Isolation Of Cholesterol Oxidase-producing Enterobacter Sp. And Purification Of Cholesterol Oxidase

Cholesterol is an important sterol in organism and it exhibited many physiologic function,but too much cholesterol in vivo is one of the dangerous factors that cause coronary artery disease, arteriosclerosis and heart infarction. Cholesterol oxidase(COD,EC1.1.3.6) is a kind of flavoprotein and belongs to GMC oxidation reduction enzyme,which catalyzes the oxidation of cholesterol (5-cholesten-3β-ol) to cholest-4-en-3-one and hydrogen peroxide, is the first and the key enzyme in the pathway of cholesterol metabolism.It may determine the cholesterol concentration in serum quickly and exactly and may be used to diagnose angiosclerosis and other ester derangement disease; it may decrease cholesterol content in food and benefit for health ;it may kill pest of Coleoptera, Lepidoptera, Diptera, Orthoptera and Homoptera and is a kind of safe and useful bioinsecticide; besides, cholest-4-en-3-one which formed by oxidation of cholesterol has the application perspective to heal obesity.The objective of this work is to obtain an abundant source of cholesterol oxidases to meet the needs of industry and medicinal field. Fourteen bacteria strains that produce high level of extracellular cholesterol oxidase (COD) were isolated from different environment. One of these strains,named COX8-9, was found to produce the highest level of cholesterol oxidase,according to the strain morphology,colony character and physiological and biochemistric character , 16SrDNA gene of COX8-9 sequencing results,it can be identified as Enterobacter.The shake flask fermentation condition and medium of strain COX8-9 producing cholesterol oxidase was optimized by single factor and orthogonal experiment.The results indicated that ,the optimal composition of fermentation medium were: cholesterol 0.2%,yeast 0.5%,amidulin 0.5%,CuSO4·5H2O 0.001%,Tween-80 0.3%, NH4NO30.1%,KH2PO40.025%,MgSO4·7H2O 0.025%, FeSO40.0001%,pH 7.0,the optimal cultivation conditions were:5% inoculum size ,15h~18h bacterium age ,volume of substrate was 30ml medium/250ml erlenmeyer flask, 30℃,200r/min,48h fermentation time, cholesterol oxidase activity reached at 748U/L.COD from strain COX8-9 was purified from the culture supernatant by ultra filtration followed with two consecutive Q-sepharose chromatographies in different pH values, and then by superdex-75 gel filtration. The purified enzyme was a monomer with a molecular weight of 58 kDa, and exhibited maximum absorption at 280 nm. The Km value for oxidation of cholesterol by this enzyme was 1.19×10-4 mol/L, with optimum activity at 25℃and pH7.0,it was stable under 40℃and at pH9.0; enzymatic activity was enhanced 3-fold in the presence of metal ion Cu2+, and the enzyme was stable during long-term aqueous storage under 30℃, indicating its potential as a clinical diagnostic reagent. Preparation and characterization of cholesterol oxidases from the other selected strains are under way.