Expression And Purification Of Recombinant Cholesterol Oxidase

Cholesterol oxidase (EC1.1.3.6，ChOx), which catalyzes the oxidation and isomerizationof cholesterol into4-cholesten-3-one and H2O2, is widely used in disease diagnosis. Besides, italso has the enormous potential in degrading food cholesterol.At present, the enzyme production level of wild-type stains producing ChOx is low (300U/L). So in the beginning of this study, we used an effective screening strategy andsuccessfully gained a strain with880U/L, which was identified as Rhodococcus equi andnamed as Rhodococcus equi WGC1. However, due to the inherent problems of all wild-typestrains, such as instable enzyme output, fussy purification operation and unsuited enzymaticproperties, we borrowed the trends of other enzymes and then finished cloning ChOx gene(ChoE) from Rhodococcus equi WGC1. Next we used bioinformatics methods to analyze andforecast the primary, secondary, tertiary structures of ChOx. In the third stage, the ChoE wasfused with a dual-tag, L2(252-273)-SUMO, and transformed into Escherichia coli BL21(DE3).After lactose induction at25°C, over90%of the fusion protein were kept in the supernatant asa soluble form. The forth stage was focused on the purification of recombinant ChOx. Usingthe cheap diatomite as adsorption materials, the adsorption equilibrium was quickly achievedwithin30min at pH5.0and the saturated capacity of diatomite was about5.8mg protein per gof diatomite. Then SUMO proteases were adding into diatomite mixtures. Only therecombinant ChOx could be cleaved down and flow into the supernatant. The final activityrecovery and purification fold were87.3and13.24, respectively. Compared with otherpurification methods, the dual-tag purification system simplified procedures, avoided high-saltelution, and gained recombinant ChOx with a natural N-terminal. Thus the dual-tagpurification system could obtain good one-step purification results. Finally, the main charactersof recombinant ChOx (pH and temperature) were investigated. The enzyme activity of therecombinant ChOx was maximal at pH7.5and50°C and the purified enzyme retainedapproximately50%of activity at55°C for2h.