Katsos N E, Labrou N E, Clonis Y D
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece.
J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Aug 5;807(2):277-85. doi: 10.1016/j.jchromb.2004.04.032.
Glutamate oxidase (GOX, EC 1.4.3.11) from Streptomyces catalyses the oxidation of L-glutamate to alpha-ketoglutarate. Its kinetic constants for L-glutamate were measured equal to 2 mM for Km and 85.8 s(-1) for kcat. BLAST search and amino acid sequence alignments revealed low homology to other L-amino acid oxidases (18-38%). Threading methodology, homology modeling and CASTp analysis resulted in certain conclusions concerning the structure of catalytic alpha-subunit and led to the prediction of a binding pocket that provides favorable conditions of accommodating negatively charged aromatic ligands, such as sulphonated triazine dyes. Eleven commercial textile dyes and four biomimetic dyes or minodyes, bearing a ketocarboxylated-structure as their terminal biomimetic moiety, immobilized on cross-linked agarose gel. The resulted mini-library of affinity adsorbents was screened for binding and eluting L-glutamate oxidase activity. All but Cibacron Blue 3GA (CB3GA) affinity adsorbents were able to bind GOX at pH 5.6. One immobilized minodye-ligand, bearing as its terminal biomimetic moiety p-aminobenzyloxanylic acid (BM1), displayed the higher affinity for GOX. Kinetic inhibition studies showed that BM1 inhibits GOX in a non-competitive manner with a Ki of 10.5 microM, indicating that the dye-enzyme interaction does not involve the substrate-binding site. Adsorption equilibrium data, obtained from a batch system with BM1 adsorbent, corresponded well to the Freundlich isotherm with a rate constant k of 2.7 mg(1/2)ml(1/2)/g and Freundlich isotherm exponent n of 1. The interaction of GOX with the BM1 adsorbent was further studied with regards to adsorption and elution conditions. The results obtained were exploited in the development of a facile purification protocol for GOX, which led to 335-fold purification in a single step with high enzyme recovery (95%). The present purification procedure is the most efficient reported so far for L-glutamate oxidase.
来自链霉菌的谷氨酸氧化酶（GOX，EC 1.4.3.11）催化L-谷氨酸氧化为α-酮戊二酸。其对L-谷氨酸的动力学常数经测定，米氏常数（Km）为2 mM，催化常数（kcat）为85.8 s(-1)。通过BLAST搜索和氨基酸序列比对发现，它与其他L-氨基酸氧化酶的同源性较低（18 - 38%）。穿线法、同源建模和CASTp分析得出了关于催化α亚基结构的某些结论，并预测出一个结合口袋，该口袋为容纳带负电荷的芳香族配体（如磺化三嗪染料）提供了有利条件。11种商业纺织染料和4种仿生染料或微型染料，其末端仿生部分带有酮羧化结构，被固定在交联琼脂糖凝胶上。对所得的亲和吸附剂微型文库进行筛选，以检测其结合和洗脱L-谷氨酸氧化酶活性的能力。除了汽巴克隆蓝3GA（CB3GA）亲和吸附剂外，所有亲和吸附剂在pH值为5.6时都能结合GOX。一种固定化的微型染料配体，其末端仿生部分为对氨基苄基氧杂环酸（BM1），对GOX表现出更高的亲和力。动力学抑制研究表明，BM1以非竞争性方式抑制GOX，抑制常数（Ki）为10.5 microM，这表明染料与酶的相互作用不涉及底物结合位点。从使用BM1吸附剂的分批系统获得的吸附平衡数据与弗罗因德利希等温线拟合良好，速率常数k为2.7 mg(1/2)ml(1/2)/g，弗罗因德利希等温线指数n为1。针对吸附和洗脱条件，进一步研究了GOX与BM1吸附剂的相互作用。所得结果被用于开发一种简便的GOX纯化方案，该方案在一步操作中实现了335倍的纯化，酶回收率高（95%）。目前的纯化方法是迄今为止报道的对L-谷氨酸氧化酶最有效的方法。
Zotero 插件