Production, purification and characterization of cholesterol oxidase from a newly isolated Streptomyces sp.

Abstract

Cholesterol oxidase production (COD) by a new isolate characterized as Streptomyces sp. was studied in different production media and fermentation conditions. Individual supplementation of 1 % maltose, lactose, sucrose, peptone, soybean meal and yeast extract enhanced COD production by 80-110 % in comparison to the basal production medium (2.4 U/ml). Supplementation of 0.05 % cholesterol (inducer) enhanced COD production by 150 %. COD was purified 14.3-fold and its molecular weight was found to be 62 kDa. Vmax (21.93 μM/min mg) and substrate affinity Km (101.3 μM) suggested high affinity of the COD for cholesterol. In presence of Ba(2+) and Hg(2+) the enzyme activity was inhibited while Cu(2+) enhanced the activity nearly threefold. Relative activity of the enzyme was found maximum in triton X-100 whereas sodium dodecyl sulfate inactivated the enzyme. The enzyme activity was also inhibited by the thiol-reducing reagents like Dithiothreitol and β-mercaptoethanol. The COD showed moderate stability towards all organic solvents except acetone, benzene and chloroform. The activity increased in presence of isopropanol and ethanol. The enzyme was most active at pH 7 and 37 °C temperature. This organism is not reported to produce COD.

Purification, characterization and amino acid content of cholesterol oxidase produced by Streptomyces aegyptia NEAE 102

“…Similar result was reported for the cholesterol oxidase from Streptomyces sp. which was most active at pH 7.0 [35]. The optimum pH of cholesterol oxidase reported from different sources were ranged from 5.0 to 8.5 [10, 21, 22].…”

“…had an optimum pH for enzyme activity at pH 7.0 and 80% of its activity was retained at pH 6–8 for 24 h, but activity rapidly declined at higher or lower pH, and at pH 10, only 10.16% of its activity was retained [35]. On the other hand, it was found that the optimum pH for the stability of cholesterol oxidase produced by Pseudomonas aeruginosa was pH 6.5.…”

Cholesterol Oxidase: Source,Properties and Applications.

“…For the maximum production of CHOx by Streptomyces sp. addition of 0.05% of cholesterol as inducer was found to be most suitable [51]. 0.15% (w/v) cholesterol concentration in the production medium is reported for optimum CHOx production from Rhodococcus sp.…”

Hybrid Dual Mode Sensor for Simultaneous Detection of Two Serum Metabolites

“…The Km values were calculated and found to be 428 ± 133 µM (reaction rate) and 350 ± 167 µM (end value) for glucose-hexokinase and 103 ± 39 µM (reaction rate) and 186 ± 49 µM (end value) for cholesterol-cholesterol oxidase respectively. This can be compared with the literature value of 150 µM [38] for hexokinase and 96 µM [39] for cholesterol oxidase. The Km values determined using a CMOS-based device are slightly higher than measured conventionally, this may be due to the pH and/or ionic concentration, which are not optimized for individual reaction but for the two reactions.…”