PURIFICATION AND CHARACTERIZATION OF 6-AMINOHEXANOIC-ACID-OLIGOMER HYDROLASE OF FLAVOBACTERIUM SP-KI72
PURIFICATION AND CHARACTERIZATION OF 6-AMINOHEXANOIC-ACID-OLIGOMER HYDROLASE OF FLAVOBACTERIUM SP-KI72
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DOI：
10.1111/j.1432-1033.1981.tb05371.x
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
OKADA, H
中科院分区：
文献类型：
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作者：
KINOSHITA, S;TERADA, T;OKADA, H
黄杆菌属（Flavobacterium sp.）KI 72的6-氨基己酸低聚物水解酶经3次柱层析和2次制备型聚丙烯酰胺凝胶电泳纯化至均一。纯化的酶具有以下特性。Sephadex G-200分子筛层析测得其分子量为84，000。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳结果表明，该酶由2个42，000个同源亚基组成。最适pH为8 - 9，最适温度为40 ℃。C反应1小时。6-氨基己酸二聚体和三聚体的Km常数和周转数分别为5.9 mM和2.4 s-1，以及6.2 mM和2.0 s-1。该酶被0.37 mM DFP [二异丙基氟磷酸盐]和0.013 mM对氯汞苯甲酸盐抑制。该酶对6-氨基己酸低聚体从二聚体到六聚体和二十聚体都有活性，但对六聚体没有活性，且活性随低聚体聚合数的增加而降低。将低聚物水解，以便从氨基末端依次除去6-氨基己酸残基。该酶不水解其他线性酰胺、环状酰胺、二肽、三肽和酪蛋白。6-氨基己酸低聚物水解酶（6-Aminohexanoic-acid-oligohydrolase，6-AMH）属于线性酰胺酶（linearamidases，EC 3.5.1.-）。
6-Aminohexanoic-acid-oligomer hydrolase of Flavobacterium sp. KI72 was purified to homogeneity by column chromatography 3 times, and by preparative polyacrylamide gel electrophoresis twice. The purified enzyme had the following characteristics. The MW was estimated to be 84,000 by Sephadex G-200 molecular sieve chromatography. The enzyme consisted of 2 homologous subunits of 42,000, judged from sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The optimum pH for activity was between 8 and 9, the optimum temperature was 40.degree. C for a 1 h reaction. The Km constants and turnover numbers for the 6-aminohexanoic acid dimer and trimer were 5.9 mM and 2.4 s-1, and 6.2 mM and 2.0 s-1, respectively. The enzyme was inhibited by 0.37 mM DFP [diisopropylfluoro-phosphate] and by 0.013 mM p-chloromercuribenzoate. The enzyme was active on 6-aminohexanoic acid oligomers from dimer to hexamer and icosamer but not on hectamer, and the activity decreased with the increase of the polymerization number of the oligomer. The oligomers were hydrolyzed so as to remove the 6-aminohexanoic acid residue successively from the amino terminus. The enzyme could not hydrolyze other linear amides, cyclic amides, dipeptides, tripeptides or casein. 6-Aminohexanoic-acid-oligomer hydrolase was classified as a new member of the linear amidases (EC 3.5.1.-).