Characterization of a Yeastd-Amino Acid Oxidase Microbiosensor ford-Serine Detection in the Central Nervous System

Abstract

d-Serine is an endogenous ligand for N-methyl-d-aspartate (NMDA) receptors, and alterations in its concentration have been related to several brain disorders, especially schizophrenia. It is therefore an important target neuromodulator for the pharmaceutical industry. To monitor d-serine levels in vivo, we have developed a microbiosensor based on cylindrical platinum microelectrodes, covered with a membrane of poly-m-phenylenediamine (PPD) and a layer of immobilized d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). By detecting the hydrogen peroxide produced by enzymatic degradation of d-serine, this microbiosensor shows a detection limit of 16 nM and a mean response time of 2 s. Interferences by ascorbic acid, uric acid, l-cysteine, and by biogenic amines and their metabolites are rejected at more than 97% by the PPD layer. Although several d-amino acids are potential substrates for RgDAAO, d-serine was the only endogenous substrate present in sufficient concentration to be detected by our microbiosensor in the central nervous system. When implanted in the cortex of anesthetized rats, this microbiosensor detected the increase in concentration of d-serine resulting from its diffusion across the blood-brain barrier after an intraperitoneal injection. This new device will make it possible to investigate in vivo the variations in d-serine concentrations occurring under normal and pathological conditions and to assess the pharmacological potency of new drugs designed to impact d-serine metabolism.

Creation of biocatalysts with prescribed properties

“…These mutants are perfectly suitable for the development of biosensors for the selective determination of D serine in brain tissue since it is the high activity with respect to D alanine that is the major disadvantage of the biosensor derived from recombinant DAAO from R. gracilis. 52 In addition, the improvement of kinetic parameters in the oxidation of cephalosporin С is observed for one of the mutants.…”

Separation and sensitive detection of D‐amino acids in biological matrices

“…A new sensitive enzyme microelectrode was developed for D-ser recently using DAAO [28] (1) unknown (mislabeled by authors), (2) arginine, (3) hydrolysis products, (4) lysine, (5) phenylalanine, (6) leucine, isoleucine, and tyrosine, (7) GABA, (8) valine, (9) citrulline, (10) a-ABA, methionine, R-ABA, and b-alanine, (11) histidine, (12) taurine, (13) alanine and glutamine, (14) threonine, (15) glycine, (16) asparagine, (17) L-serine, (18) D-serine, (19) phosphoethanolamine, (20) internal standard, and (21) glutamate. (Adapted from ref.…”

“…The production of H 2 O 2 by this enzyme has been used to estimate D-ser levels in mammalian brain by several approaches which indirectly detect peroxide generation. Recently, Pernot et al developed a yeast D-amino acid oxidase microbiosensor (enzyme microelectrode) that is highly selective for D-ser compared with L-amino acids and offers a mean response time of 2 s and detection limit of 16 nM in vivo [28]. A schematic of the microelectrode as well as data representing its response time to D-ser is given in Fig.…”

Immobilization of Genetically-Modified d-Amino Acid Oxidase and Catalase on Carbon Nanotubes to Improve the Catalytic Efficiency

“…It has been proposed that evolved H 2 O 2 is eliminated during the production of 7ADCA [6]. DAAOs can also be used to catalyze the production of α-keto acids from essential D-amino acids [7,8], resolution of racemic mixtures of amino acids [9], and quantification of the D-amino acid content in biological fluids [10][11][12][13][14][15]. Cooperating with other enzymes, DAAOs promote the catalysis processes [16,17].…”