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、荧光光谱及FT-IR光谱对修饰效果进行了初步表征,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%,未对酶的活性基团-SH发生修饰,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修饰还明显提高了酶的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.
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 than those of native papain（2.28 s-1,1.15 s·L·g-1） and activation energy（Ea） for catalysis of modified papain（25.4 kJ·mol-1） were lower than that of native papain（29.3 kJ·mol-1）.pH and thermal stability of papain were enhanced obviously and the half-life time（t1/2） of papain became longer and activation energy（Ea,d） for denaturation raised from 77.0 kJ·mol-1 to 94.5 kJ·mol-1 after native papain was modified by PA.So chemical modification by PA was an effective method to reform catalysis and stability of papain.
出处
《分子催化》
EI
CAS
CSCD
北大核心
2010年第6期529-536,共8页
Journal of Molecular Catalysis(China)
基金
华南农业大学"211工程"重点学科建设项目(2009B010100001)
国家自然科学基金(青年科学基金项目)(20906034B0604)
广东省科技计划项目(2008B080701011)
