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。Fe2+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。

A keratinase-producing strain of Bacillus isolated from the alimentary tracts of snakes was classified as Bacillus cereus YSQ08. The alkaline protease aprA gene (1194 bp) of this strain was studied by analyzing the genome and proteinase families of Bacillus cereus ATCC 14579, and the purified keratinase from Bacillus cereus YSQ08 was characterized. The aprA from Bacillus cereus YSQ08 was cloned, optimized, and expressed in recombinant Pichia pastoris X33 for further studying its potential keratinase abilities. After 10-fold concentration, the keratinolytic activity of the recombinant Pichia pastoris X33 was found to be 122.60 U/mL. The aprA protein displayed on the cell surface of Pichia pastoris X33 showed higher keratinolytic activity of 295.78 U/g. The results of the characterization indicated that the optimum conditions for the enzymatic reaction were pH 8.0 at 55 ℃. The presence of Fe2+ significantly increased the keratinase activity of the recombinant aprA protein by 329.08%. The surface-displayed aprA protein was immobilized as a whole-cell catalyst; this had higher thermostability at a lower cost of purification and recycling.

深圳市战略新兴产业发展专项资金资助项目（CXZZ20140418101933991）