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 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.
Key words: Papain; Chemical modification; Stability; Kinetics; Activation Energy
E-mail: xiongyahong@scau.edu.cn
Journal of Molecular Catalysis, Vol. 24, Issue 6, 2010, 529~536
