慈菇(Sagittaria safittifolia)α-半乳糖苷酶的纯化与性质

Purification and Properties of α-galactosidase from Arrowhead (Sagittaria sagittifolia)

以对硝基苯糖苷基为底物，测定了慈菇的１２种糖苷酶，其中α－甘露糖苷酶、α－和β－半乳糖苷酶活力较高；经硫酸铵分级沉淀，ＳｅｐｈａｄｅｘＧ－１５０分子筛层析，ＣｏｎＡＳｅｐｈａｒｏｓｅ４Ｂ亲和层析，ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换层析，从慈菇抽提液纯化了α－半乳糖苷酶。纯化酶的比活提高１０７２倍，活力回收１５．６％，在圆盘聚丙烯酰胺凝胶电泳和ＳＤＳ－ＰＡＧＥ上均显示１条蛋白质带，在α－半乳糖苷酶浓度为１５０ｍＵ／ｍｌ的溶液中测不到其他糖苷酶的活力。慈菇α－半乳糖苷酶的分子量用ＳｅｐｈａｄｅｘＧ－１００凝胶过滤柱测定或在ＳＤＳ－ＰＡＧＥ上测定均为６０ｋＤ，酶反应的最适ｐＨ在５．８附近，最适温度为６０℃。该酶分解对硝基苯基－α－半乳糖苷的Ｋ＿ｍ值为３．７×１０￣（－４）ｍｏｌ／Ｌ，Ｖ＿ｍ值为２．１×１０￣（－４）ｍｏｌ／Ｌ。银离子、汞离子显著抑制酶活力，Ｄ－半乳糖和密二糖均竞争性地抑制该酶水解对硝基苯基α－Ｄ－半乳糖苷的活力，根据Ｄｉｘｏｎ作图求得其Ｋ＿ｉ值分别为０．９２×１０￣（－３）ｍｏｌ／Ｌ和１．９８×１０￣（－３）ｍｏｌ／Ｌ。２－脱氧－Ｄ－半乳糖和Ｌ－岩藻糖为酶活力的非竞争性抑制剂。化学修饰?

The activity of twelve glycosidases has been determined in arrowhead extract using pnitrophenyl glycoside as a substrate. Among themα-mannosidase ,α- and β-galactosidase showed higher activity. α-Galactosidase has been purified 1072 fold with an activity recovery of 15.7% from arrowhead extract through ammonium sulfate fractionafion (20%-40% saturation) .Sephadex G-150 gel filtration, Con A-Sepharose affinity chromatography and DEAE-Sepharose CL-6B ion-exchange chromatography. The purified enzyme gave a single band on disc-and SDS-polyacrylamide gel electrophoreses. The molecular weight of agalactosidase was 60 kD by gelfiltrtion on Sephadex G-100 and by SDS-PAGE. The enzyme activity was optimum at PHS. 8 and 60℃ . The K_m and V_m values with p-nitrophenyl-α-galactoside was 3. 7 × 10￣(-4)mol/L and 2. 1×10￣(-4) mol/L respectively. The enzyme was markedly inhibited by Ag￣+ and PCMB. Galactose and melibiose both competitively inhibited the enzyme activity. The K_i value calculated by Dixon plots was 0. 92 mmol/L and 1. 98 mmo/L respectively. The enzyme was non-competitively inhibited by 2-deoxy-D-galactose and D-fucose. Modification of purified α-galactosidase by several chemical reagents indicated that Trp and His residue and carboxyl groups were involved in the active part of the enzyme.