Screening Of A Red Mutant Of Cholesterol Oxidase Producer From Brevibacterium Sp. And The Study Of Related Enzyme Stability

A higher producer of cholesterol oxidase(COD),red mutant DGCC N-25, was isolated from the Brevibacterium mutant DGCDC-82 mutated by NTG under Ultrasonication . The COD production of the mutant DGCCN-25 was 140% higher than the initial strain. And the red substance was extracted and analyzed, named as Brevibacterium chrysoidine pigment. The fermentation condition for COD production of mutant DGCCN-25 was improved by adjusting cholesterol content from 3 g/l to 4g/l and yeast extract from 8 g/l to 9g/l, the COD activity reached 1.512U/ml.The preparation of COD from a mutant DGCCN-25 was studied. The broth free of cells was attained after centrifugation with isopropanol 4.4%(V/V), the lost of COD reduced from 25% to 8%. Sephadex G-25 gel chromatography and ion-exchange chromatograph are adopted respectively for further purification. After Sephadex G-25 gel purified, 2.58-fold purified COD was obtained with a Specific activity of 4.95U/mg.After Ion-exchange chromatography purified, 2.67-fold purified COD was obtained with a Specific activity of 13.26U/mg. Then, used Sepha- dex G-25 gel, and yielded COD with a Specific activity is 15.31 U/mg, the total yield is 41.3%.The crude COD from mutant DGCCN-25 showed unstable when it was treated higher than 40℃, activity lost of 70% at 4℃after 350 day's storage, and easy denaturalization when it is prepared through vacuum freeze-dried. The research showed adding potassium sodium tartrate-manrose to the enzyme system improving the stability of COD : reducing enzyme lost from 30% to 10% when it was treated at 40℃for 3h, rising enzyme receive from 20% to 70% when it was saved at 4℃for 340 days; and reducing enzyme lost from 30% to 10% when it was vacuum freeze-dried.