University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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l-Malate

  • IUPAC Name: (2S)-2-hydroxybutanedioic acid
  • Molecular Formula: C4H4O5-2
  • CAS Registry Number: NA
  • InChI: InChI=1S/C4H6O5/c5-2(4(8)9)1-3(6)7/h2,5H,1H2,(H,6,7)(H,8,9)
  • InChI Key: BJEPYKJPYRNKOW-REOHCLBHSA-N

@ ChemSpider@ NIST@ PubChem

Citations 15

"Biosensing For Determination Of Food Components And Estimation Of Their Functions"
Nippon Shokuhin Kagaku Kogaku Kaishi 2001 Volume 48, Issue 2 Pages 157-163
Kiyoshi MATSUMOTO, Hiroyuki UKEDA

Abstract: A review on methodologies of food analysis by combination of using immobilized biocatalysts and flow injection analysis. Rapid evaluation of food functional properties, e.g., super oxide dismutase (SOD)-like activity and inhibition of a-glucohydrolase (AGH) and angiotensin I-converting enzyme (ACE) activities is also discussed. 22 references.
Wine Immobilized enzyme Reactor

"Flow Injection Determination Of Malate With Immobilized Malate Dehydrogenase"
Anal. Chim. Acta 1989 Volume 221, Issue 2 Pages 337-340
Ala'ddin M. Almuaibed and Alan Townshend

Abstract: Malate dehydrogenase was immobilized on controlled-pore glass and packed in a glass column (2.5 cm x 2.5 mm); the column was used in a flow injection system. The carrier stream (2 mL min-1) consisted of 0.1 M phosphate buffer (pH 11.5) - 3.6 mM NAD+ (1:1); detection was at 340 nm. The detection limit was 7 µM and calibration graphs were rectilinear for 0.9 mM malate. The coefficient of variation (n = 10) was 1.5% for 0.44 mM. The column was stable for 1 month without loss in activity. The sampling rate was 50 h-1. Malate dehydrogenase was immobilized on controlled-pore glass and packed in a glass column (2.5 cm x 2.5 mm); the column was used in a flow injection system. The carrier stream (2 mL min-1) consisted of 0.1 M phosphate buffer (pH 11.5) - 3.6 mM NAD+ (1:1); detection was at 340 nm. The detection limit was 7 µM and calibration graphs were rectilinear for 0.9 mM malate. The coefficient of variation (n = 10) was 1.5% for 0.44 mM. The column was stable for 1 month without loss in activity. The sampling rate was 50 h-1.
Immobilized enzyme Injection technique Calibration Buffer pH Controlled pore glass

"Determination Of L-malate In Wine By Flow Injection With Co-immobilized Malate Dehydrogenase-oxaloacetate Decarboxylase"
Talanta 1993 Volume 40, Issue 8 Pages 1163-1165
Nobutoshi Kiba, Mie Oguchi and Motohisa Furusawa

Abstract: Oxaloacetate decarboxylase (used as a trapping enzyme) and malate dehydrogenase were co-immobilized on to aminated poly(vinyl alcohol) beads (GS 520; 13 µm) packed into a column (5 cm x 4 mm), using glutaraldehyde (method described). White or red (decolorized using nylon beads) wine was diluted with water, then portions (50 µL) were injected into a carrier stream of glycine buffer of pH 10 containing 0.1 M MgCl2/8 mM NAD+ (0.6 ml/min) and carried to the enzyme reactor (30°C) where the NADH produced was monitored fluorimetrically at 465 nm (excitation at 340 nm). The conversion efficiency of the reactor was 44%, and the relative activities for L- and D-malate were 100 and 9, respectively. Other carboxylic acids did not interfere at concentration. 2 mM. The calibration graph was rectilinear from 0.4-300 µM-L-malate, and the RSD at 20 µM was 1%; the detection limit was 0.2 µM. Recoveries of spiked red wine samples were 97-102%. One reactor could be used to analyze >1400 samples.
Wine Red Fluorescence Interferences Immobilized enzyme Poly vinyl alcohol beads

"Chemiluminometric Flow Injection Method For Determination Of Free L-malate In Wine With Co-immobilized Malate Dehydrogenase/NADH Oxidase"
Talanta 1995 Volume 42, Issue 11 Pages 1751-1755
Nobutoshi Kiba*, Junko Inagaki and Motohisa Furusawa

