Assessment of alkaline cholesterol oxidase purified from Rhodococcus sp. PKPD-CL for its halo tolerance, detergent and organic solvent stability Pramod J. Kasabe, Geetanjali T. Mali, Padma B. Dandge ⇑ Department of Biochemistry, Shivaji University, Kolhapur 416004, Maharashtra, India article info Article history: Received 20 June 2015 and in revised form 9 August 2015 Accepted 10 August 2015 Available online 11 August 2015 Keywords: Halo tolerant Detergent and organic solvent tolerant Alkaline cholesterol oxidase Brackish water abstract The novel bacterium, Rhodococcus sp. PKPD-CL was isolated and identified from the ‘Chilika Lake’ located at Odisha state of India, which is a largest brackish water habitat in Asia. Rhodococcus sp. PKPD-CL pro- duces extracellular halo tolerant, detergent and organic solvent stable alkaline cholesterol oxidase. It has apparent molecular weight of 60 kDa and was purified 59 fold by using 60% saturated ammonium sulfate fractionation, anion exchange followed by size exclusion chromatographic techniques with 37% recovery. It showed substrate specificity for 3b-hydroxysteroids with K m of 1.1  10 4 M for cholesterol. The pH, 8.0 and the temperature, 37 °C were required for its optimum activity. Enzyme is considerably stable at pH 6.0–8.5 and temperature up to 50 °C. At 4 and 30 °C it maintained its 100% activity up to 60 days. The isoelectric point of the enzyme was 9.5. It showed 80% residual activity with 20% NaCl (3.42 M) and 83% relative activity with 12% NaCl (2.05 M) concentration. The metal ions like Zn 2+ , Cu 2+ , Ag+, Fe 3+ , Ba 2+ inhibited the enzyme activity >60% while Hg 2+ served a potent inhibitor whereas Mg 2+ found to be a good enhancer for it. The enzyme was stable in presence of chemical reagents (NaN 3 , EDTA), detergents (Tween-80, Tween-20, Triton X-100, sodium cholate) and various organic solvents (isopropanol, ethanol, benzene, chloroform, methanol, toluene, ethyl acetate, butanol and dimethylsulfoxide). Such a multi stress tolerant and versatile enzyme produced by Rhodococcus sp. PKPD-CL may serve as a good choice for industrial applications. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction Cholesterol oxidase (CHOX) is an enzyme which oxidizes the cholesterol and converts cholesterol into 4-cholesten-3-one and H 2 O 2 [1]. In eukaryotes, cholesterol is an essential steroid molecule used for production of hormones, bile salts and also in the develop- ment of plasma membranes of the cells [2]. The systemic distribu- tion of cholesterol occurs through the blood plasma of all mammals. However, high cholesterol deposition in the blood ves- sels leads to cardiovascular diseases and other disorders associated with it and further its secondary complications. Therefore the level of cholesterol in the food and blood serum has to be monitored and regulated time to time for avoiding such kind of complications. Most of the efforts have been made to develop sensitive in vitro assessment techniques for monitoring and estimating cholesterol in biological samples. Among these cholesterol determining meth- ods, enzymatic method employing the use of three enzymes, cholesterol esterase (EC 3.1.1.13), cholesterol oxidase (EC 1.1.3.6), and peroxidase (EC 1.11.1.7) is common [3]. In addition to its clin- ical use, the cholesterol oxidase also has other industrial and biotechnological applications. It acts as a signaling protein for biosynthesis of the polyene macrolides [4] and has an insecticidal activity as well [5]. The enzyme can also be used to produce pre- cursors for chemical synthesis of steroid hormones [6]. It is also responsible for virulence of certain pathogenic species such as Mycobacterium sp. [7] and Rhodococcus equi [8]. Variety of bacterial genera such as Arthrobacter [9], Brevibacterium [10], Corynebacterium [11], Mycobacterium [12], Nocardia [13], Pseudomonas [14], Rhodococcus [15], Schizophyllum [16], Streptomyces [17], Streptoverticillium [18] etc. producing choles- terol oxidase have been reported. It has been shown that Bacillus sp. SFF34 could produce a high level of extracellular cholesterol oxidase [19,36]. Recently some other bacterial species have been reported for the production of CHOX but the present crucial need is to have maximum production of this versatile enzyme with good sensitivity and extreme stability. Such needs might be satisfied by improving the existing cholesterol oxidase producer strains by using modern and recombinant biotechnologies. Another way is to hunt for some novel producers possessing the potential to deli- ver enhanced production of cholesterol oxidase with maximum http://dx.doi.org/10.1016/j.pep.2015.08.011 1046-5928/Ó 2015 Elsevier Inc. All rights reserved. ⇑ Corresponding author. E-mail addresses: pbd_biochem@unishivaji.ac.in, pjk.biochem@gmail.com (P.B. Dandge). Protein Expression and Purification 116 (2015) 30–41 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep