【作者】 苑琳；

【导师】 戚薇；

【作者基本信息】 天津科技大学 ， 发酵工程， 2004， 硕士

【摘要】 本文对一株碱性蛋白酶高产菌株的发酵培养基及发酵条件进行了优化，并对酶的分离纯化工艺及酶的主要性质进行了研究。本文研究的主要内容和结论如下： 1、通过对菌株发酵培养基主要成分的逐个替代实验，实现了生产原料的国产化，并确定其培养基配方为：棉籽饼粉（80目）3％，酵母浸粉1.75％，麦芽糊精（DE≥30％）10％，柠檬酸钠0.3％，CaCl₂ 0.3％，K₂HPO₄ 1％。 2、对菌株的发酵条件进行了优化，确定了其最适摇瓶发酵条件：种龄12h，接种量2％，装液量50ml／250ml，摇床转速200r／min，培养温度34℃，发酵54h，碱性蛋白酶的发酵单位可达33985u／ml，达到进口原料的生产水平。在此基础上进行了7L发酵罐发酵工艺的优化，确定其工艺参数为：装料系数0.65，接种量5％，温度34℃，转速200～500r／min，通风量1：0.5～1：2.5，溶氧维持在30～40％。采用自动流加工艺，控制发酵液pH值不低于7.0，发酵50h。采用该工艺连续发酵三批，碱性蛋白酶产量稳定，平均为36471u／ml。 3、采用盐析及离子交换柱层析等方法对酶进行了纯化，SDS-PAGE表明纯化后的样品已达到电泳纯，分子量为28kDa。经过纯化，酶的比活力达18620.20u／mg，纯化了5.23倍，酶活力回收率为27.54％。 4、对酶的性质进行了研究。当以酪蛋白为底物时，其最适作用pH为10～11，最适作用温度为40～60℃。此外，该酶在pH7～12的范围内稳定，在45℃左右有较好的热稳定性。更多还原

【Abstract】 The fermentation medium and the conditions of a high yield strain producing high alkaline protease were studied, the purification and the characterization of the alkaline protease were also studied. The main contents and the results of the study were as follows:1. The main components of the medium were substituted one by one, and all of the producers’ goods were homemade. The optimum medium were consist of: cotton seed meal 3%, yeast extract 1.75%, maltrin (DE 30%) 10%, Nacitrate 0.3%,CaCl20.3%, K2HPO4l%.2. The optimum fermentation conditions were confirmed: seed age 12h, inoculum volume 2%, a 250ml flask containing 50ml medium, 200r/min, 34 C, and the highest enzyme activity was 33985u/ml after cultivated 54h. The experiment was also scaled up in the 7L fermentor. Controlling the fermented broth pH value over 7.0 by the feeding process, the alkaline protease activity was about 36579u/ml of the crude enzyme liquor.3. The alkaline protease was purified using ammonium sulphate precipitation and ion exchange chromatography. The specific activity was 18620.20u/mg, 27.54% of the alkaline protease was recovered with 5.23-fold purification.4. The optimum pH and temperature toward the hydrolysis of casein were pH 10-11 and 40~60 C. The alkaline protease was stable at pH 7~12 and about 80% of the initial enzymatic activity was retained after a 120min incubation period at pH 10.5 and 45 C.更多还原