Abstract: Aminated poly(vinyl alcohol) beads were packed into a stainless-steel column (5 cm x 4 mm i.d.) and activated with 2.5% glutaraldehyde in 0.1 M phosphate buffer of pH 7. Enzyme solution (5 mg malate dehydrogenase; 600 iu and 5 mg NADH oxidase; 250 iu, in 10 mL 0.1 M phosphate buffer of pH 7) was circulated through the column for 6 h at a flow rate of 0.2 ml/min. A schematic diagram of the flow injection method is presented. Wine was introduced into the system and the H2O2 produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear from 30 µM- to 25 mL-malate and the detection limit was 0.08 µM. RSD (n = 7) was 0.54%. Recoveries were 74-101%. Sample throughput was 30/h without carryover. The reactor was renewed every two weeks. A flow injection system with a co-immobilized malate dehydrogenase/reduced nicotineamide adenine dinucleotide (NADH) oxidase reactor and a chemiluminometer is described for the determination of free L-malate in wine. Malate dehydrogenase and NADH oxidase were co-immobilized on poly(vinyl alcohol) beads and packed into a stainless-steel column (5 cm times 4 mm i.d.). The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear from 3 x 10^-7 M to 2.5 x 10^-4 M (the linear correlation coefficient was 0.9998); the detection limit (signal-to-noise ratio, 3) was 8 x 10^-8 M. The sample throughput was 30 h-1 without carryover. The reactor was renewed every 2 weeks.
Wine Chemiluminescence Immobilized enzyme Poly vinyl alcohol beads

"A Bienzyme Electrode Probe For Malate"
Anal. Chem. 1996 Volume 68, Issue 2 Pages 360-365
M. C. Messia, D. Compagnone, M. Esti, and G. Palleschi

Abstract: A new amperometric malate enzyme electrode probe has been constructed using a hydrogen peroxide-based sensor coupled with malic and pyruvate oxidase enzymes. The first enzyme catalyzes the oxidation of malic acid, which in the presence-of NADP(+) yields pyruvate as product. The oxidation of pyruvate is catalyzed by pyruvate oxidase, which yields H2O2 as product in the presence of O-2 and phosphate as cosubstrates and thiamine pyrophosphate and Mg2+ as cofactors. The H2O2 is then detected by the electrochemical transducer, and the output current changes are correlated to the concentration of malic acid in solution. Analytical parameters such as pH, temperature, buffer, substrate and cofactor concentrations, and response time have been optimized, Probe stability and reproducibility have been evaluated. The malic enzyme was used first in solution and then coimmobilized with pyruvate oxidase, Coimmobilization of the oxidase and dehydrogenase enzymes has been performed both randomly and asymmetrically on different supports, Calibration curves for malate have been constructed with all the analytical parameters optimized. The detection limit for this newly designed probe was 5 x 10^-7 mol/L, with a broad linear range between 10^-6 and 5 x 10^-4 mol/L. Recovery studies of malate in a wine matrix have been carried out. Malic acid has been determined in grape musts during grape maturation, Results correlated well when compared with those from a spectrophotometric procedure.
Wine Amperometry Electrode Interferences Immobilized enzyme Method comparison

"Indirect Flow Injection Assays For Glucose-6-phosphate Dehydrogenase - Glucose-6-phosphate And Malate Dehydrogenase - L-malate Using Immobilized Bacterial Luciferase"
Anal. Lett. 1989 Volume 22, Issue 8 Pages 1861-1871
Nabi, A.;Worsfold, P.J.

