Current Biotechnology

Pabulo Henrique Rampelotto  
Center of Biotechnology and PPGBCM
Federal University of Rio Grande do Sul
Porto Alegre, RS
Brazil

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An Efficient Immobilization of Cholesterol Oxidase from Bacillus sp. COX-T3 onto Psyllium Husk Fibers

Author(s): Lata Kumari, Tanu Johari, Shamsher S. Kanwar.

Graphical Abstract:


Abstract:

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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Article Details

VOLUME: 3
ISSUE: 4
Year: 2014
Page: [295 - 304]
Pages: 10
DOI: 10.2174/2211550104666150224234927
Price: $58