Development of a high-performance liquid chromatographic system with enzyme reactors for the determination of N-acetyl branched-chain amino acids

Abstract

Immobilized enzymes were used as column reactors in a high-performance liquid chromatographic system for the specific detection of N-acetyl branched-chain amino acids (AcBCAs) such as N- acetyl- l-valine (AcVal), N- acetyl- l-leucine (AcLeu) and N- acetyl- l-isoleucine (AcIle). Aminoacylase and leucine dehydrogenase were immobilized onto poly(vinyl alcohol) beads. The AcBCAs were separated as three peaks on a Capcell C 1 SG120 column with 0.03 M phosphate buffer (pH 8.0). Aminoacylase was capable of hydrolysing the AcBCAs to amino acids, which react with β-nicotinamide adenine dinucleotide (NAD +) in the presence of leucine dehydrogenase. The reduced nicotinamide adenine dinucleotide (NADH) produced was monitored fluorimetrically. The calibration graphs were linear from 4 to 200μ M for AcVal and AcLeu, and from 5 to 300μ M for AcIle; detection limits for AcVal, AcLeu and AcIle were 2, 2 and 3μ M, respectively. The immobilized aminoacylase reactor should be renewed every 5 days owing to a poor stability of aminoacylase.