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Bioconversion of non-water-soluble compounds has been hindered because of their low solubility in an aqueous medium. Aqueous medium-organic solvent two-phase systems are likely to be advantageous for bioconversion of water-immiscible substrates at high concentrations. However, organic solvents often affect enzyme stability and activity (2, 6). Enzymes displaying high stability and activity under such conditions would be very useful for technological applications in which organic solvents are employed.

We have isolated a cyclohexane-tolerant and cholesterol-converting bacterium, Pseudomonas sp. strain ST-200. This organism effectively oxidizes cholesterol dissolved in an organic solvent overlaying the medium and less effectively oxidizes cholesterol suspended in the medium (3, 4, 7). The oxidized products remained stable in the organic solvent phase. We found cholesterol oxidase activity in the culture supernatant of strain ST-200. In this study, the cholesterol oxidase was purified to examine involvement of this activity in the cholesterol oxidation mediated by strain ST-200 in the presence of organic solvent.

Cholesterol oxidase activity was found in the culture supernatant when strain ST-200 was grown in LB medium, consisting of 1% Bacto Tryptone (Difco Laboratories, Detroit, Mich.), 0.5% Bacto Yeast Extract (Difco), and 1% NaCl. A sonicated lysate of ST-200 cells showed no cholesterol oxidase activity (results not shown). The amount of cholesterol oxidase activity expressed by strain ST-200 grown without cholesterol was the same as that when the strain was grown in LB medium containing cholesterol. The enzyme is not likely to be inducible by cholesterol.

ST-200 was grown at 30°C for 17 h in LB medium, and then the culture was centrifuged (8,000 × g, 15 min, 4°C). Proteins in the supernatant (12 liters) were extracted with (NH₄)₂SO₄ (70% saturation) at 4°C overnight. The precipitate was recovered by centrifugation (10,000 × g, 30 min, 4°C) and dissolved in 10 mM Tris-HCl (pH 8.0). This solution was dialyzed against the same buffer at 4°C and loaded on a column (2.5 by 10 cm) of DEAE-cellulose DE52 (Whatman, Maidstone, England) that had been equilibrated with 10 mM Tris-HCl (pH 8.0) buffer. The column was washed with 100 ml of the Tris-HCl buffer at a flow rate of 72 ml/h. The cholesterol oxidase activity passed through the column. This step was the most effective to purify the enzyme; it yielded a 10-fold purification (see Table 1).

The cholesterol oxidase-positive fractions were pooled and centrifuged (7,000 × g, 15 min, 4°C) after addition of (NH₄)₂SO₄ (45% saturation). The supernatant was loaded on a column (2.5 by 20 cm) of Butyl-Toyopearl 650S (Tosoh, Tokyo, Japan) that was equilibrated with 45% saturated (NH₄)₂SO₄-10 mM Tris-HCl (pH 8.0). The column was washed with 150 ml of the Tris buffer containing (NH₄)₂SO₄ (45% saturation) and then eluted with a decreasing linear gradient of (NH₄)₂SO₄ in 300 ml of the Tris-HCl buffer at a flow rate of 70 ml/h. The cholesterol oxidase eluted at 10% saturation of (NH₄)₂SO₄, suggesting that the enzyme is comparatively hydrophobic. The cholesterol oxidase was precipitated with (NH₄)₂SO₄ (80% saturation) from the positive fraction, dissolved in 5 ml of 10 mM Tris-HCl (pH 8.0), and dialyzed twice against the same buffer at 4°C.

The solution was loaded on a column (2.5 by 98 cm) of Sephadex G-100 (Pharmacia, Uppsala, Sweden) that was equilibrated with a buffer consisting of 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 5 mM sodium cholate. The column was eluted with this buffer at a flow rate of 60 ml/h. In the absence of sodium cholate, the oxidase eluted at the void volume of the column and was not separated from impurities. The cholesterol oxidase-positive fractions were pooled and dialyzed against 10 mM Tris-HCl buffer (pH 8.0).

Table 1 summarizes the purification steps employed to purify the cholesterol oxidase. The cholesterol oxidase was purified 36-fold from the culture supernatant. The purified enzyme preparation gave a single band upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1). Its molecular mass was estimated to be 60 kDa. The enzyme exhibited a specific activity of 15.2 U/mg of protein at 30°C and pH 7. The activity was assayed by measuring H₂O₂ generation (1) as follows: the reaction mixture (total of 3 ml) consisted of 50 mM sodium potassium phosphate buffer (pH 7.0), 64 mM sodium cholate, 0.34% Triton X-100, 1.4 mM 4-aminoantipyrine, 21 mM phenol, 0.89 mM cholesterol, and 5 units of horseradish peroxidase (Toyobo Co., Ltd., Tsuruga, Japan) per ml. Development of red color in the assay mixture was tracked by monitoring the absorbance at 500 nm at 30°C for 5 min. The enzymatic activity was calculated from the extinction coefficient of the red dye quinoneimine. One unit of enzymatic activity was defined as the amount required to oxidize 1 µmol of cholesterol per min at 30°C.

View larger version (67K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of cholesterol oxidase during the purification procedure. Samples containing 0.01 U of cholesterol oxidase were electrophoresed on an SDS-7.5% polyacrylamide gel. The gel was stained with Coomassie brilliant blue R250 as described by Laemmli (10). Lane 1, precipitate obtained with 70% saturated (NH4)2SO4; lane 2, fraction from the DEAE-cellulose DE52 column; lane 3, fraction from the Butyl-Toyopearl 650S column; lane 4, final preparation obtained following Sephadex G-100 gel chromatography. M, Molecular size markers (kilodaltons).

