Optimized extraction and kinetic study of cholesterol oxidase from a newly isolated Escherichia fergusonii strain from local whey samples: insights through a combined experimental study and artificial neural network modeling

Abstract

Abstract Background Microbial cholesterol oxidase (ChoX) has wide clinical and industrial applications; therefore, many efforts are being made to identify promising sources. This study aimed to isolate a novel ChoX-producing bacterial strain from whey samples. Results The most efficient strain was selected based on extracellular ChoX-producing ability and characterized as Escherichia fergusonii (E. fergusonii) through molecular and biochemical analysis. The maximum production of ChoX was obtained at the optimum condition of 48 h of incubation under shaking conditions (130 rpm) at 35 °C in a basal medium adjusted to pH 6.5, including 1.4 g/L cholesterol as a sole carbon. The crude product was purified by ammonium sulfate precipitation and followed by ion exchange chromatography utilizing Q-Sepharose, resulting in 5.35-fold and 13.86-fold purification, respectively, with a final specific activity of 15.8 U/mg. Additionally, molecular weight was determined by SDS-PAGE to be 49.0 kDa. The optimum conditions required for the higher cholesterol decomposition ability of purified ChoX were suggested to be 30 °C and pH 7.5 in the presence of MgSo4 with a K m value of 0.71 mM. However, other case studies of metal ions showed an unfavorable effect on enzymatic performance. The enzyme retained almost 72.0% of its initial activity after 80 days of storage at 4 °C. Furthermore, the ChoX enzyme revealed acceptable stability at a pH value of 6.5 to 8.5, maintaining its initial activity of more than 50.0%. Finally, an artificial neural network (ANN) was designed to predict the most effective factor in the fermentation process for enzyme production and the purified ChoX activity. Conclusions Considering the properties of the extracted enzyme from E. fergusonii, it would be regarded as a potential ChoX source for commercial applications.

Keywords

• Escherichia fergusonii
• Cholesterol oxidase
• 16S rRNA gene
• Artificial neural network
• Optimization