诺卡氏菌属GS-17耐热茁霉多糖酶的提纯和性质

PURIFICATION AND PROPERTIES OF THERMOSTABLE PULLULANASE FROM NOCARDIA SP. GS-17

诺卡氏菌属GS-17(Nocardia sp.GS-17)的耐热茁霉多糖酶(Pullulanase EC.3.2.1.41)的粗酶液经中空纤维柱超滤浓缩、羟基磷灰石柱层析和Pullulan-Sepharose 6B亲和层析,得到凝胶电泳均一的纯酶,比活提高264倍.酶作用最适温度为55℃,最适PH6.2,分子量140000,等电点pI为6.0.该酶水解茁霉多糖、支链淀粉和可溶性淀粉,但不水解糖原.酶在50℃作用于茁霉多糖的米氏常数K_m为0.90mg/ml,最大反应速度V_(max)为57μmol·min^(-1)·mg^(-1).Zn^(2+)、Fe^(3+)、Hg^(2+)、Cu^(2+)、Pb^(2+)和环状糊精对酶有抑制作用,Ca^(2+)对酶有激活作用.经蛋白质侧链化学修饰研究表明,色氨酸残基位于酶的活性位区.该酶是由1129个氨基酸残基组成的单肽链,酶的N末端序列经测定为:Ala-Gly-His-Gly-Pro-Asp-Val-Gln-Asp-Gly-

A thermostable pullulanase(EC. 3. 2. 1. 41)from a thermophile,Nocardia sp. GS-17,was purified to homogeneous by ultrafiltration, hydroxyapatite chromatography and affinity chromatography on pullulan-Sepharose 6B. The enzyme was purified 264-fold. The enzyme had a molecular weight of 140 000 by SDS-PAGE. The isoelectric point was pH6. 0, The enzyme hydrolizied α-1,6 glucosidic linkages of pullulan,amylopectin, soluble starch,but not glycogen. The Km and Vmal values for enzyme activity on pullulan at 50℃ were 0. 90mg/ml and 57/μmol · min-1 · mg-1, respectivily. The optimum temperature and pH for activity were 55℃ and 6. 2, respectivily. The enzyme was activated by Ca2+ and strongly inhibitied by Zn2+, Fe3+ , Hg2+ , Cu2+, Pb2+ and γ-cyclodextrin. Modification of the enzyme with 10μmol/L NBS led to loss of the activity and substrate could protect the enzyme from NBS inactivation. These suggested that tryptophan residue may be located at the binding site of the enzyme. The amino acid composition and N-terminal sequence of the enzyme was determined. The N-terminal sequence was Ala-Gly-His-Gly-Pro-Asp-Val-Gln-Asp-Gly-.