Expression And Affinity Purification Of Cholesterol Oxidase From Brevibacterium Sp.DCCDC-82

In this paper, in order to promote the yield of the Cholesterol Oxidase（COD）, and obtaina simple and efficient purification process, considering that the expression level of the proteincan be improved by His-tag, three different recombinant enzymes had been expressed inEscherichia coli: the untagged enzyme（BCO）, and two His-tag enzymes（BCOc/BCOn）,identical to the untagged enzyme except that they carried the additional C or N terminalHis-tag. Then the recombinant enzymes were purified by IMAC or coenzyme medium R-2affinity chromatography, and the properties were studied.ChoB was representive for the gene of COD, using it as the template from theBrevibacterium sp. DGCDC-82, three applified fragments ChoB, ChoBc and ChoBn wereobtained, then linked with pET28a （+）, and expressed in E.coli BL21（DE3） successfully.After fermentation promotion, BCOn showed the highest express level and activity, andspecific activity could reach to6.5U/mg. This revealed that the expression level could beimproved with a fusion gene in its N terminal.Two methods were used to purify them: metal ion affinity chromatography （IMAC） andcoenzyme medium affinity chromatography. By HisTRap FF, tagged COD（BCOc and BCOn）were purified, and recovery rate was60%, purity was more than90%; Due to the lacking ofHis-tag for BCO, we made a new affinity medium, which included3parts: sepharose CL4Bas chromatography medium, riboflavin as affinity ligand, cyanuric chloride andethylenediamino as spacer. And then BCO, BCOc and BCOn were purified, and recovery ratecan reach to70%, meanwhile, the purity was more than95%. Compared with the IMAC(Ni²⁺), the coenzyme medium affinity chromatography for flavin enzymes could get a betterpurification effect without any purification label.Mg²⁺, Na+, Ca²⁺and Zn²⁺had little effect, but Ag+and Hg²⁺had strong inhibition on theseenzymes. It was found that the enzyme acitivity of BCOn was strongly increased, but notBCOc and BCO, when Cu²⁺was added to the assay system. The kinetic parameters VmaxofBCO, BCOc and BCOn were0.027mmol/L·min,0.031mmol/L·min and0.038mmol/L·min,the Kmwere0.36mmol/L,0.25mmol/L and0.33mmol/L respectively.