Screening The High Yield Stains Of Cholesterol Oxidase (COD) And Researching The Properties Of COD

More and more attention has been paid on cholesterol oxidase for its significant function on food processing,medical assay and biological pest-resistance which showed a great applied potential.The main research in this paper are as follow:the high yield strain which could producing cholesterol oxidase was screened;optimization fermentation conditions of producing cholesterol oxidase by Pseudomonas stutzeri were investigated;cholesterol oxidasc from Pseudomonas stutzeri was isolated and purified;cholesterol oxidasc properties were studied.Major results were as follows:(1)Forty strains using cholesterol as the sole carbon source were isolated from eleven samples collected from different locations.One strain with high yield of extracellular cholesterol oxidase was screened.The highest activity of cholesterol oxidase was 839.35 U/L.The strain was identified as Pseudomonas stutzeri through analyzing its morphology,physiological and biochemical characteristics and sequence analysis of 16S rDNA.(2)The culture condition of Pseudomonas stutzeri was optimized in this paper.The optimal medium is:Glucose 3%,yeast extract paste 0.75%,cholesterol 0.04%,Triton-1000.05%,NaCl 0.1%,K2HPO4 0.02%,MgSO4 0.005%,FeSO4 0.001%,CaCl2 0.01%;the optimal cultrue condition is:pH7.5,the inoculums volume was 2%,medium volume 50mL in 250mL flask and 200r/min,the tempreture is 41?.Under the optimal cultivation condition,the cholesterol oxidase activity of Pseudomonas stutzeri was increased to 1691.722U/L,improve 1.02 times than before.(3)The cholesterol oxidase of Pseudomonas stutzeri was preliminary purified using saturated ammonium sulfate and dialysis before subjected to the Sephadex G-75.The results are as follows:specific activity of the purified enzyme reached 24.95 U/mg,the purification multiples is 9.18.The specific activity of 24.95 U/mg improve 8.18 times than the specific activity of the fermentation supernatant with 2.718 U/mg,and improve 1.93 times than the specific activity of the crude enzyme after salting with 8.521 U/mg.(4)Properties of cholesterol oxidase which has purified were studied.The optimum temperature is 37? and optimum pH is 7.5.The temperature stability range is from 20? to 40? and the pH stability range is from 6 to 8.The activity of cholesterol oxidase kept more than 70%in the buffer environment of pH6?pH8 after 1h.