深海角蛋白降解菌Bacillus aquimaris JM7的发酵条件优化及功能基因的克隆表达

发布时间：2018-05-13 20:45

  本文选题：海水芽孢杆菌 + 发酵优化　； 参考：《国家海洋局第三海洋研究所》2016年硕士论文

【摘要】：随着养殖业规模持续扩大,羽毛作为家禽的副产物的大量堆积不仅造成环境破坏、病原微生物滋生,还会造成角蛋白资源的严重浪费。本研究采用条件温和、工艺环保的微生物法降解羽毛角蛋白,利用液体发酵法探究菌株降解羽毛的最优化条件,并对角蛋白酶基因进行克隆,进一步在大肠杆菌系统中进行融合表达,拟实现角蛋白酶的外源表达。1.以羽毛为唯一碳、氮源筛选到一株高效降解角蛋白的菌株海水芽孢杆菌Bacillus aquimaris JM7。对Bacillus aquimaris JM7以羽毛为底物的发酵条件进行了单因素及正交试验优化,实验结果显示:Bacillus aquimaris JM7降解羽毛最适发酵条件为:培养基初始pH值为9.0;菌接种量4%(v/v);培养温度40℃;摇床转速150rpm;羽毛粉添加量为0.2%(m/v);额外添加氮源及蔗糖对菌株降解羽毛粉均有抑制作用。上述条件下培养30h后,羽毛粉降解率达74.5%,高于多数文献报道水平。Bacillus aquimaris JM7降解羽毛主要产生谷氨酸、丝氨酸、脯氨酸等17种氨基酸。2.对角蛋白酶基因Ker02562进行了克隆、表达和纯化。采用PCR技术扩增了角蛋白酶基因,其全长为963 bp,编码了一个由320个氨基酸残基组成、预计分子量为34.181kDa的蛋白质。构建了重组表达质粒并转化大肠杆菌BL21(DE3)进行诱导表达,SDS-PAGE分析表明,重组角蛋白酶E.coli-pCold I-Ker02562的分子量为38kDa左右(角蛋白酶34kDa,pCold I自身标签4kD左右),和预测分子量一致。3.对纯化后的角蛋白酶酶学性质研究结果表明:(1)酶的最适反应温度为40℃,在10-50℃条件下热稳定性良好;最适pH为7.0-10.0,该条件下酶能保持90%以上的活性;(2)金属离子Ca2+,Fe2+,Sr2+对酶活具有促进作用;(3)浓度为5mM的PMSF和1mM、5mM的EDTA对酶活有抑制作用,说明该酶为丝氨酸蛋白酶,其活性中心可能含有金属离子;(4)有机溶剂DMSO、异丙醇和乙腈对酶活均有抑制作用,浓度越高,抑制作用越大。
[Abstract]:As the scale of aquaculture continues to expand a large number of feathers as by-products of poultry not only cause environmental damage pathogenic microorganisms breeding but also cause serious waste of keratin resources. In this study, a mild and environmentally friendly microbial method was used to degrade feather keratin, and liquid fermentation was used to explore the optimum conditions for degradation of feather, and the keratinase gene was cloned. Further fusion expression was carried out in Escherichia coli system to realize the extraneous expression of keratinase. A highly efficient keratin degrading strain, Bacillus aquimaris JM7, was isolated from the feather as the sole carbon source. The fermentation conditions of Bacillus aquimaris JM7 with feather as substrate were optimized by single factor and orthogonal experiment. The results showed that the optimum fermentation conditions were as follows: initial pH value of culture medium was 9. 0; inoculation amount was 4% / v / v; culture temperature was 40 鈩，

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