摘要
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.
关键词
Α-半乳糖苷酶
慈菇
分离纯化
性质
α-Galactosidase
Arrowhead (Sagittaria safittifolia)
Purification
Properties