【作者】 张洋；

【导师】 刘树滔；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 本文是关于利用地衣芽孢杆菌(Bacillus licheniformisFBK-1)发酵产生角蛋白酶粗酶液,对其理化性质进行研究,将其应用到羽毛角蛋白的生物加工过程中,并分析酶解效果的研究。通过微生物发酵得到含角蛋白酶的发酵液,研究了发酵液的最适作用pH、最适作用温度以及金属离子的影响,然后在最适条件下酶解羽毛角蛋白,测定酶解产物的分子量大小、可溶性蛋白质含量、胃蛋白酶消化率及氨基酸组成。地衣芽孢杆菌FBK-1(Bacillus licheniformis FBK-1)在以羽毛为唯一碳源和氮源的液体培养基中会产生含有角蛋白酶的发酵液(粗酶液),发酵周期为72h,发酵液中角蛋白酶酶活单位在600-700U/ml之间。发酵产生的粗酶液最适作用pH为9.0,最适作用温度为50℃,在pH 5.0-9.0、温度在40℃以下时角蛋白酶活性较稳定,一定浓度的Na+、K+、Ca2+、Mg2+对粗酶液中的角蛋白酶酶活有促进作用。将整根鸡毛润湿,在121℃下处理1hr,冷却至室温,加入粗酶液,40℃缓慢震摇3hr,烘干粉碎制成羽毛粉。其中粗蛋白含量80%,可溶性蛋白含量10%,胃酶消化率78%,与市售羽毛粉具有相同品质。将鸭毛粉碎成粉、润湿,在121℃下处理1hr,冷却至室温,加入适量粗酶液,40℃缓慢震摇28h,过程控制pH 9.0,离心分离上清与沉淀。酶解沉淀中可溶性蛋白含量为17.96%,胃酶消化率88%,除三种氨基酸(頼氨酸、蛋氨酸、组氨酸)含量低于鱼粉,其余14 种氨基酸含量均高于鱼粉或处于同一水平,可作为饲料添加剂替代鱼粉;酶解液上清经凝胶柱Super Peptide 10/300 GL 分离,发现酶解液上清中的组分均为分子量在6kD 以下的多肽和氨基酸;经过氨基酸组成分析,酶解液中含有17 种氨基酸,可作为多肽与氨基酸产品加工的原料。综上所述,地衣芽孢杆菌FBK-1(Bacillus licheniformis FBK-1)的发酵液为一种新型的角蛋白酶酶液,它可酶解羽毛角蛋白,酶解产物可被加工成羽毛粉、氨基酸与多肽等多种产品。更多还原

【Abstract】 Keratinase was fermented by Bacillus licheniformis FBK-1. The effects of pH , temperature and metal ions on enzymic activity were investigated. The enzyme was applied on feather degradation, the character of product including molecular weight distribution, protein solubility, pepsin Digestion rate and Amino acid was analyzed. The crude enzyme from Bacillus licheniformis FBK-1 was produced when the feather is the sole source of Carbon and Nitrogen. The time of fermentation is 72 hours, the activity of keratinase is between 600 and 700 unit per milliliter, the optimum conditions of activity were pH9.0 and temperature50℃. The enzyme was stable at pH between 8.0-9.0 and temperature is up to 40℃. The activity was enhanced by Na+、K+、Ca2+、Mg2+, activity is dosc dependent. The wetting chicken feather incubated at 121℃for 1 hour, then hydrolyzed by keratinase production, the dried enzymolysis product powdered, was subject to further analysis, the result indicated 80 percent crude protein , 10 percent of soluble protein and 78 percent of Pepsin Digestion rate. The product possessed same quality with feather meal in the market. The wetting duck feather meal treated in 121℃1 hour was hydrolyzed at 40℃, pH 9.0 for 28 hours by compatible keratinase production, then supernate and precipitate was separated by centrifuge. The precipitate contain 17.96 percent of soluble protein and 88 percent of Pepsin Digestion rate. Except three kinds of amino acid (Lys,Arg,His),the content of rest 14 kinds of amino acid was higher than fish meal. The precipitate can replace fish meal as feedstuff additive. The supernate was applied to a Super Peptide column 10/300 GL, most fraction were eluted behind 6KD. Both supernate and precipitate were subjected into amino acid analysis, the supernate contains 17 kinds of amino acid,which can be material of amino acid and peptide productions. Therefore,the enzyme fermented by Bacillus licheniformis FBK-1 was a new kind of keratinase. It can be used to hydrolyze feather and the enzymolysis product can be applied to amino and peptide productions.更多还原