Kinetic and thermodynamic properties of a trypsin isoform GT-A from the grass carp (Ctenopharyngodon idellus) intestines

Chinese. 研究草鱼肠道胰蛋白酶的化学组成和酶学性质，以便深入了解该酶的特性并探讨其工业应用潜力。用生物化学方法结合仪器分析，测定了草鱼消化道胰蛋白酶同工酶GT-A的氨基酸组成，分析了其动力学和热力学性质。GT-A的最大反应速度（Vmax）和米氏常数（Km）值分别为2 000 min－1和21.21 μmol，生理转化效率（Vmax/Km）较高，为94.3 min－1•μmol－1；其Arrhenius活化能很低，为17.22 kJ/mol。差示扫描量热分析（DSC）表明，其热变性温度为66.3 ℃，Ca2+的存在可将该酶的热变性温度提高到73.0 ℃。氨基酸分析表明，草鱼胰蛋白酶GT-A的芳香族氨基酸含量很低，形成二硫键的潜力极低。紫外扫描发现，GT-A在205 nm附近有最大紫外吸收，而在280 nm附近的吸收很微弱。GT-A具有冷水性鱼消化蛋白酶的部分特性，且其生理转化效率高而活化能低，具有很好的利用前景。_

English. Investigating the chemical composition and enzymological properties of trypsin from the grass carp (Ctenopharyngodon idellus) intestine is helpful to intensively understand its property and to probe into its potential industrial and commercial application. By biochemical and instrumental analysis,amino acid composition as well as kinetic and thermodynamic properties of the grass carp trypsin isozyme GT-A were determined. Vmax and Km values were determined to be 2 000 min－1 and 21.21 μmol, respectively.And its physiological efficiency (Vmax/Km) was calculated to be 94.3 min－1•μmol－1. GT-A had a low Arrhenius activation energy of 17.22 kJ/mol. By differential scanning calorimetry (DSC), the thermo denaturation temperature of GT-A was determined to be 66.3 ℃. And in the presence of Ca2+, the thermo denaturation temperature of GT-A was increased by 72.9 ℃. The amino acid composition analysis result showed that the content of both aroma amino acid and cysteine of GT-A was low. GT-A had the strongest absorbance near 205 nm, but weak absorbance near 280 nm. These results showed that GT-A possessed some properties of the digestive proteinases from cold-adapted fish, such as high physiological efficiency and low Arrhenius activation energy,and exhibited potential industrial and commercial applications.