南极大磷虾（Euphausia superba Dana）蛋白酶的纯化与特性分析

Purification and characterization of a proteinase from Euphausia superba Dana (Antarctic krill).

作者信息

Turkiewicz M, Galas E, Kalinowska H, Romanowska I, Zielińska M

出版信息

Acta Biochim Pol. 1986;33(2):85-99.

Abstract

The thiol-dependent serine proteinase (inhibited by DFP, PMSF, pCMB and iodoacetate) was isolated from the whole krill specimens and from the content of the krill digestive tract. The enzyme was purified to homogeneity using a seven-step procedure. Its specific activity with denatured haemoglobin as a substrate was about 6.0 unit/mg. The molecular weight of the enzyme, as determined by gel exclusion chromatography was 33 000 and by polyacrylamide gel electrophoresis with SDS 31 600 (12.5% gel) and 27 000 (7.5% gel). The enzyme is an acidic glycoprotein (pI below 2.9) containing about 5% of carbohydrate. The pH optimum of the enzyme with haemoglobin was 6.0 at the optimal temperature of 40 degrees C in 15-min reaction. The enzyme showed the esterase activity (hydrolysis of BAEE) and was inactive with carbobenzoxy- and benzoyl-dipeptides with the following C-terminal amino acids: Phe, Tyr, Lys, Gly and Leu.

摘要

硫醇依赖性丝氨酸蛋白酶（被二异丙基氟磷酸酯、苯甲基磺酰氟、对氯汞苯甲酸和碘乙酸抑制）是从整个磷虾标本以及磷虾消化道内容物中分离得到的。该酶通过七步纯化程序被纯化至同质。以变性血红蛋白为底物时，其比活性约为6.0单位/毫克。通过凝胶过滤色谱法测定，该酶的分子量为33000，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳法测定，在12.5%凝胶中为31600，在7.5%凝胶中为27000。该酶是一种酸性糖蛋白（等电点低于2.9），含约5%的碳水化合物。在15分钟反应中，以血红蛋白为底物时，该酶的最适pH为6.0，最适温度为40℃。该酶具有酯酶活性（水解苯甲酰-L-精氨酸乙酯），对以下C末端氨基酸的苄氧羰基和苯甲酰二肽无活性：苯丙氨酸、酪氨酸、赖氨酸、甘氨酸和亮氨酸。