角蛋白酶论文：枯草芽孢杆菌角蛋白酶基因kerC在毕赤酵母中表达的研究.docVIP

角蛋白酶论文：枯草芽孢杆菌角蛋白酶基因kerC在毕赤酵母中的表达研究 【中文摘要】角蛋白酶(keratinase)是一种可特异降解角蛋白的蛋白酶类。近年来角蛋白酶在多个领域中具有良好的应用前景,特别是在饲料生产中,通过角蛋白酶作用将废弃羽毛降解后作为动物饲料的添加剂,充分利用废弃的羽毛蛋白资源,这将改善蛋白质饲料短缺的现状。本研究利用DNA重组技术将来自于枯草芽孢杆菌B-3的角蛋白酶基因kerC表达在巴斯德毕赤酵母X-33中,成功实现了角蛋白酶基因kerC在真核表达系统中的高效表达,并对其酶学性质进行了研究,主要研究结果如下：1.根据枯草芽孢杆菌B-3角蛋白酶基因kerC(GenBank No.:JN021789),设计去除信号肽并含有EcoRI和XbalI酶切位点的上下游引物,PCR扩增角蛋白酶基因kerC表达片段。测序表明,扩增kerC基因表达片段中3个碱基发生错义突变,致使3个氨基酸发生改变。对编码的角蛋白酶蛋白空间结构进行分析,发生改变的3个氨基酸部位未在其活性中心。2.将重组表达载体pPICZaA-kerC线性化后电击转化巴斯德毕赤酵母X-33感受态细胞,转化子经Zeocin的抗性筛选、PCR鉴定和酶活力测定,确定6株阳性转化子。重组菌株P.X.KC-9在以1%浓度的甲醇诱导培养108 h后,其酶活力最高,达到32.31U/mL,比活力为95.43 U/mg。3.测定该重组角蛋白酶的酶学性质,结果表明,重组角蛋白酶kerC的最适反应温度为60℃,在30℃-65℃范围内能保留最高酶活的97%以上。kerC的最适反应pH为pH7.5,在中性和弱碱性环境中对羽毛粉具有较强的降解能力,在pH7-8范围区间内能保持最高酶活的80%以上。在0.33 mM的离子作用浓度环境中,Fe2+、Co2+和Ca2+能增强kerC角蛋白酶的活力,其中Fe2+提高36.44%。Fe3+完全抑制重组角蛋白酶的酶活力,Na+、Mn2+、Zn2+、Mg2+和Cu2+也均15.56%-77.78%范围内,不同程度上抑制了重组角蛋白酶的酶活力。经SDS分析,重组菌株P.X.KC-9表达的角蛋白酶的分子量约为31 KDa,与预测值相近。 【英文摘要】Keratinases are a group of proteolytic enzymes that can hydrolyze insoluble keratins.In recent years,keratinases have been used in the fields of leather processing,detergent manufacture and feed production.Therefore,keratinases have displayed satisfactory application performance and economic value. Particularly in animal feed additives,the bioconversion of chicken feather keratin into animal feedstuffs will ameliorate the shortage of protein feed.The keratinase gene kerC without leader sequence from Bacillus subtilis B-3 was cloned into vector pPICZaA for extracellular expression in Pichia pastoris X-33.KerC was expressed successfully in the Eukaryotic expression system, and the expression condition was highly improved.1.According to the sequence of keratinase gene kerC from Bacillus subtilis B-3,a pair of primers containing the restriction sites of EcoRI and Xball was designed and synthesized.The sequence of kerC without peptide sequence was amplified by PCR and cloned into vector pPICZaA,recombinant plasmid pPICZaA-kerC was constructed successfully.The amp