The enzyme solution exhibited two absorption maxima at 355 and 450 nm, like a typical flavoprotein. Most cholesterol oxidases contain 1 mol of tightly bound flavin adenine dinucleotide (FAD) per mol of protein as a prosthetic group (8, 9, 16), although some of the enzymes, such as Nocardia erythropolis oxidase, lack this cofactor (13). A solution of ST-200 cholesterol oxidase (1.2 mg/ml) showed an absorbance of 0.198 at 450 nm. In this solution, the molarity of the protein was estimated to be 20 µM based on the molecular mass measured by SDS-PAGE. The molar adsorption coefficient of FAD ( = 1.13 × 10⁷ cm²/mol) was employed to calculate the concentration of FAD, 17.5 µM. This result indicated that the enzyme contained 1 mol of FAD per mol of protein.

The enzyme was active at pH 5.0 to 8.5 and was most active at pH 6.8 to 8.0 when cholesterol oxidation activity was assayed by measuring O₂ consumption (17) stoichiometrically accompanying the oxidation of the substrate with a dissolved-oxygen meter (model 53; Yellow Springs Instrument Co., Yellow Springs, Ohio). Most cholesterol oxidases show optima around pH 7.0 to 7.5 (12, 13, 15, 17). The enzyme activity reached a maximum at 60°C. Most cholesterol oxidases show optimal activity at temperatures in the range of 50 to 60°C (12, 13, 15, 17). Thermal stability was examined by incubating the enzyme in sodium potassium phosphate (pH 7.0) buffer at various temperatures for 30 min. The enzyme was stable at temperatures from 4 to 50°C, retained 73% of its activity after the incubation at 60°C, and lost almost all activity at 70°C.

The enzyme oxidized various 3-hydroxysteroids (Table 2). The enzyme was not reactive with 3-hydroxysteroids, such as epicholesterol. These results indicate a high specificity of the enzyme for 3-hydroxysteroids. The length of the side chain attached to position 17 seems to affect the oxidation rate. The enzyme displayed low reactivity with 3-hydroxysteroids having short side chains attached to position 17, such as pregnenolone, dehydroepiandrosterone, and epiandrosterone. The activity of Nocardia erythropolis cholesterol oxidase is dependent on the chain length (14), but the activity of the oxidases from Brevibacterium sterolicum and Streptomyces violascens is not (15, 17). In addition, the degree of saturation of the B ring affects the enzyme activity.

Cholesterol is poorly soluble in water. Thus, enzymatic H₂O₂ generation reactions were carried out using cholesterol emulsified with a surfactant, 0.03% Triton X-100. K_m and V_max values of the cholesterol oxidase were estimated from Lineweaver-Burk plots. Table 3 shows the values together with those of commercially available cholesterol oxidase preparations derived from a Streptomyces sp., a Brevibacterium sp., another Pseudomonas sp., and Nocardia erythropolis. The V_max values were similar to one another. The K_m constants of the oxidases of ST-200 and Nocardia erythropolis were relatively low among the enzymes. The ST-200 oxidase V_max/K_m ratio was the highest among those of the enzymes examined. ST-200 cholesterol oxidase would be active against a low level of cholesterol, such as an aqueous cholesterol solution in which the solubility is approximately 11 µM.

The enzymatic cholesterol oxidation efficiencies were evaluated upon consumption of cholesterol dissolved in organic solvents with various log P_ow values (i.e., partition coefficients of the organic solvent between n-octanol and water) (Table 4). Organic solvents containing a comparatively low concentration of cholesterol were added to the assay solution to monitor the decrease in its amount. Residual cholesterol was determined by reverse-phase chromatography on a column of ODS-1201-H (4.6 by 200 mm; Senshu Science, Tokyo, Japan) attached to a high-pressure liquid chromatography (HPLC) apparatus. The column was eluted with n-hexane-isopropanol (1:0.02, vol/vol) at a flow rate of 1.0 ml/min. The elution was monitored by measurement of the A₂₁₅. When ST-200 cholesterol oxidase was examined, the cholesterol consumption rate was high in the presence of benzene, toluene, p-xylene, propylbenzene, or diphenylmethane and was 3.0- to 3.5-fold higher than that found in the absence of organic solvent. The oxidation was extremely low when cholesterol was dissolved in chloroform. Cholesterol oxidation rates with the oxidases of Nocardia erythropolis and the other Pseudomonas sp. were not so high in the presence of organic solvents. The oxidation rates with the oxidases of the Streptomyces sp. and the Brevibacterium sp. were not enhanced by the organic solvents.

We previously reported that strain ST-200 grown with cholesterol or several 3-hydroxysteroids dissolved in appropriate organic solvents effectively oxidized them (3, 4, 7). The extracellular cholesterol oxidase purified in this study oxidized cholesterol dissolved in the organic solvents and less effectively oxidized cholesterol suspended in the assay solution (Fig. 2). This high activity is probably due to the stability in the presence of various organic solvents and the low K_m constant appropriated for a low level of cholesterol (Table 3). The substrate specificity (Table 2) of the oxidase was the same as that found in the ST-200 culture. It is likely that early steps in the cholesterol conversion reactions found in the ST-200 culture are mediated by the oxidase.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Conversion of cholesterol dissolved in organic solvent by ST-200 oxidase. Cholesterol (10 mg) was dissolved in 0.25 ml of a solvent mixture (diphenylmethane and p-xylene; 7:3, vol/vol) () or cyclooctane () and added to 2.5 ml of the assay solution consisting of ST-200 cholesterol oxidase (0.2 U/ml)-15 mM sodium cholate-50 mM phosphate buffer (pH 7.0). As a control, 10 mg of cholesterol was suspended in 2.5 ml of the assay solution (). These reaction mixtures were shaken at 30°C. Samples (10 µl) were withdrawn from the organic solvent layers. The uniphasic reaction mixture was extracted with chloroform.