Production of cholesterol oxidase by a newly isolated Rhodococcus sp. M. Tabatabaei Yazdi 1, *, F. Malekzadeh 2 , Gh. Zarrini 1 , M.A. Faramarzi 1 , N. Kamranpour 1 and Sh. Khaleghparast 1 1 Department of Biotechnology, College of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Microbiology, College of Science, Tehran University, Tehran, Iran *Author for correspondence: Fax: 98-21-646-1178, E-mail: mtabataba@yahoo.com Received 7 December 2000; accepted 11 August 2001 Keywords: cholesterol oxidase, medium development, Rhodococcus sp. Summary Fifteen strains of microorganisms with ability to degrade cholesterol were isolated. Among them a Gram-positive, non-motile, non-sporing bacterium with meso-DAP in the cell wall and with a rod-coccus cycle showed the highest ability for cholesterol degradation. It was identi®ed as Rhodococcus sp. strain 2C and was deposited by code 1633 in Persian type culture collection (PTCC). This strain was able to produce high levels of both extracellular and cell- bound cholesterol oxidases in media containing cholesterol as a sole carbon source. The eects of medium composition and physical parameters on cholesterol oxidase production were studied. The optimized medium was found to contain cholesterol 0.15% (w/v), yeast extract 0.3% (w/v), diammonium hydrogen phosphate 0.1% (w/v), Tween 80 (0.05%). The optimum pH and temperature for cholesterol oxidase production in optimized medium were found to be 8±30 °C respectively. Triton X-100 showed the greatest eect in releasing the cell-bound enzyme. The ®rst and most probably the main metabolite of cholesterol degradation was puri®ed and identi®ed as 4-cholestene-3-one. Introduction 3b-Hydroxysteroid oxidase catalyses the conversion of the D 5 -3b-hydroxyl group of sterol to the corresponding D 4 -3-ketone with concomitant reduction of oxygen to hydrogen peroxide. The enzyme is commonly known as cholesterol oxidase (cholesterol: oxygen oxidoreductase, EC 1.1.3.6) (Kreit et al. 1992a). Cholesterol oxidase (COX) has become one of the most important diagnostic enzymes because of its utility in the determination of cholesterol in food and blood serum. It may also be used for the production of a starting material for chemical synthesis of pharmaceu- tical steroids (Watanabe et al. 1989; Sojo et al. 1997) and also to degrade dietary cholesterol, which has been implicated in cardiovascular disease, and hence to improve human health (Kaunitz 1978). This enzyme has also been used as a potent insecticidal protein active against boll weevil larvae (Purcell et al. 1993). Many microorganisms have been isolated from soil and food of animal origin such as butter, bacon, chicken fat and pork fat and have been shown to produce COX including Brevibacterium sterolicum (Uwajima et al. 1974), Streptomyces species (Kamei et al. 1978), Strep- toverticillium cholesterolicum (Inouye et al. 1982), Schizophyllum commune (Fukuya & Miyake 1979; Sojo et al. 1997), Nocardia (Richmond 1973) and Rhodococ- cus species (Watanabe et al. 1986, 1989; Kreit et al. 1992b; Lee et al. 1997a,b; Chou et al. 1999). COX in Nocardia and Rhodococcus strains were shown to be an intrinsic membrane-bound type which can be extracted from cells by treatment with non-ionic detergents such as Triton X-100 (Smith & Brooks 1976; Kreit et al. 1992a; 1994). However the COXs of Brevibacterium, Streptomyces and Streptoverticillium are only found extracellularly (Uwajima et al. 1974; Tomioka et al. 1976; Inouye et al. 1982). Furthermore, some Rhodo- coccus species produce both secreted and membrane- bound forms of COX (Watanabe et al. 1989; Kreit et al. 1992a,b; Sojo et al. 1997). This paper describes the result of our screening program for isolation of microorganisms producing high levels of COX from environmental sources, iden- ti®cation of one of the most potent isolates, optimiza- tion of the conditions for COX production, puri®cation and characterization of the main metabolite of choles- terol degradation. Materials and methods Isolation of microorganisms Various soil and water samples from soap-producing factories, tanneries and compost were collected. To isolate microorganisms, 1 g or 1 ml of each sample was World Journal of Microbiology & Biotechnology 17: 731±737, 2001. 731 Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.