Abstract: The assay involves the use of alkanal monooxygenase (FMN-linked) - oxidoreductase co-immobilized on CNBr-activated Sepharose 4B (0.1 g in a 6 cm x 2.5 mm coil). Reagent streams consisted of phosphate buffer solution (pH 7.5) containing (A) 1 µM-glucose-6-phosphate dehydrogenase (I), 0.1 mM NADP, 0.1 mM glucose 6-phosphate (II) and 0.1 mM dithiothreitol and (B) 1 µM-flavine mononucleotide, decaldehyde (10 ppm) and Triton X-100 (1 ppm). Bioluminescence was detected at 490 nm. For determination of II, carrier stream (A) contained II instead of I, carrier stream (B) was unchanged and the serial glass coils contained immobilized I and alkanal monooxygenase (FMN-linked) - oxidoreductase (0.1 g of each). Analogous methods were used to assay malate dehydrogenase (III) and determine L-malate (IV). Detection limits were 15 fmol of I, 10 nM-II, 30 fmol of III and 1 µM-IV; the coefficient of variation (n = 5) were 5%.
Bioluminescence Immobilized enzyme Buffer Triton X Glass Detection limit Sepharose beads Indirect Surfactant

"L-Malate Determination In Wines And Fruit Juices By Flow Injection Analysis. Adaptation Of A Coupled Dehydrogenase - Transferase System"
Anal. Lett. 1989 Volume 22, Issue 15 Pages 2897-2913
Chemnitius, G.C.;Schmid, R.D.

Abstract: Malate dehydrogenase and aspartate aminotransferase were co-immobilized on controlled-pore glass; the carrier stream (0.1 M K phosphate of pH 7.0) contained NAD+ to generate oxalacetate (I) and NADH from L-malate and the first enzyme, and L-glutamate to trap I with the second enzyme. The NADH was detected fluorimetrically. The rectilinear range was 5 to 100 µM-L-malate, but up to 50 mM L-malate could be determined directly by zone sampling, a flow injection configuration which is described. From 12 to 20 samples could be analyzed per hour. Results agreed with those obtained by an official method.
Wine Fruit Fluorescence Controlled pore glass Immobilized enzyme Method comparison Standard method Zone sampling

"Simultaneous Flow Injection Analysis For L-lactate And L-malate In Wine Based On The Use Of Enzyme Reactors"
Electroanalysis 1992 Volume 4, Issue 5 Pages 545-548
Shuichi Yoshioka, Hiroyuki Ukeda, Kiyoshi Matsumoto, Yutaka Osajima

Abstract: Malate dehydrogenase and diaphorase were co-immobilized on CNBr-activated Sepharose 4B; lactate oxidase was immobilized on Amino-Cellulofine. The product was packed into a glass tube (10 cm x 2 mm) to form a reactor for malate or lactate, respectively, which was incorporated into one line of a flow injection system operated at 20°C. The sample solution was diluted with 0.05 M pyrophosphate buffer of pH 9.0 and saturated with menadione, then pumped through the sample loop (150 µL) of the lactate line and mixed with 5 mM NAD+ in the same buffer before reaching the sample loop (150 µL) of the malate line. By means of a 16-way switching valve, sample solution was simultaneously injected into the two lines and transported by a stream (2.0 and 1.0 mL min-1 in the lactate and the malate line, respectively) of the pyrophosphate buffer saturated with menadione to the enzyme reactors and thence to an O electrode operated at -0.8 V for amperometric measurement. Peak currents were rectilinearly related to concentration. for 0.05 to 1.2 mM L-malate and 0.01 to 0.5 mM L-lactate. Sampling frequency was 15 h-1. The method was used for determining both analytes in wine; red wines were pre-treated with gelatin to remove polyphenols. Results agreed with those obtained by the conventional F-kit method. The simultaneous determination of L-malate and L-lactate, by enzyme-supported flow injection analysis was developed using 2 enzyme reactors in parallel and a single O2 electrode. NADH formed in the reaction of malate dehydrogenase (MDH) was regenerated to NAD+ with dissolved O2, using vitamin K3 and diaphorase (DI). L-Lactate was determined using the enzyme lactate oxidase (LOD). When sample solutions were simultaneously injected into the 2 reactors (the MDH-DI-reactor and the LOD-reactor) with a controlled residence time, a train of 2 peaks corresponding to L-lactate and L-malate was seen. The peak currents were linearly related to the L-malate and L-lactate concentration. in the range 0.05-1.2 mM and 0.01-0.5 mM, respectively. The present system was applied to the determination of L-lactate and L-malate in wine. The results showed good agreement with those obtained using a conventional method (F-kit method), suggesting that this system may be applicable to the monitoring of malolactic fermentation during wine prodn.
Wine Amperometry Electrode Reactor Immobilized enzyme Sepharose beads Merging zones Method comparison

