Klomklao Sappasith, Benjakul Soottawat, Visessanguan Wonnop, Kishimura Hideki, Simpson Benjamin K
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.
J Agric Food Chem. 2006 Jul 26;54(15):5617-22. doi: 10.1021/jf060699d.
Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.
通过硫酸铵沉淀，随后进行一系列色谱分离，将长鳍金枪鱼（Thunnus tonggol）脾脏中的胰蛋白酶纯化至402倍。通过尺寸排阻色谱法和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）估计胰蛋白酶的分子量为24 kDa。在天然PAGE上呈现为单一条带的胰蛋白酶在pH 8.5和65℃时显示出最大活性。它在6 - 11的宽pH范围内稳定，但在高于50℃的温度下不稳定。该酶需要钙离子来保持热稳定性。其活性受到1.0 g/L大豆胰蛋白酶抑制剂和5 mM甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）的强烈抑制，并受到2 mM乙二胺四乙酸的部分抑制。随着NaCl浓度（0 - 30%）的增加，活性降低。该酶对盐酸Nα-对甲苯磺酰-L-精氨酸甲酯的Km为0.25 mM，Kcat为200 s-1。胰蛋白酶的N端氨基酸序列被确定为IVGGYECQAHSQPHQVSLNA，与其他胰蛋白酶高度同源。
