Overexpression,one-step purification,and characterization of a type II cholesterol oxidase from a local isolate <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. PTCC 1633 |
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| Authors: | Abdollah Ghasemian Mojtaba Tabatabaei Yazdi Zargham Sepehrizadeh Zohreh Tabatabaei Yazdi Gholamreza Zarrini |
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| Institution: | (1) Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran;(2) Department of Animal Biology, Faculty of Natural Sciences, Tabriz University, Tabriz, Iran |
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| Abstract: | A 1.6 kb gene encoding a cholesterol oxidase (choR) from a local isolate, Rhodococcus sp. PTCC 1633 was cloned into pET23a and the highly expressed recombinant enzyme was purified from the cell lysate of IPTG-induced
Escherichia coli BL21(DE3)pLysS with one-step absorption on cholesterol. The purified protein had a molecular mass of 55 kDa, isoelectric
point at about pH 9.0 and absorption peaks at 280, 380 and 460 nm, indicating that the enzyme is a flavoprotein. The optimum
pH and temperature for the recombinant enzyme were 7.0 and 50°C, respectively. Steady-state kinetic revealed that the cholesterol
oxidase had a K
m
of 32 μM. This study is the first report concerning expression and one-step purification of a gene encoding cholesterol oxidase
from Rhodococcus spp. This study revealed that this enzyme is a type II cholesterol oxidase. |
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| Keywords: | Cholesterol oxidase Overexpression Rhodococcus One step-purification |
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