摘要
用制备凝胶电泳从焦曲霉(Aspergilluts ussus)菌株的培养液中分离纯化了右旋糖酐酶(EC 3.2.1.11)。纯酶经聚丙烯酰胺凝胶电泳分析为均一的,比活力为820 u/mg,提高20.5倍。酶作用最适 pH 为5.0—5.5,最适温度为50℃,在 pH 4.5—7.5和40℃以下稳定,在50℃保温30分钟酶活力损失55%。用 SDS-凝胶电泳测定酶的分子量为68000,用聚丙烯酰胺凝胶薄层等电聚焦电泳测得 pI 值为5.5。紫外吸收光谱在274nm 有最大吸收值,低谷在252 nm。纯酶只作用于α-1,6-葡萄糖苷键,水解右旋糖酐的产物为异麦芽糖、异麦芽三糖、葡萄糖和少量的较高聚合度的异麦芽寡糖,该酶为内切型的。作用于交联葡聚糖凝胶(Sephadex)时,随其交联度的增加酶作用能力减弱。纯酶作用于不同分子量的右旋糖酐(MW:2×10~4、1×10^(?)、4×10~4)的K_m值分别为0.30%、0.61%和0.70%。Hg^(2+)、Ag^+对酶完全抑制,Cu^(2+)、Mn^(2+)、Pb^(2+)、Be^(2+)、Fe^(3+)和 F^-对酶有一定的抑制作用,金属螯合剂 EDTA 对酶活性无影响,N-溴代琥珀酰亚胺(NBS)对酶强烈抑制。
An extracellular dextranase(EC.3.2.1.11)was purified approximately 20-foldfrom culture broth of A ustus by preparative gel electrophoresis.The purifiedenzyme was demonstrated by PAGE to be a homogeneous protein.Optimum condi-tions for the enzyme reaction were pH 5.0—5.5 and 50℃,respectively.The enzymewas stable over a pH range of 4.5 to 7.5 and a temperature lower than 40℃.TheHalf-live at 50℃ was 30 min.The molecular weight estimated by SDS-PAGE was68,000.The isoelectric point determined hy PAGIF was pH 5.5 Michaelis consta-nts(K_m)for three kinds of dextra (MW:2×10~6,1×10^(?),4×10~4)were 0.30%,0.61% and 0.70%,respectively.The ultraviolet absorbtion spectrum showeda maxi-mum at 274nm and a minimum at 252nm.The enzymatic products of dextranT-2000 analysed by TLC were mainly isomaitose,isomaltotriose,glucose and lesshigher oligomers,thus.the enzyme was proved to be an endo-type.The enzymeattacked only α-1,6 glycosidic linkage.Enzyme activity decreased with increase ofcross-linking degree of sephadex.The enzyme activity was inhibited completely byHg^(2+),Ag^+ and partially by Cu^(2+),Mn^(2+),Be^(2+),Fe^(3+),Pb^(2+),F^-.The enzyme wasstrongly inhibited by N-bromosuccinimide,and partially 2-hydroxy-5-nitrobenzylbromide,which suggested that tryptophan residue was essential for enzyme activity.
出处
《微生物学报》
CAS
CSCD
北大核心
1992年第3期218-226,共9页
Acta Microbiologica Sinica
关键词
焦曲霉
右旋糖酐酶
纯化
Aspergtllus ustus
Dextranase
Purification and properties
