Purification and characterisation of the extracellular cholesterol oxidase enzyme from Enterococcus hirae

Recently many efforts are being carried out to reduce cholesterol in foods. Out of the 50 selected isolates that were tested using the agar well diffusion method to assess their ability to decompose cholesterol, 24 bacterial isolates were screened based on their cholesterol-decomposition ability in liquid media.

The bacterial isolate that displayed the highest cholesterol oxidase activity was identified as Enterococcus hirae. The maximal growth and cholesterol decomposition were achieved with a 1-day incubation under static conditions at 37 °C in cholesterol basal medium adjusted to pH 7 supplemented with 1 g/l cholesterol as the substrate, no additional carbon or nitrogen sources and 0.5 % CaSO4. The cholesterol oxidase enzyme (ChoX) produced by E. hirae was extracted at an (NH4)2SO4 saturation level of 80 % and purified with 79 % yield, resulting in 2.3-fold purification. The molecular weight of (ChoX) was 60 kDa. The optimal conditions required for the maximal activity of the purified COD enzyme produced by E. hirae were 30 min, 40 °C, pH 7.8, substrate concentration of 1 g/l and 200 ppm of MgCl2. The enzyme maintained approximately 36 % and 58.5 % of its activity after 18 days of storage at 4-8 °C. Also, the enzyme loss its activity by gradual thermal treatment, but it maintained 58.5 % of its activity at 95 °C for 2 hr.

E. hirae Mil-31 isolated from milk had a great capacity to decompose cholesterol in basal medium supplemented with cholesterol under its optimal growth conditions. Decomposition process of cholesterol by this strain results from its production of cholesterol oxidase enzyme (ChoX). The highest specific enzyme activity and highest purification fold of purified enzyme were achieved after using Sephadex G-100.