"Flow Injection Analysis Using Enzyme-immobilized Latex Suspended In Carrier Solution"
Anal. Sci. 1988 Volume 4, Issue 6 Pages 653-654
T. KOJIMA, T. SUZUKI and F. MORISHITA

Abstract: Lactate dehydrogenase, malate dehydrogenase and horse-radish peroxidase were separately immobilized on Estapor PSI68 (Rhone Poulenc) latex paricles (0.3 µm). Samples, e.g., lactate, malate and H2O2, were injected into a carrier suspension of the immobilized-enzyme latex particles and the products were detected fluorimetrically. Preliminary results are discussed.
Fluorescence Immobilized enzyme Latex

"Measurement Of L-malate Using Immobilized Enzyme Reactors Comparison Of Results Obtained With Four Different Enzymatic Systems"
Biosci. Biotechnol. Biochem. 1996 Volume 60, Issue 5 Pages 847-851
MATSUMOTO Kiyoshi HIGUCHI Seiichi TSUKATANI Tadayuki

Abstract: For the measurement of malate by an enzyme sensor, we did a comparative study using malate dehydrogenase (MDH) alone, MDH and glutamate oxaloacetate transaminase (GOT) together, a malic enzyme (ME) that requires NADP as a cofactor, and MDH and NADH oxidase together, With respect to the response of each reactor to 0.5 mM L-malate, the systems using ME alone and MDH plus NADH oxidase gave high values, The ranges of measurements were 0.05-1.00 mM (MDH alone), 0.01-0.05 mM (MDH plus GOT), 0.01-0.50 mM (ME alone) and 0.02-1.00 mM (MDH plus NADH oxidase), In the system with MDH alone, however, reducing sugars in the sample interfered with measurements and it was impossible to use this system for practical analysis of fruit samples, By contrast, the systems using ME alone or MDH plus NADH oxidase were unaffected by the presence of reducing sugars and were suitable for analysis of samples, Thus, the MDH-NADH oxidase system is recommended for practical analyzes of samples.
Wine Fruit Amperometry Enzyme Interferences Immobilized enzyme

"Simultaneous Determination Of L-malate And Ethanol In Wine By A Sensor Based On Oxygen Consumption Including Parallel Configuration Of Enzyme Columns"
Bunseki Kagaku 1990 Volume 39, Issue 11 Pages 723-727
Ukeda, H.;Nakada, Y.;Matsumoto, K.;Osajima, Y.

Abstract: A flow injection analysis system (illustrated) was developed consisting of two parallel enzyme reactors with a single O electrode. L-Malate (I) was determined by injecting sample solution into a carrier stream of 0.05 M pyrophosphate buffer (pH 9.0) saturated with vitamin K3 (0.5 mL min-1). The stream was mixed with NAD solution and the mixture was passed through a malate dehydrogenase - diaphorase enzyme reactor. Ethanol (II) was determined by injecting sample solution into a stream of 0.05 M phosphate buffer (pH 8.0; 1.0 mL min-1) and the mixture was passed through an alcohol oxidase enzyme reactor. Sample solution was injected simultaneously into both reactors. Calibration graphs were rectilinear from 90 to 900 µM I and from 18 to 50 mM II. The method was applied in the simultaneous determination of I and II in wine. Results were compared with those by HPLC and the F-kit method.
Wine Electrode HPLC Sensor Enzyme Column Buffer pH Simultaneous analysis

"Simultaneous Flow Injection Determination Of Malate And Ethanol Based On Oxygen Consumption With Dehydrogenase And Oxidase Reactors"
GBF Monogr. Ser. 1991 Volume 14, Issue 1 Pages 203-208
Hiroyuki UKEDA, Yuji NAKADA, Kiyoshi MATSUMOTO and Yutaka OSAJIMA

Abstract: The simultaneous determination system of L-malate and ethanol was developed by using two enzyme reactors in parallel and a single oxygen electrode. NADH formed in the reaction of malate dehydrogenase was regenerated to NAD with dissolved oxygen using vitamin Ka and diaphorase. Ethanol was determined by use of alcohol oxidase. When a sample solution was simultaneously injected to two reactors, a train of two peaks corresponding to ethanol and L-malate appeared in an FIA-gram. The peak current was linearly related to ethanol and L-malate concentration in the range 18-50 mM and 0.09-0.9 mM, respectively. The present system was applicable to the determination of ethanol and L-malate in wine.

