海枣曲霉β-D-岩藻糖苷酶的研究

STUDIES ON THE β-D-FUCOSIDASE FROM ASPERGILLUS PHOENICIS

虽然β-D-岩藻糖苷酶(EC3.2.1.38)已从多种动植物中分离纯化,但因它们的专一性均不高而难以确证。我们从海枣曲霉(Aspergillus phoenicis)培养物中提取,经过 PEG6000-磷酸缓冲液双水相分离,相继用 DEAE-sephadex A-50、羟基磷灰石、Sephadex G-100等柱层析分离,获得了凝胶电泳均一的β-D-岩藻糖苷酶,比活力提高500倍。酶反应的最适条件为40℃和 pH 6.0,在35℃以下和 pH5.5—6.5稳定。凝胶过滤法测分子量为50000—60000,用 SDS-PAGE 测出分子量为57000。酶的 K_m 值为2.4mmol/L,V_(max)为12.8μmol·min^(-1)·mg^(-1)。金属离子 Ag^+、Hg^(2+)对酶有强抑制作用,巯基乙醇和牛血清清蛋白以及甘油和多种糖能提高酶活力。化学修饰结果表明,-SH、—COOH 基团和组氨酸、色氨酸残基为酶活力所必需。该酶只能水解 pNP-β-D-岩藻糖苷,有严格的底物专一性,并能专一地受 D-岩藻糖和 D-岩藻糖酸-γ-内酯所抑制,为迄今为止最专一的β-D-岩藻糖苷酶,堪称该酶的典型代表。

Although β-D-fucosidase(β-fucosidefucohydrolase,EC 3.2.1.38)has been isolatedfrom various sources,the identity of this en-zyme is still not settled.We have purified aspecific β-D-fucosidase in electrophoreti-cally homogeneous form crude extracts ofAspergillus phoenicis by polyethyleneglycol6000-phosphate buffer aqueous two-phase se-paration,and successive chromatography onDEAE-Sephadex A-50,hydroxyapatite andSephadex G-100 columns.The molecular wei-ght of the enzyme was estimated to be 57000by SDS-polyacrylamide gel electrophoresisand 50000 to 60000 by gel filtration on Se-phadex G-100.The enzyme showed optimumcoside were 2.4mmol/L,and 1.28 μmol min^(-1)the pH range 5.5—6.5 and below 35℃.TheKm and the Vmax values for pNP-β-Ⅰ)-fu-coside were 2.4mmol/L,and 1.28 μmol·min^(-1).mg^(-1)respectively.The enzyme was stronglyinhibited by sulfhydryl group reagents,PCMB,NEM and iodoacetate.It was also in-hibited by EDC,DEP and NBS.Thus,-SH,-COOH groups,histidyl and tryptophyl re-sidues were essential for enzyme activity.Thepurified β-D-fucosidase showed high speci-ficity toward p-nitrophenyl β-D-fucoside.The enzyme was inhibited by D-fucose andD-fucono-γ-lactone,but not by D-galactose,D-galactono-γ-lactone,D-glucose or D-glu-cono-γ-lactone;the latter compounds are spe-cific inhibitors of β-D-galactosidase and β-D-glucosidase respectively.Thus,this enzy-me is the most strictly specific β-D-fucosidasewhen compared with those previously repor-ted.