【作者】 杨明琰；

【导师】 郭爱莲；

【作者基本信息】 西北大学 ， 应用微生物学， 2005， 硕士

【摘要】 从50株不同种属的酿酒酵母中筛选出一株生物量及SOD含量均较高的酵母菌B94,以其为出发菌进行了原生质体紫外线诱变,亚硝基胍及⁶⁰CO-γ射线复合诱变育种,结果得到了一株高产突变株CNU94,其SOD含量为1872u/g湿菌体,较出发菌提高134%,且遗传性能稳定,是—株有生产潜力的高产SOD菌株。 对突变株CNU94的发酵工艺条件进行了正交试验及单因子条件实验,确定了其发酵产酶的优化培养条件为:培养基组成为葡萄糖2%,蛋白胨2%,酵母膏1%,7°Be麦芽汁9%（V/V）,CuSO₄75mmol/ml,ZaSO₄25mmol/ml,pH5.5,摇瓶装量为75ml/500ml三角瓶,接种量15%,培养20小时加H₂O₂至终浓度为1%,继续培养至24小时。在此条件下,细胞生物量为5.26g/100ml,SOD含量为2662u/g湿菌体,SOD产量为14002u/100ml,产量较出发菌提高99%。 酿酒酵母CNU94经发酵培养后,离心收集菌体,将菌体用甲苯法破壁得到粗酶液,粗酶液经50℃加热,调节pH除杂蛋白,丙酮分级沉淀后,上DEAE纤维素-32柱层析四步纯化工艺,可得到纯化的SOD,其比活为3500u/rag,收率为58.3%,纯化倍数为30.43倍。PAGE电泳后的活性染色结果显示,酵母超氧化物歧化酶具四条同功酶,蛋白染色结果显示酶纯度已达电泳纯。该酶经酶类型鉴定为Cu/ZnSOD。更多还原

【Abstract】 The strain of Saccharomyces cerevisiae B94 was obtained from more than 50 strains of different species and genera of yeasts. It’s SOD content is more than 800u/g fresh cell. Based on this strain, a SOD high-producing mutant was obtained through compound mutagenic of UVtreatment to protolast, NTG and ⁶0COγ-rays .The content of the mutant reached 1872u/gfreshcell, which was 134 high than original strains. Genetic stability test showed it was an excellent SOD producer.Based on orthogonal and monofactorial test, the medium and fermentation condition wasstudied.It showed the optimal medium consisted of 2% glucose, 9%（V/V） malt wort, 2%peptone, 1%yeast extract, CuSO₄ 75mmol/ml, ZnSO₄25mmol/ml; the optimal culture condition were as follows: initial pH 5.5,medium cubage was 75ml/500ml, 15% concentration wasinoculated; After 20 hours, H₂O₂ was added to final concentration 1%, then culture to 24 hours. In this condition, the biomass was 5.26g/100ml, SOD activity was 2662u/g fresh cell, SOD production was 1.40×10⁴u/100ml, which was 99% high than original strain.S.cerevisiae CNU94 was purified by procedures consisting of heat, pH regulation,two-steps acetone precipitation and chromatography of DEAE cellulose -32 column. The purified enzymatic specific activity and recovery was 3500u/mg and 58.3%, respectively,the purification multiple was 30.43-fold as much.The activity staining showed Sxervisiace had fourisoenzyme, protein staining showed it reached homogeneous.The types identification showed it was a kind of Cu/Zn SOD.更多还原