蜡样芽胞杆菌YSQ08碱性蛋白酶aprA基因在毕赤酵母中的表达

发布时间：2018-04-04 04:18

  本文选题：角蛋白酶　切入点：aprA　出处：《现代食品科技》2016年12期

【摘要】：从蛇消化道内容物分离出的一株产角蛋白酶菌株为蜡样芽孢杆菌YSQ08。通过分析Bacillus cereus ATCC 14579的基因组和蛋白酶家族,对比分析蜡样芽孢杆菌YSQ08纯化角蛋白酶的酶活特性,确定碱性蛋白酶aprA基因(1194 bp)为目标角蛋白酶。为深入研究角蛋白酶的潜在能力,对蜡样芽孢杆菌YSQ08碱性蛋白酶aprA基因在毕赤酵母X33上进行克隆、表达和优化重组。结果其10倍浓缩的角蛋白酶酶活达到122.60 U/mL。经优化后的aprA基因在表面展示重组酵母中得到高效表达,并且具有较高的角蛋白酶活性,酶活最高可达到295.78 U/g干细胞重。酶学性质分析结果表明,表面展示表达的重组aprA蛋白酶性质与天然型相比具有更高的热稳定性,最适反应温度为55℃,最适pH为8.0。Fe~(2+)可显著提高重组蛋白的酶活性,最高可达329.08%。表面展示表达的重组角蛋白酶可作为全细胞固定化催化剂,具有更好的热稳定性并能降低酶的纯化回收成本。
[Abstract]:A keratinase producing strain isolated from the contents of snake digestive tract was Bacillus cereus YSQ08.By analyzing the genome and protease family of Bacillus cereus ATCC 14579, and comparing the activity of keratinase purified by Bacillus cereus YSQ08, we confirmed that aprA gene of alkaline protease (aprA) was the target keratinase.In order to study the potential ability of keratinase, the YSQ08 alkaline protease aprA gene of Bacillus cereus was cloned, expressed and recombined with Pichia pastoris X33.Results the activity of 10 times concentrated keratinase was 122.60 U / mL.The optimized aprA gene was highly expressed in the surface display recombinant yeast and had high keratinase activity, the highest activity of which could reach 295.78 U / g stem cell weight.The results of enzymatic analysis showed that the properties of recombinant aprA protease expressed on the surface had higher thermal stability than that of the natural type. The optimum reaction temperature was 55 鈩，

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