Centro de Investigación en Alimentación y Desarrollo (CIAD), Carretera a La Victoria Km. 0.6, Apdo. Postal 1735, Hermosillo, Sonora 83000, Mexico.
Food Chem. 2013 Nov 15;141(2):940-5. doi: 10.1016/j.foodchem.2013.03.078. Epub 2013 Apr 6.
Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS-PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)). Trypsin activity decreased continuously as NaCl concentration increased (0-30%). Km and kcat values were 0.13mM and 1.46s(-1), respectively. Results suggest the enzyme have a potential application where room processing temperatures (25-35°C) or high salt (30%) concentration are needed, such as in fish sauce production.
高体革鯻内脏胰蛋白酶经硫酸铵分级沉淀、凝胶过滤、亲和层析和离子交换层析（DEAE-琼脂糖）纯化。SDS-PAGE 显示，胰蛋白酶的分子量约为 27.5kDa，在同工酶凝胶中显示为单一带。它在 pH9.5 和 40°C 时表现出最大活性，使用 N-苯甲酰基-dl-精氨酸对硝基苯胺（BAPNA）作为底物。酶被苯甲基磺酰氟（PMSF）（100%）、N-α-对甲苯磺酰基-l-赖氨酸氯甲基酮（TLCK）（85.4%）、苯甲脒（80.2%）和大豆胰蛋白酶抑制剂（75.6%）有效抑制，部分被 N-对甲苯磺酰基-l-苯丙氨酸氯甲基酮（TPCK）（10.3%）、乙二胺四乙酸（EDTA）（8.7%）和胃蛋白酶抑制剂 A（1.2%）抑制。酶活性受到金属离子（Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)）的轻微影响。胰蛋白酶活性随 NaCl 浓度的增加而持续下降（0-30%）。Km 和 kcat 值分别为 0.13mM 和 1.46s(-1)。结果表明，该酶在需要室温处理温度（25-35°C）或高盐（30%）浓度的情况下具有潜在的应用，例如在鱼露生产中。
