Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1.
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2019
2019
Historique:
received:
16
10
2018
accepted:
29
01
2019
entrez:
14
2
2019
pubmed:
14
2
2019
medline:
14
11
2019
Statut:
epublish
Résumé
Cholesterol oxidase is a bifunctional bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This valuable enzyme has attracted a great deal of attention because of its wide application in the clinical laboratory, synthesis of steroid derived drugs, food industries, and its potentially insecticidal activity. Therefore, development of an efficient protocol for overproduction of cholesterol oxidase could be valuable and beneficial in this regard. The present study examined the role of various parameters (host strain, culture media, induction time, isopropyl ß-D-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature) on over-expression of cholesterol oxidase from Chromobacterium sp. DS1. Applying the optimized protocol, the yield of recombinant cholesterol oxidase significantly increased from 92 U/L to 2115 U/L. Under the optimized conditions, the enzyme was produced on a large-scale, and overexpressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver-Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were determined. This study reports a straightforward protocol for cholesterol oxidase production which can be performed in any laboratory.
Identifiants
pubmed: 30759160
doi: 10.1371/journal.pone.0212217
pii: PONE-D-18-27497
pmc: PMC6373949
doi:
Substances chimiques
Bacterial Proteins
0
Cholesterol Oxidase
EC 1.1.3.6
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0212217Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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