Evaluation of low-intensity laser radiation on stimulating cholesterol degrading activity of Streptomyces fradiae Part III cholesterol oxidase purification and application

Abstract

as chromatography - mass spectrometry has proved that the only by-product, resulted from cholesterol decomposition in the reaction mixtures containing the enzyme and cholesterol (as substrate), was 4-cholesten-3-one. By using the high performance liquid chromatography (HPLC), it was found that the highest amount of this product in the reaction mixtures occurred when Streptomyces fradiae was exposed to 210 mW Nd-YAG laser delivered for 8 min in presence 0.5 mg/ml chlorophyllin as photosensitizer. Purification of cholesterol oxidase, recovered from non-irradiated and laser-irradiated photosensitized culture of S. fradiae, was performed through fractional precipitation with ammonium sulfate and by applying DEAE-cellulose column followed by Sephadex G-200 column chromatography. The specific activity of the enzyme recovered from radiated culture (11.21 units/mg) was significantly higher than that recovered from non-irradiated one (7.45 units/mg). The purity of the enzymes was judged by the appearance of only one band for each enzyme using SDS-polyacrylamide gel electrophoresis. The molecular weights of the enzymes recovered from non-irradiated and laser-irradiated cultures were 55.0 and 52.1 kDa, respectively. In in vitro experiment, a decrease in the total and free human serum blood cholesterol contents by 38.79 and 80.90% was induced in the case of pure enzyme treatment recovered from irradiated culture as compared to 16.36 and 59.43% in the case of the corresponding non-irradiated control, respectively. Experimental data indicated that injection of white chickens with cholesterase enzyme recovered from laser-irradiated culture of S. fradiae, resulted in a remarkable serum and egg yolk cholesterol reduction reaching 27.2 and 19.4% as compared to 19.4 and 12.7% for the enzyme recovered from non-irradiated culture, respectively. Neither mortalities nor disease symptoms occurred within all birds by the end of one month observation. Key words: Cholesterol degradation, Nd-YAG laser, photosensitizer, Streptomyces fradiae.