A mesophilic Bacillus sp. initially isolated from tiger excreta and later identified as a Bacillus subtilis strain was used to produce an extracellular cholesterol oxidase (COX) in cholesterol-enriched broth. This bacterial isolate was studied for the production of COX by manipulation of various physicochemical parameters. The extracellular COX was successfully purified from the cell-free culture broth of B. subtilis by successive salting out with ammonium sulfate, dialysis, and riboflavin-affinity chromatography. The purified COX was characterized for its molecular mass/structure and stability. The enzyme possessed some interesting properties such as high native Mr (105 kDa), multimeric (pentamer of ∼21 kDa protein) nature, organic solvent compatibility, and a half-life of ∼2 h at 37 °C. The bacterial COX exhibited ∼22 % higher activity in potassium phosphate buffer (pH 7.5) in the presence of a nonionic detergent Triton X-100 at 0.05 % (v/v). The K m and V max value of COX of B. subtilis COX were found to be 3.25 mM and 2.17 μmol min ml(-1), respectively. The purified COX showed very little cytotoxicity associated with it.

Purification and Characterization of an Extracellular Cholesterol Oxidase of Bacillus subtilis Isolated from Tiger Excreta

To examine the effect of purified cholesterol oxidase (COX) of a Bacillus sp. COX-T3 for treating high-lipid diet-induced hypercholesterolemia, an experimental mouse model was established. The enzyme was purified by using a Riboflavin-Sephadex G-25 affinity column. After keeping mice assigned to Group 1 to 5 on a high fat diet, the mice showed 14.4, 20.6, 24.4, 19.4 and 17.0 % body weight gain, respectively (mean body weight gain of ~19.0 %) over 9 weeks of intake of high-fat feed. The maximum body weight was attained in the mice assigned to Group 3 (32.73 ± 2.11 g) in comparison to the control regimen kept on normal diet for the same period of time. The results reflected the successful establishment/development of a near obese mouse model by intake of high-fat diet for 9 weeks. The cholesterol level in the high-fat diet fed mice significantly increased with age up to 6th week. For the Group 1 to 5, the high fat diet caused a significant increase of serum cholesterol. The purified COX was intra-muscularly administered to the mice at 0.1 to 0.5 U/dose (100 μl) on alternate days for 5 weeks. The mice assigned to treatment group(s) 3, 4 and 5 showed 2.2, 2.2 and 3.5 % decline in their average body weight(s) that indicated a dose/ concentration dependent effect of administration of Bacillus sp. COX-T3 in bringing a reduction in the body weight(s) of these mice. The reduction of serum cholesterol level by 45.3, 40.9, 22.4, 28.4 and 40.7 % in Group 1 to 5, respectively, was achieved in comparison to the serum cholesterol levels recorded after 9 weeks of intake of high fat diet in mice. Interestingly, the addition of the bacterial COX at 1.2 U.ml-1 concentrations to the Hep2C cells slightly promoted the cell viability while the COX at 1.8 U.ml-1 exerted a cytotoxic effect on the cell growth as reflected by ~10.6 % decline in the cell viability recorded in MTT assay. The observed data indicated a successful therapeutic application of the purified COX of Bacillus sp. COX-T3 in treating an obese hypercholesterolemic murine model over a reasonable period of enzymatic treatment. 

Application of Bacillus sp. COX-T3 Cholesterol Oxidase in Reduction of Serum Cholesterol and Body Weight Management in a Murine Model

In this paper we optimized liquid fermentation conditions of Monascus X-1 on cholesterol oxidase (COD) production, and then the COD was isolated by ammonium sulfate deposition and its properties were studied. The results showed that COD produced by Monascus X-1 was a kind of extracellular enzyme. The yields came to maximum at the 6th day. The most suitable medium for Monascus X-1 generating COD was the rice medium, the highest enzyme activity was obtained at 140r/min, inoculum concentration 6%, when 60mL of the culture in 250mL shake flasks grown in rice medium. Under the optimized cultivation conditions, the enzyme activity reached 1168U/L, which increased 1.52 times compared to the previous fermentation conditions. The enzyme had favorable pH and thermal stability, while the optimum pH and temperature of enzyme activity was 7.0 and 40, respectively.

Optimal Conditions for Cholesterol Oxidase Production and Enzymatic Properties Study Produced by Monascus X-1

Production of carnation pigments in submerged culture of Monascus spp.

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Optimal Conditions for Cholesterol Oxidase Production and Enzymatic Properties Study Produced by Monascus X-1

Production of carnation pigments in submerged culture of Monascus spp.