Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d'Ingénieurs de Sfax, Tunisia.
J Agric Food Chem. 2010 May 12;58(9):5763-9. doi: 10.1021/jf100534a.
Trypsin from the intestine of smooth hound (Mustelus mustelus) was purified by fractionation with ammonium sulfate, Sephadex G-75 gel filtration, and DEAE-cellulose ion exchange chromatography, with a 65-fold increase in specific activity and 15% recovery. The molecular weight of the purified trypsin was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme showed esterase-specific activity on N(alpha)-p-tosyl-L-arginine methyl ester hydrochloride (TAME) that was four times greater than its amidase-specific activity on Nalpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the trypsin activity were pH 8.5 and 50 degrees C, respectively, using TAME as a substrate. The enzyme was extremely stable in the pH range of 7.0-9.0 and highly stable up to 40 degrees C after 1 h of incubation. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK), specific inhibitors for trypsin. In addition, smooth hound trypsin showed higher proteolytic activity at high NaCl concentration, demonstrating its potential for protein hydrolysis at high salt content. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECKPHSQ. This sequence showed high homology with trypsins from marine vertebrates and invertebrates. Purified trypsin had a Michaelis-Menten constant (K(m)) and catalytic constant (K(cat)) of 0.387 +/- 0.02 mM and 2.62 +/- 0.11 s(-1), respectively, when BAPNA was used as a substrate. For the hydrolysis of TAME, K(m) and K(cat) were 0.156 +/- 0.01 mM and 59.15 +/- 2.2 s(-1), respectively.
从平滑无须鳕（Mustelus mustelus）的肠道中提取的胰蛋白酶，经过硫酸铵分级沉淀、Sephadex G-75 凝胶过滤和 DEAE-纤维素离子交换层析纯化，比活提高了 65 倍，回收率为 15%。用凝胶排阻色谱法和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估计纯化胰蛋白酶的分子量为 24 kDa。该酶对 N(alpha)-对甲苯磺酰-L-精氨酸甲酯盐酸盐（TAME）具有酯酶特异性活性，比其对 Nalpha-苯甲酰-DL-精氨酸对硝基苯胺（BAPNA）的酰胺酶特异性活性高 4 倍。用 TAME 作为底物时，胰蛋白酶的最适 pH 和温度分别为 pH8.5 和 50°C。该酶在 pH7.0-9.0 范围内极为稳定，在孵育 1 小时后高达 40°C 仍高度稳定。该纯化酶被大豆胰蛋白酶抑制剂（SBTI）和 N-对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）强烈抑制，这两种抑制剂都是胰蛋白酶的特异性抑制剂。此外，平滑无须鳕胰蛋白酶在高 NaCl 浓度下表现出更高的蛋白水解活性，表明其在高盐含量下具有蛋白质水解的潜力。纯化胰蛋白酶的 N 端前 12 个氨基酸的序列为 IVGGYECKPHSQ。该序列与来自海洋脊椎动物和无脊椎动物的胰蛋白酶具有高度同源性。当 BAPNA 作为底物时，纯化胰蛋白酶的米氏常数（K(m)）和催化常数（K(cat)）分别为 0.387 +/- 0.02 mM 和 2.62 +/- 0.11 s(-1)。对于 TAME 的水解，K(m)和 K(cat)分别为 0.156 +/- 0.01 mM 和 59.15 +/- 2.2 s(-1)。
