Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination
Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination
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DOI：
10.1007/s00253-015-7198-5
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发表时间：
2016-05-01
影响因子：
5
通讯作者：
Sugimori, Daisuke
中科院分区：
文献类型：
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作者：
Hirano, Yoshitaka;Chonan, Keisuke;Sugimori, Daisuke
我们的筛选研究从总状合头藻中获得了一个铜胺氧化酶(SrAOX)，它对乙醇胺(EA)的亲和力和催化效率远远高于其他任何胺氧化酶(AOX)。将该酶从无细胞提取液中纯化至电泳均一，在pH 7.2-7.5，温度为45℃时，对EA的活性最高。这种酶起同源二聚体的作用。其推导的氨基酸序列与Delemar根霉菌AOX的同源性为55.3%，并含有铜-AOX、NYDY和HHQH两个共同序列，表明该基因是一种铜-AOX类型。结构同源性模拟表明，残基(ML113)-M-112、(141)FADTWG(146)M158和N318是唯一的，T144可能表征了SrAOX的底物专一性。重组酶(RSrAOX)是用大肠杆菌生产的。RSrAOX活性对EA(pH7.5和45A℃)的稳态动力学分析得出K(M)和k(CAT)值分别为0.848 A+/-0.009 mm和9.11A+/-0.13 S(-1)。标准曲线在0.1~2 mM EA范围内呈线性关系，用发色链霉菌磷脂酶D分别检测10 mU/m L~2.5 mg m L(-1)(15 mU M~3.6 mM)磷脂酰乙醇胺，可满足临床应用的要求。
Our screening study yielded a copper amine oxidase (SrAOX) from Syncephalastrum racemosum, which showed much higher affinity and catalytic efficiency toward ethanolamine (EA) than any other amine oxidase (AOX). Following purification of the enzyme to electrophoretic homogeneity from a cell-free extract, the maximum activity toward EA was detected at pH 7.2-7.5 and 45 A degrees C. The SrAOX complementary DNA (cDNA) was composed of a 2052-bp open reading frame encoding a 683-amino acid protein with a molecular mass of 77,162 Da. The enzyme functions as a homodimer. The deduced amino acid sequence of SrAOX showed 55.3 % identity to Rhizopus delemar AOX and contains two consensus sequences of Cu-AOX, NYDY, and HHQH, suggesting SrAOX is a type 1 Cu-AOX (i.e., a topaquinone enzyme). Structural homology modeling showed that residues (ML113)-M-112, (141)FADTWG(146) M158, and N318 are unique, and T144 possibly characterizes the substrate specificity of SrAOX. The recombinant enzyme (rSrAOX) was produced using Escherichia coli. Steady-state kinetic analysis of rSrAOX activity toward EA (pH 7.5 and 45 A degrees C) gave K (m) and k (cat) values of 0.848 A +/- 0.009 mM and 9.11 A +/- 0.13 s(-1), respectively. The standard curves were linear between 0.1 and 2 mM EA, and 10 mu g mL(-1)-2.5 mg mL(-1) (15 mu M-3.6 mM) phosphatidylethanolamine using Streptomyces chromofuscus phospholipase D, respectively, was sufficiently sensitive for clinical use.