Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1
| Autor: | Samaneh Sadat Rasi Varaei, Abolfazl Golestani, Mostafa Lakzaei, Mahdi Aminian, Aliakbar Fazaeli |
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| Rok vydání: | 2019 |
| Předmět: |
Lysis
Cholesterol oxidase medicine.medical_treatment Protein Expression Enzyme Purification Biochemistry chemistry.chemical_compound Recombinant Protein Purification Overproduction chemistry.chemical_classification 0303 health sciences Multidisciplinary biology Chemistry Lipids Recombinant Proteins Laboratory Equipment Cholesterol Bioassays and Physiological Analysis Recombination-Based Assay Medicine Engineering and Technology Biological Cultures Research Article Protein Purification Science Equipment Library Screening Research and Analysis Methods Steroid 03 medical and health sciences Bacterial Proteins Affinity chromatography Gene Expression and Vector Techniques medicine Molecular Biology Techniques Molecular Biology Enzyme Assays 030304 developmental biology Molecular Biology Assays and Analysis Techniques Cholesterol Oxidase 030306 microbiology Chromobacterium Biology and Life Sciences Proteins Enzyme assay Culture Media Enzyme biology.protein Biochemical Analysis Purification Techniques |
| Zdroj: | PLoS ONE PLoS ONE, Vol 14, Iss 2, p e0212217 (2019) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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