بهینه سازی بیان، تخلیص و تعیین ویژگی های آنزیم کلسترول اکسیداز از کروموباکتریوم sp. DS1

فضایلی, علی اکبر and گلستانی, ابوالفضل and لکزایی, مصطفی and راثی ورعی, سمانه سادات and امینیان, مهدی (1397) بهینه سازی بیان، تخلیص و تعیین ویژگی های آنزیم کلسترول اکسیداز از کروموباکتریوم sp. DS1. PloS ONE ــ 14 (2). e0212217. شاپا 1932-6203

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Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1

English Abstract

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.

Item Type:Article
زبان سند : انگلیسی
نویسنده اول :علی اکبر فضایلی
نویسنده :ابوالفضل گلستانی
نویسنده :مصطفی لکزایی
نویسنده :سمانه سادات راثی ورعی
نویسنده مسئول :مهدی امینیان
Additional Information:IF: 2.74 Indexed in: ISI, Scopus, PubMed/Medline, Embase, DOAJ
کلیدواژه ها (انگلیسی):cholesterol oxidase , isopropyl thiogalactoside , bacterial protein , cholesterol oxidase
Subjects:QU Biochemistry
Divisions:Faculty of Medicine > Department of Basic Sciences > Department of Biochemistry
ID Code:13928
Deposited By: Dr. Aliakbar Fazaeli
Deposited On:13 Nov 1399 10:35
Last Modified:13 Nov 1399 10:58

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