Purification And Stability Of Superoxide Dismutase Frome Wine Yeast

Superoxide dismutase(SOD) is distributed in the biosphere, it was the main biologicalfree radical scavenger and nature antioxidant. The SOD preparations are widely applized inthe medical service, foods and the cosmetics field. but natural SOD enzyme with poor stability,short half-life and other shortcomings, which limits its wider uses.Gansu is rich in wine yeastresources, it can viewed as better material of extracting SOD. This test is based on wine yeastas research material, determined the best optimization of extraction process of SOD,ion-exchange chromatography was applied to further purified the enzyme and immobilizedthe SOD by chitosan.finally compared the stability between nature SOD and immobilizedSOD. The main results are as follows:1. SOD was extracted from wine yeast by ultrasonic wave and ultrasonic-assistedsnailase technology, SOD specific activity was identified as a measure indicate. By a series ofsingle-factor and orthogonal tests, the optimum process of ultrasonic wave wasmaterial-liquid ratio1:2.5, ultrasonic power400W, ultrasonic time16min, thermaldenaturation temperature45℃, under these conditions, the specific activities of SOD was169.32U/mg; SOD was extracted from it with ultrasonic-assisted snailase technology, thebest process conditions was ultrasonic power400W, ultrasonic treatment time12min,concentration of Snailase2.0%, extraction pH6.6, treatment temperature37℃. Underthese optimal conditions, the specific activity of SOD could grow up to192.27U/mg.2. Waste wine yeast as the research object, The SOD was extracted from it withultrasonic-assisted snailase technology. After heat treatment and DEAE-Sephadex A50ion-exchange chromatography, purification fold was27.48and the yield ratio was15%. The purityof SOD measured by SDS-PAGE, One electrophortic band was detected that corresponds tomolecular mass of32kDa.3. With the purified SOD as test material, chitosan as a fixed carrier, the processparameters were optimized by single-factor and orthogonal experiment. The results showedthat5mL SOD(1219.3U/mg) was added in0.4g chitosan which was activated by0.6%glutaraldehyde at25℃for1.5h, specific activity of immobilized enzyme was280.79U/mg.4. The stability of nature SOD was compared with immobilized SOD, the results showed that thethermostability, acid-base stability and half-life of immobilized SOD were higher than thenatural SOD, improved by26.16%,43.61%and77.5d, respectively. In addition,Na+and Ca2+were not significance to nature enzyme and immobilized SOD, Cu2+and Zn2+with a low density could have more significant influence on nature and immobilized SOD than that withhigh concentration, in addion, Zn2+with high concentration could increase the activity ofSOD and Fe2+showed different inhibition on nature and immobilized SOD.