扭曲相思树种子芳基酰胺酶的纯化及部分特性分析

Purification and partial characterization of Enterolobium contortisiliquum seed arylamidase.

Costa R H, Stella R C, Alves K B

Departamento de Bioquímica, Escola Paulista de Medicina, São Paulo, Brasil.

Braz J Med Biol Res. 1991;24(4):337-44.

Abstract

Arylamidase activity was isolated from Enterolobium contortisiliquum seeds (2 U/g) using L-Leu-2-naphthylamide as substrate to monitor the purification. 2. The enzyme preparation was purified 733-fold by ammonium sulfate precipitation, and by ion exchange and gel filtration chromatography, in 6.6% yield. 3. SDS-Polyacrylamide gel electrophoresis after fast protein liquid chromatography on a Mono Q column, showed only one protein band with a molecular weight of 35 kDa. 4. The optimum pH for arylamidase activity was 6.5. Taking the hydrolysis rate of Lys-2-naphthylamide as one, the relative rates at which the other substrates were hydrolyzed were: Leu-2-naphthylamide, 30, Met-2-naphthylamide, 18, Arg-2-naphthylamide, 2, Ala-2-naphthylamide, 1.5, and L-Leu-p-nitroanilide, 26. 5. The arylamidase activity was inhibited 50% by 0.1 mM HgCl2, 0.1 mM MnCl2, 0.1 mM ZnCl2, 0.13 mM NiCl2, 0.2 mM o-phenanthroline and 1 microM sodium p-hydroxymercuribenzoate, and activated 35% by 5.0 microM EDTA. Iodoacetate (0.67 mM), dithioerythritol and 2-mercaptoethanol (3.3 mM), and chloride ions (0.2 M) had no effect on the enzyme activity.

摘要

以L-亮氨酸-2-萘酰胺为底物，从扭叶苏木种子中分离出芳基酰胺酶活性（2 U/g），以监测纯化过程。
通过硫酸铵沉淀、离子交换和凝胶过滤色谱法，该酶制剂纯化了733倍，产率为6.6%。
在Mono Q柱上进行快速蛋白质液相色谱后，SDS-聚丙烯酰胺凝胶电泳显示只有一条分子量为35 kDa的蛋白带。
芳基酰胺酶活性的最适pH为6.5。以Lys-2-萘酰胺的水解速率为1，其他底物的相对水解速率分别为：Leu-2-萘酰胺，30；Met-2-萘酰胺，18；Arg-2-萘酰胺，2；Ala-2-萘酰胺，1.5；L-亮氨酸对硝基苯胺，26。
0.1 mM HgCl2、0.1 mM MnCl2、0.1 mM ZnCl2、0.13 mM NiCl2、0.2 mM邻菲罗啉和1 microM对羟基汞苯甲酸钠可抑制芳基酰胺酶活性50%，5.0 microM EDTA可激活35%。碘乙酸盐（0.67 mM）、二硫苏糖醇和2-巯基乙醇（3.3 mM）以及氯离子（0.2 M）对酶活性无影响。