Interaction of l-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography

Glutamate oxidase (GOX, EC 1.4.3.11) from Streptomyces catalyses the oxidation of l-glutamate to α-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 α-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 μM, 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 mg1/2 ml1/2/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.