Determination of L-Malate Using Immobilized Malate Dehydrogenase and Aspartate Aminotransferase in a Flow System and its Application to Analyze the L-Malate Content of Beverages

Abstract

The quantity of L-malate was determined using apparatus comprised of a reactor with immobilized malate dehydrogenase (MDH) and aspartate aminotransferase (AST) in a flow line. NADH formed by an enzymatic reaction was fluorometrically detected. The optimal concentration of NAD + in the carrier containing 0.1 M glutamate was determined. The maximum peak areas due to NADH were observed at pH 8.0 when the pH of the carrier consisting of Tris buffer ranged from 7.0 to 8.5. Various buffer types were also examined as carrier media at pH 8.0 and Tris buffer showed the maximum peak area. When the carrier composed of Tris buffer (0.1 M, pH 8.0) was used, the calibration curve for malate was linear in the range of 0.05-50 µM (r = 1.000). The detection limit (S/N = 3) was 0.03 µM. Relative standard deviations of the peak area at 1 µM and 10 µM were 1.5% (n = 7) and 0.36% (n = 7), respectively. Thirty samples of malate (10 µM) were analyzed for 1 hr. This method was applied to the analysis of malate in several beverages, and malate content determined by this method agreed with that determined by a commercially available test-kit method.

Determination of Citrate Using Immobilized Citrate Lyase and Malate Dehydrogenase in a Flow System and Its Application to Analyze Citrate Content of Beverages

“…This lower limit of detection is comparable with that observed in the study 9) where soluble CL and MDH were used, detecting the decrease of NADH fluorometrically. This value is larger than that (0.03 µM) observed in the analysis of D-malate 13) where NADH formed by immobilized enzymatic reactions was fluorometrically detected. The relative standard deviation of the peak area at 10 µM was 2.2% (n = 7).…”

“…This decrease of peak area would be due to the decreased activity of immobilized CL, since immobilized MDH had shown comparative stability, as reported in the previous study. 13) It was observed in the analysis of the samples 10 days after the first analysis. The enzyme reactor containing carrier was stored in the refrigerator (about 5 • C) for the period between the first and the second analysis.…”

“…Co-immobilization of CL and MDH to activated aminopropyl glass was first performed in the buffer of pH 6.0 according to previous studies; [11][12][13] however, CL was not immobilized at this pH. The reaction of enzymes with activated aminopropyl glass in the buffer of pH 7.0 resulted in the immobilization Values are peak areas relative to that obtained with phosphate buffer, and are the averages of triplicate determinations.…”

“…The pH of the buffer for immobilization to activated aminopropyl glass differs from that in the previous studies. [11][12][13] To aminopropyl glass beads (0.4 g) in 3.6 ml of 0.1 M phosphate buffer (pH 10.0) was added 0.4 ml of a 25% aqueous solution of glutaraldehyde. The mixture was bubbled with N 2 gas for 1 hr at room temperature.…”

“…We have utilized [11][12][13] immobilized enzymes in a flow system to determine the component of drinks. In these methods, enzymes can be used repeatedly.…”