Oshikawa K, Aoki K, Yoshino Y, Terada S
Department of Chemistry, Faculty of Science, Fukuoka University, Japan.
Biosci Biotechnol Biochem. 2000 Oct;64(10):2186-92. doi: 10.1271/bbb.64.2186.
A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60 degrees C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.
通过用水提取、硫酸铵沉淀以及在DEAE - Toyopearl 650M、丁基 - Toyopearl 650M和G - 3000 SW柱上连续进行色谱分离，从刀豆种子中纯化出一种肽酶。该酶的表观分子量为41,000。在pH 9和60℃时活性最高。与牛胰蛋白酶相比，该酶能更快速地水解Arg - X和Lys - X键处的合成底物，且不能切割蛋白质或酯类底物。该酶受到烷基胺和几种丝氨酸蛋白酶抑制剂如二异丙基氟磷酸酯、抑糜酶素、亮抑酶肽和苯甲脒的抑制。半胱氨酸蛋白酶抑制剂、金属蛋白酶抑制剂和蛋白质类胰蛋白酶抑制剂无效。烷基胺的抑制作用取决于烷基链的长度。从底物特异性和对化学物质的敏感性来看，该酶是一种具有类胰蛋白酶特异性的独特肽酶。
