Abstract

The structure and bio-activity of an endogenous cholesterol oxidase fromBrevibacterium sp. was compared to the same enzyme exogenously expressed inEscherichia coli BL21 (DE3) with and without N- or C-terminal his-tags. The different proteins were purified with affinity and subtractive protocols. The specific activity of the natural enzyme from Brevibacterium sp. was 17.5 ± 0.2 U/mg, while the activities of the exogenously expressed forms were 16 ± 0.3 U/mg for non-tagged enzyme from E. coli, 12 ± 0.1 U/mg for the N-terminal his-tagged enzyme, and 4 ± 0.3 U/mg for C-terminal his-tagged enzyme. Circular dichroism revealed that the added histidine residues altered the natural folding of the enzyme. The natural cholesterol oxidase was composed of 39% α-helix, 40% β-sheet, and 20% random coil, while the non-tagged enzyme was composed of 40% α-helix, 35% β-sheet, and 24% random coil. In contrast, the N-terminal his-tagged enzyme was composed of 45% α-helix, 29% β-sheet, and 25% random coil, and the C-terminal his-tagged enzyme was composed of 55% α-helix, 16% β-sheet, and 28% random coil. Hydrophobic fluorescence analysis revealed that the hydrophobicity of the enzyme was reduced by his-tags. Coenzyme-like fluorescent probe binding analysis indicated that the coenzyme binding site should be blocked by his-tags. The his-tag method for protein isolation can disrupt the catalytic activity of the cholesterol oxidase.

Key words: Cholesterol oxidase; Brevibacterium sp.; Escherichia coli; structural disruption, His-tags.