Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Belgrade, Serbia.
Arch Insect Biochem Physiol. 2010 Aug;74(4):232-46. doi: 10.1002/arch.20371.
Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5-9.5) and temperature optimum of 45 degrees C with the K(M) ratio of 0.065 mM for benzoyl-arginine-p-nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (K(I) value of 0.012 mM, Ic(50) value of 0.204 mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5 kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38 kDa, suggesting that the enzyme is a monomer.
从黄粉虫幼虫前中肠提取的胰凝乳蛋白酶样酶（TLE）经阴离子交换层析和凝胶过滤层析纯化，并进行了特性分析。粗肠提取物经纯化后，TLE 的比活增加了 322 倍。该酶的最适 pH 值为 9.0（最适 pH 值范围为 8.5-9.5），最适温度为 45°C，对苯甲酰精氨酸对硝基苯胺（BApNA）的 K(M)值为 0.065 mM。在测试的多种抑制剂中，苯甲脒的抑制效果最强（K(I)值为 0.012 mM，Ic(50)值为 0.204 mM），而 SBTI、TLCK 和 PMSF 对 TLE 活性的抑制作用是部分的。几乎所有测试的二价阳离子都能增强酶的活性，其中 Co2+和 Mn2+使 TLE 活性提高了 2.5 倍。经 Superose 12 柱凝胶过滤纯化后的 TLE（TLE）具有 37.5 kDa 的分子量和超过 9.3 的等电点。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示出一条 38 kDa 的带，表明该酶是单体。