"A Bienzyme Electrode For L-malate Based On A Novel And General Design"
J. Biotechnol. 1998 Volume 61, Issue 2 Pages 129-133
Nenad Gajovic*, Axel Warsinke and Frieder W. Scheller

Abstract: The coimmobilization of a NAD(P)(+)-dependent dehydrogenase with salicylate hydroxylase (SHL, EC 1.14.13.1) in front of a Clark-electrode yields a flexible new design for dehydrogenase based biosensors. The feasibility of the approach has been tested with malic enzyme (MDH; EC 1.1.1.40) as the dehydrogenase, resulting in a novel L-malate sensor. It had substantial advantages over the biosensor approaches reported earlier: effective re-oxidation of NADPH by SHL yielded an extended linear range from 0.01 to 1.2 mmol L-1 L-malate and strongly reduced NADP(+)-requirement (< 0.025 mmol l-1), while the working stability was increased to more than 30 days. The results obtained from six real samples showed a close correlation with the standard enzymatic method. The presented scheme with SHL and the Clark-electrode can be employed together with any NAD(P)(+)-dependent dehydrogenase.
Electrode Electrode Apparatus Linear dynamic range

"Comparison Of Two Enzyme Sequences For A Novel L-malate Biosensor"
J. Chem. Technol. Biotechnol. 1997 Volume 68, Issue 1 Pages 31-36
Nenad Gajovic*, Axel Warsinke, Frieder W. Scheller

Abstract: Two novel amperometric biosensors for the determination of L-malic acid in food samples have been compared. Both sensors make use of a Clark-type O-2-electrode but differ in the enzymes used. The first sensor is composed of malate dehydrogenase (decarboxylating), also known as 'malic enzyme' (MDH(dec.), EC 1.1.1.40) and pyruvate oxidase (POP, EC 1.2.3.3). It covers a linear detection range from 1 µmol L-1 to 0.9 mmol L-1 L-malate, with a response time of 1.5 min (t(90)) and a relative standard deviation of 3.5%. Measurements with real samples offered a good correlation with the standard enzymatic assay (difference±7%) Stored at room temperature, the response of the sensor is constant for 8 days. The second biosensor is based on the three enzyme sequence malate dehydrogenase (MDH, EC 1.1.1.37), oxaloacetate decarboxylase (OAC, EC 4.1.1.3) and pyruvate oxidase (POP, EC 1.2.3.3). It has a non-linear calibration curve. Concentrations from 5 µmol L-1 to 1 mmol L-1 L-malate can be detected, within a response time of 1.5 min and with a relative standard deviation of 20%. The lower detection limit for L-malate is 2 µmol L-1. The response is constant for 10 days when the sensor is stored at room temperature. 11 References
Food Potentiometry Electrode Sensor Detection limit Immobilized enzyme Method comparison

"Potentiometric Biosensor For Detection Of L-malate And D-isocitrate Employing A Carbonate Selective Electrode And Enzyme Immobilization For Flow Injection Analysis"
Prev. Nutr. Food Sci. 1998 Volume 3, Issue 1 Pages 36-42
In-Sook Kwun, Meera Kim

Abstract: Ion-selective eleltrodes(ISEs) are simple electrodechemical devices for the direct measurement of ions in the samples. A novel potentiometric biosensor for the determination of L-Malate or D-isocitrate has been developed by using CO2-3 -ISE-FIA system was composed of a pump, an injector, a malic enzyme or isocitric dehydrogenase enzyme reactor, a CO2-3 -ISE, a pH/mV meter, and an integrater. The various factors, such as buffer capacity types of plstericizer and polymer, were optimized for the CO2-3 selectivity. In this novel CO2-3 --ISE-FIA system, the potential difference due to the amount of CO2-3 produced from each enzyme reaction was proportional to the amount of L-malate or D-isocitrate.
Food Potentiometry Sensor Electrode Immobilized enzyme