A Simple and Rapid Method for Screening of Nocardioides simplex Mutant with High Cholesterol Oxidase Activity

CHO (EC 1.1.3.6) is a widely used enzyme that has industrial and clinical uses related to hypertension and obesity. This enzyme is used for the clinical determination of total or free serum cholesterol by coupling with the related enzymes [Allain et al., 1974]. CHO catalyzes the first step of the cholesterol degradation, oxidizing the cholesterol to 4-cholestene-3-one through the reduction of the flavin adenine dinucleotide cofactor [Uwajima et al., 1974]. The reduced cofactor is recycled to the oxidized form by the reduction of oxygen to hydrogen peroxide (Fig. 1A). The hydrogen peroxide is then coupled to the horseradish peroxidase as a substrate, and oxidizes a color reagent into a colored product [Murooka and Yamashita, 2001; Xiang and Sampson, 2004]. This colored product has been used as the basis for the measurement of CHO activity. The CHOs have been isolated from various microorganisms, including Arthrobacter [Liu et al., 1988], Brevibacterium [Uwajima et al., 1974], Corynebacterium [Shirokane et al., 1977], Nocardia [Cheethan et al., 1982], Pseudomonas [Lee et al., 1989; Doukyu and Aono, 2001], Rhodococcus [Johnson and Somkuti, 1991], Schizophyllum [Fukuyama and Miyake, 1979], Streptomyces [Tomioka et al., 1976], Streptoverticillium [Inouye et al., 1982], and Burkholderia [Doukyu and Aono, 1998], and their properties have been reported. To increase the CHO production from Nocardioides simplex, we performed mutagenesis by treatment with EMS. However, to obtain mutants with increased CHO activity, the EMS-treated cells needed to be screened on a large scale. Therefore, the establishment of a simple and rapid screening method was important. In this study, we describe a simple, rapid method for screening mutant strains with increased CHO activity. N. simplex (ATCC6946), obtained from the Korean culture center of microorganisms (Seoul, Korea), was grown in a YM medium containing 0.3% (w/v) yeast extract, 0.3% (w/v) malt extract, 0.5% (w/v) peptone, and 1% (w/v) D-glucose and incubated at 30°C on a rotary shaker (200 rpm). The ODA stock solution (3%, w/v) was prepared by dissolving 150 mg ODA in 5 mL deionized water. The cholesterol stock solution (1%, w/v) was prepared as follows; 1 mL Triton X-100 was added to a 50-mL tube and stirred constantly in a water bath (about 80oC). After the Triton X-100 was warmed up, 100 mg cholesterol was added and stirred thoroughly until all cholesterol was dissolved. Subsequently, 9 mL deionized water was slowly added to the tube with stirring for complete mixing. When the solution became opaque, it was warmed again in the same water bath for about 1 min. For EMS mutagenesis, the cells were grown in 1 mL YM broth for 15 to 18 h until the optical density reached 0.7 at 600 nm. The cells that had been incubated in 1 mL broth were pelleted by centrifugation at 10,770×g for 2 min, and were resuspended in 1 mL of 0.1 M sodium phosphate buffer (pH 7.0). EMS at the final concentration of 0.08% (v/v) to 0.64% (v/v) was added to each cell suspension, which was then mixed by gentle vortexing. The mixture was allowed to stand for 1, 10 or 30 min at room temperature, and was washed four times with 0.1 M sodium phosphate buffer. After the EMS treatment, the cells were plated on the YM agar medium and incubated at 30oC for about 2 days. Each colony was transferred onto the master YM agar plate using sterilized toothpicks, and the plate was then incubated at 30oC for 24 h. After the colony formation, paper discs (6 mm in diameter, Advantec, Toyo, Japan) were placed on each colony, and 25 μL reaction cocktail (0.5% ODA, 0.2% cholesterol, and 0.75 unit peroxidase in 0.1 M sodium phosphate buffer) was dropped per paper disc. The plates with the paper disc-loaded colonies were incubated for additional 3 h at 30oC. As the CHO of each colony reacted with the *Corresponding author Phone: +82-31-222-6514; Fax: +82-31-222-6552 E-mail: jmjeong@suwon.ac. kr