| Cholesterol oxydase(EC1.1.3.6) catalyzes the oxidation of cholesterol tocholest-4-en-3-one with the reduction of oxygen to hydrogen peroxide. Thecholesterol oxydase is widely applied to diagnosis,drugs manufacture industry,food industry,biologicalagricultural and so on,it has the widespread applicationprospect.This research through UV+LiCl,UV+8-mop processing Paenibacillus spX534, then XUM-10 swain was obtained, enzyme activity is 221.20 U.L-1,itsenzyme activity high 2.5 times than parent strain.In addition, the CLO was optimized, and fermentation technology wasimproved. After 50h's fermentation in shake flask under the condition of 30℃and the initial pH 8.0, rotation speed 180r/min,the enzyme activity in themedium reached to 275.41 U.L-1.We purified the cholesterol oxydase enzyme by circumrotate evaporation,sucrose concentration, DEAE—cellulose ion exchange, Sephadex G—75gel filtration. The activity of the enzyme was improved to 6.83U/mg. Themolecular mass estimated by SDS-PAGE was 58 kDa. Its kinetic parameter Km is 3.3×10-5mol/L and Vmax is 4.60 mmol/(L·min) for cholesterol.Thepurified product was identified as cholest-4-en-3-one with thin steel platechromatographic analysis.Compared with the many kinds of fixed enzymes method, finallydetermined the fixed enzyme the material is PVA+H3BO3+Na2CO3. And theetermination fixed enzyme optimum temperature is 60℃, optimum pH is 7.0,its thermostability and the pH stability relativeand free enzyme to have thevery great degree enhancement, passed SEMS scanning,there are many smalland the dense porous network structure in the immobilizated enzyme interior,so the free enzyme can be very good embedded in the immobilizated material.Through the conversionrate experiment of cholesterol in milk byimmobilizated enzyme, we know the maximum conversion rate is75.64％.
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