海洋胶红酵母菌CD-008产超氧化物歧化酶发酵条件优化及酶分离纯化

在单因素试验基础上，结合Plackett-Burman、Box-Behnken设计及响应面分析法进行回归分析以确定海洋胶红酵母菌Rhodotorula sp. CD-008产超氧化物歧化酶最佳发酵条件，并通过盐析、超滤离心、Sephadex G-100凝胶过滤层析研究了酶的分离纯化条件。结果表明，pH、转速、温度对酶活力有显著影响，最优发酵条件为pH 5.34、转速150 r/min、温度21.40 ℃，优化后发酵液酶活力达到6 430.52 U/g湿菌体，比优化前提高了1.53倍。粗酶液经65%饱和度的硫酸铵盐析、超滤离心、Sephadex G-100凝胶过滤层析后获得的SOD比酶活达到419.90 U/mg，纯化倍数为9.60倍，蛋白回收率为8.2%。SDS-PAGE凝胶电泳显示，纯化后的SOD的分子质量约为37.5 ku。

Optimization of fermentation conditions of SOD-producing Rhodotorula sp. CD-008 and separation and purification of the SOD

On the basis of single factor experiments, the optimum fermentation conditions of SOD-producing Rhodotorula sp. CD-008 were determined by Plackett-Burman, Box-Behnken design and response surface analysis, and the separation and purification conditions of SOD were research by salting out, ultrafiltration centrifugation and Sephadex G-100 gel filtration chromatography. Results indicated that pH, rotate speed and temperature had a significant effect on enzyme activity. The optimum fermentation conditions was pH 5.34, rotate speed 150 r/min and temperature 21.40 ℃. Under the conditions, the enzyme activity in fermentation liquor was 6 430.52 U/g fresh cells, which was 1.53 times higher than that before optimization. The crude enzyme liquid was purified by ammonium sulfate with degree of saturation 65%, ultrafiltration centrifugation and Sephadex G-100 gel filtration chromatography, specific enzyme activity of SOD obtained was 419.90 U/mg, purification fold was 9.60 and protein recovery rate was 8.2%. SDS-PAGE electrophoresis showed that molecular weight of SOD purified was about 37.5 ku.