溜曲霉β-N-乙酰氨基己糖苷酶的纯化与性质

PURIFICATION AND CHARACTERIZATION OF β-N-ACETYLHEXOSAMINIDASE FROM ASPERGILLUS TAMARII

溜曲霉(Aspergillus tamarii)S215除去菌体的培养液经硫酸铵及聚乙二醇分级沉淀、Se-phadex G-25脱盐、DEAE-Sephadex A-25离子交换柱层析及羟基磷灰石吸附柱层析等步骤,得到了凝胶电泳均一的样品,纯化了170倍左右,得率24.7%。在纯化过程中,β-GlcNAcase 和β-GalNAcase 活力始终不能分开,其活力比保持在2.5左右。用浓度梯度 PAGE 测定分子量为140000,用 SDS-PAGE 测定亚基分子量为72000。用 PAGIEF 测定等电点为4.2。β-GlcNAcase 反应最适 pH 为5.5—6.5,β-GalNAcase 为5.0—6.0。在 pH5.5—8.3范围内稳定。两者反应最适温度为60℃,在50℃保温8h,β-GlcNAcase 剩余活力为52.7%,GalNAcase 为44.9%;62℃10min,两酶活力仅剩1%。MB^(2+)和 Fe^(2+)件对酶活力微有激活作用,Hg^(2+)强烈抑制酶活力;Ag^+、CB^(2+)、Pb^(2+)、Cd^(2+)及 Zn^(2+)对酶活力有一定抑制作用。测定了氨基酸组成,天门冬氨酸和谷氨酸(包括酰胺)含有24.2%,三种碱性氨基酸占14.9%,而甲硫氨酸仅有0.6%左右。

A β-N-acetylhexosaminidase from my-celium-free culture filtrate of Asp tamariiS215 was purified to PAGE homogenous byammonium sulfate and polyethylene glycolfractionation precipitation followed by Sepha-dex G-50 desalt,DEAE-Sephadex A-25 chro-matography,hydroxyapatite chromatographyand DEAE-Sephadex A-25 rechromatographywith 170-fold purification and 24.7% reco-very.The ratio of the β-GlcNAcase andβ-GalNAcase was 2.5 and remained constantthroughout the purification.The Mr estimat-ed with concentration gradient PAGE was140000 and subunit Mr determined withSDS-PAGE was 72000,the number of subunitwere 2.The pl was 4.2 determined byPAGIEF.The optimum pH was 5.5—6.5 and 5.0—6.0 for β-GlcNAcase and β-GalNAcase re-spectively with stable pH range 5.5—8.3 forboth.The optimum temperature was 60℃for β-GlcNAcase and β-GalNAcase.Theresidual activity of β-GlcNAcase was 52.7%after treated at 50℃ for 8h and it was44.9% for β-GalNAcase.The residual ac-tivities of both were down to 1% after treatedat 62℃ for 10min.The activity was slightly activated byMn^(2+) or Fe^(2+),while strongly inhibited byHg^(2+) and slightly by Ag^+,Cu^(2+),Pb^(2+),Cd^(2+)or Zn^(2+).Analyses of amino acids compositionshowed that the β-HexNAcase contained about24.2% acidic amino acids and 14.9% basicamino acids and only 0.6% methionine.