Laboratoire de Genie Enzymatique et de Microbiologie, Ecole Nationale d'Ingenieurs de Sfax, B.P. W, 3038 Sfax, Tunisia.
J Agric Food Chem. 2009 Nov 25;57(22):10943-50. doi: 10.1021/jf902059a.
An alkaline trypsin from the intestine of striped seabream (Lithognathus mormyrus) was purified and characterized. The enzyme was purified to homogeneity by precipitation with ammonium sulfate, Sephadex G-100 gel filtration and CM-Sephadex cation-exchange chromatography, with a 24.9-fold increase in specific activity and 13% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 27.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. The purified trypsin appeared as a single band on native PAGE. Interestingly, the enzyme was highly active over a wide range of pH from 8.0 to 11.0, with an optimum at pH 10.0 using Nalpha-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 8.0, 11.0, and 12.0 were 73%, 67% and 50.4%, respectively. The enzyme was extremely stable over a broad pH range (5.0-12.0). The optimum temperature for enzyme activity was 50 degrees C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI). In addition, the enzyme showed excellent stability toward various surfactants and bleache agents and compatibility with some commercial solid and liquid detergents. The trypsin kinetic constants, Km and kcat of the enzyme for BAPNA, were 0.29 mM and 1.36 s(-1), respectively, while the catalytic efficiency kcat/Km was 4.68 s(-1) mM(-1).
从条纹鲷（Lithognathus mormyrus）的肠道中纯化和表征了一种碱性胰蛋白酶。该酶通过硫酸铵沉淀、Sephadex G-100 凝胶过滤和 CM-Sephadex 阳离子交换层析纯化至均一性，比活度提高了 24.9 倍，回收率为 13%。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和尺寸排阻层析法估计纯化的碱性胰蛋白酶的分子量为 27.5 kDa。在天然 PAGE 上，该酶呈现出单一的条带。有趣的是，该酶在 pH 8.0 到 11.0 的较宽范围内具有高度活性，以 Nalpha-苯甲酰基-dl-精氨酸-p-硝基苯胺（BAPNA）为底物时，最佳 pH 值为 10.0。在 pH 8.0、11.0 和 12.0 下的相对活性分别为 73%、67%和 50.4%。该酶在较宽的 pH 范围内（5.0-12.0）非常稳定。酶活性的最佳温度为 50°C。该纯化酶被大豆胰蛋白酶抑制剂（SBTI）强烈抑制。此外，该酶对各种表面活性剂和漂白剂表现出优异的稳定性，并与一些商业固体和液体洗涤剂兼容。该酶对 BAPNA 的动力学常数 Km 和 kcat 分别为 0.29 mM 和 1.36 s(-1)，而催化效率 kcat/Km 为 4.68 s(-1) mM(-1)。
