化学修饰木瓜蛋白酶的酶学性质研究 熊亚红 吴巧儿 苏健鸿 （华南农业 .doc

化学修饰木瓜蛋白酶的酶学性质研究 熊亚红 吴巧儿 苏健鸿 （华南农业大学 理学院生物材料研究所，广东 广州 510642） 摘 要 采用邻苯二甲酸酐（PA）对木瓜蛋白酶进行了化学修饰，通过三硝基苯磺酸法、紫外光谱荧光光谱对修饰进行了初步表征，采用动力学方法考察了pH和温度对修饰酶水解活性和稳定性的影响，并计算了一系列动力学和热力学参数。实验结果表明： PA对木瓜蛋白酶的平均氨基修饰度为43%，修饰酶较原酶的紫外吸收峰和最大荧光发射峰均发生蓝移，紫外吸收强度降低荧光强度增大；PA修饰未改变木瓜蛋白酶的最适反应温度，但将其最适反应pH由7.0提高到8.5，且酶活力也提高了约20%；PA修饰有效提高了酶的催化水解效率和酶与底物的亲和力，如40 ℃、最适pH条件下修饰酶的转化数kcat（3.03 s-1）和亲和力kcat/Km（1.70 s·L·g-1）均较原酶的（2.28 s-1、1.15 s·L·g-1）高，修饰酶催化水解反应的活化能Ea（25.4 kJ·mol-1）较原酶的（29.3 kJ·mol-1）低；PA修饰明显提高了酶的pH稳定性和热稳定性，半衰期t1/2延长，酶分子的热变性活化能Ea,d由77.0 kJ·mol-1提高到94.5 kJ·mol-1。可见PA化学修饰法是一种有效改善木瓜蛋白酶的催化性质和稳定性的方法。 关键词：木瓜蛋白酶；化学修饰；稳定性；动力学；热力学参数 中图分类号：Q55；O643.32　　　　　　文献标识码：A Study on the Enzymatic Property of Chemical Modifi Papain XIONG Ya-hong WU Qiao-er SU Jian-hong (Institute of Biomaterial, College of Science, South China Agricultural University, guangzhou 510642, China) Abstract: Papain was modified with phthalic anhydride (PA). The modification effect was characterized by trinitrobenzene sulfonic acid method and ultra violet spectrum and fluroscence spectrum and FT-IR spectrum. Effects of pH and temperature on hydrolysis activities and stabilities of modified papain were studied by kinetic method and series of kinetic and thermodynamic parameters were calculated. The experimental results showed that the average amino modification yield was 43% and sulphydryl as an active group of papain weren’t modified. Compared with native papain, modified papain engendered blue shift of ultraviolet peak and fluroscence emission peak and decrease in ultraviolet absorbance and increase in fluroscence intensity. Although PA modification didn’t change the optimal temperature for catalysis of papain, its optimal pH for catalysis was enhanced from 7.0 to 8.5 and enzymatic activity also increased by 20%. PA modification of papain caused that catalysis efficence and substrate affinity were promoted. For example, turnover number kcat (3.03 s-1) and substrate affinity kcat/Km (1.70 s·L·g-1) of modified papain were higher tha