, which catalyze the degradation of cholesterol to cholest-4-en-3-one, are widely used in the pharmaceutical and food processing industries. The cholesterol oxidase from Pimelobacter simplex (PsChO3) was transformed into E. coil BL21(DE3), but it was expressed mainly as inclusion bodies, and any soluble PsChO3 failed to bind to Ni-NTA resin. To overcome this obstacle, we devised a simple yet efficient purification and refolding process using 8 M urea for the solubilization of PsChO3 and achieved a high yield of the enzyme in its active form. Column-bound PsChO3 was refolded in situ through a gradient of successively decreased urea concentrations and purified using Ni-affinity chromatography, ionic exchange and gel filtration. This treatment converted the denatured PsChO3 into a soluble protein exhibiting an unexpected dehydrogenation activity amounting to 9.27 U/mg - an activity not reported for enzymes with noncovalently-linked FAD to date. The product, cholest-5-en-3-one, was confirmed using TLC, GC-MS and NMR. Structural analysis revealed a distinct binding mode in both FAD and substrate domain, which may explain the enzyme's unusual catalytic behavior. (C) 2017 Published by Elsevier Inc.
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