An extracellular cholesterol oxidase (COX) from Bacillus sp. COX-T3 was successfully
purified from the cell free culture broth by successive steps of ammonium sulphate precipitation,
dialysis and Riboflavin-affinity column chromatography to ~28.4-fold with an over all yield of 6.2%.
The purified COX showed a single band of Mr 21 kDa in SDS-PAGE and ~99 kDa band in Native-
PAGE thus indicating an oligomeric nature of the enzyme. The purified COX was adsorbed on to
Psyllium husk followed by exposure to glutarayldehyde (2%; v/v) as a cross-linking agent. The Psyllium husk-bound
enzyme treated with glutaraldehyde showed an activity of 555.0 U while Psyllium husk-bound enzyme without
glutaraldehyde treatment possessed a lower activity (328 U) in comparison to the enzyme used for immobilization (365
U). Thus the Psyllium husk-bound biocatalyst showed 34.2% increase in its activity at 37°C, with pH 7.5 of the free
enzyme. None of the salt ions improved the enzyme activity. Approximately 30% of the original enzyme activity was
noticed in the presence of Ca2+ ions (0.344 U/ml) while Mn2+ ions (0.02 U/ml) drastically reduced the activity of Psyllium
husk-bound COX. The husk-bound COX retained its activity for first 4 days and lost 76.4% of its activity on the 5th day.
The purified free and Psyllium husk-immobilized COX exhibited Km and Vmax values of 14.26 mM and 5.34 U/ml/min,
and 18.84 mM and 6.28 U/ml/min, respectively. The Psyllium husk-immobilized enzyme however, showed 20.74 mM
and 7.75 U/ml/min of Km and Vmax value, respectively in the presence of 50% toluene. Interestingly, in the presence of
toluene, the husk-bound COX not only remained highly active/ stable but also provided ~45% higher rate of reaction
(Vmax) in comparison to the free enzyme.
Keywords: Characterization, Cholesterol oxidase, extracellular, Bacillus sp., Psyllium husk, purification.
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