Biotechnology Program, Faculty of Agro- and Bio-Industry, Thaksin University, Phatthalung Campus, Phatthalung 93210, Thailand.
Department of Food Science and Agricultural Chemistry, McGill University, Macdonald Campus, Ste. Anne de Bellevue, Quebec H9X 3V9, Canada.
Int J Biol Macromol. 2019 Jul 15;133:971-979. doi: 10.1016/j.ijbiomac.2019.04.122. Epub 2019 Apr 24.
Anionic trypsin from albacore tuna spleen was purified by chromatographic separations on Q-Sepharose, Superdex 75 and Arginine Sepharose 4B. The trypsin migrated as single bands in both SDS-PAGE and native-PAGE. The molecular weight of purified trypsin was estimated to be 30 kDa using SDS-PAGE. The enzyme exhibited maximal activity at pH 9.0 and 55 °C for hydrolysis of Boc-Val-Pro-Arg-MCA. pH and temperature stabilities of the trypsin were well maintained in the pH range of 6-11 and over a temperature range from 20 up to 50 °C. The enzyme was effectively inhibited by soybean trypsin inhibitor, N‑tosyl‑l‑phenyl‑alanine chloromethyl ketone (TLCK) and Pefabloc SC. The N-terminal amino acid sequence of 20 residues of the purified enzyme was IVGGYECQAHSQPHQVSLNA, which is highly homologous to other fish trypsins. The k/K of the enzyme for Boc-Val-Pro-Arg-MCA was 2.60 ± 0.07 s mM. Purified trypsin also hydrolysed fish muscle proteins, suggesting its effectiveness in degradation of food proteins.
从鲣鱼脾脏中分离纯化阴离子胰蛋白酶，通过 Q-Sepharose、Superdex 75 和精氨酸琼脂糖 4B 进行色谱分离。胰蛋白酶在 SDS-PAGE 和 native-PAGE 中均呈现单一条带。使用 SDS-PAGE 估计纯化胰蛋白酶的分子量为 30 kDa。该酶在 pH 9.0 和 55°C 下对 Boc-Val-Pro-Arg-MCA 的水解具有最大活性。该酶的 pH 和温度稳定性在 pH 6-11 范围内和 20 至 50°C 的温度范围内得到很好的维持。该酶被大豆胰蛋白酶抑制剂、N-甲苯磺酰-L-苯丙氨酸氯甲基酮（TLCK）和 Pefabloc SC 有效抑制。纯化酶的 20 个残基的 N-末端氨基酸序列为 IVGGYECQAHSQPHQVSLNA，与其他鱼类胰蛋白酶高度同源。该酶对 Boc-Val-Pro-Arg-MCA 的 k/K 为 2.60±0.07 s mM。纯化的胰蛋白酶还水解鱼肉蛋白，表明其在降解食物蛋白方面的有效性。
