Determination of Malic Acid in Real Samples by Using Enzyme Immobilized Reactors and Amperometric Detection

Manzoli, Alexandra; Tomita, Inês N.; Fertonani, Fernando Luís; Oliveira‐Neto, Graciliano de; Mascini, Marco; Yamanaka, Hideko

doi:10.1081/al-120039428

Cited by 7 publications

(8 citation statements)

References 14 publications

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“…Ascorbate oxidase from cucumber have been used to eliminate ascorbic acid interference from malate determination in analysis of citrus juice [19] and also to determine glutathione [20], organophosphorous pesticida ethyl paraoxon [21] based on enzymatic inhibition method.…”

Section: Introductionmentioning

confidence: 99%

Amperometric biosensor for ascorbic acid

et al. 2005

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A L-ascorbic acid biosensor based on ascorbate oxidase has been developed. The enzyme was extracted from the mesocarp of cucumber (Cucumis sativus) by using 0.05 mol L -1 phosphate buffer, pH 5.8 containing 0.5 mol L -1 NaCl. After the dialysis versus phosphate buffer 0.05 mol L -1 pH 5.8, the enzyme was immobilized onto nylon net through glutaraldehyde covalent bond. The membrane was coupled to an O 2 electrode and the yielding reaction monitored by oxygen depletion at -600 mV using flow injection analysis optimized to 0.1 mol L -1 phosphate buffer pH 5.8, as the carrier solution and flow-rate of 0.5 mL min -1 . The ascorbic acid calibration curve was linear from 1.2x10 -4 to 1.0x10 -3 mol L -1 . The evaluation of biosensor lifetime leads to 500 injections. Commercial pharmaceutical samples were analyzed with the proposed method and the results were compared with those obtained by high-performance liquid chromatography (HPLC).

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Section: Introductionmentioning

confidence: 99%

Amperometric biosensor for ascorbic acid

et al. 2005

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“…MDH enzymes with a preference for NAD + are classified as members of Enzyme Commission (EC) class 1.1.1.37, whereas the plant and algal enzymes with a preference for NADP + are members of EC 1.1.1.82. It is the NAD +preferring enzymes of EC 1.1.1.37 that have been used exclusively for malate biosensors (Giménez-Gómez et al, 2017, Blaedel and Engstrom, 1980, Mizutani et al, 1991, Yoshioka et al, 1992, Silber et al, 1994, Cho et al, 1995, Prodromidis et al, 1996, Gajovic et al, 1997, Katrlı ḱ et al, 1999, Miertuš et al, 1998, Maines et al, 2000, Manzoli et al, 2004, Mazzei et al, 2007, Arvinte et al, 2008, Arvinte et al, 2009, Gamella et al, 2010, Monošík et al, 2012a, Vargas et al, 2016, Mundaca-Uribe et al, 2017, Röhlen et al, 2017. The reaction (equation ( 1)) involves a hydride transfer from malate to the co-factor (Cunningham et al, 1997).…”

Section: Malate Dehydrogenasementioning

confidence: 99%

“…One study used bovine MDH (Manzoli et al, 2004) and the other three did not specify the species origins of their commercially-sourced enzymes. The ME-based sensors (Table 1.2) either used commercial preparations of the chicken enzyme (four sensors), or enzymes of unspecified origin (two sensors).…”

Section: Expanding the Diversity Of Malate Oxidoreductases Used In Biosensorsmentioning

confidence: 99%

“…Ref. Acetic acid, adipic acid, alanine, ascorbic acid, butyric acid, calcium, chloride, citric acid, glutamic acid, glycine, lactic acid, leucine, lysine, magnesium, oxalic acid, oxaloacetic acid, potassium, sodium, tartaric acid (Prodromidis et al, 1996) Ethanol, white and red wine matrices (Messia et al, 1996) Oxaloacetate, pyruvate (Gajovic et al, 1997) NADH, NADPH (Gajovic et al, 1998) Acetic acid, ascorbic acid, citric acid, L-lactate, succinic acid, sulfite, tartaric acid, white and red wine matrices (Katrlı ́k et al, 1999) Ascorbic acid (Maines et al, 2000) Ascorbic acid, citric acid, glutamic acid, tartaric acid (Arif et al, 2002) Red wine matrix (Esti et al, 2004) White wine matrix (Lupu et al, 2004) Ascorbic acid (Manzoli et al, 2004) Gallic acid, red wine matrix (Bucur et al, 2006) Gallic acid, red wine matrix (Gurban et al, 2006) Acetic acid, arabinose, ascorbic acid, citric acid, ethanol, fructose, galactose, gluconic acid, glucose, glycerol, D-lactic acid, L-lactic acid, tartaric acid (Gamella et al, 2010) Acetic acid, L-ascorbic acid, citric acid, ethanol, fructose, glucose, L-lactic acid, red wine matrix, tartaric acid (Monošík et al, 2012a) Acetic acid, L-arabinose, ascorbic acid, citric acid, ethanol, D-fructose, D-galactose, gluconic acid, D-glucose, glutamine, glycerol, D-lactic acid, L-lactic acid, tartaric acid (Vargas et al, 2016) Acetic acid, ascorbic acid, citric acid, ethanol, fructose, gluconic acid, glucose, glycerol, L-lactate, tartaric acid (Giménez-Gómez et al, 2017) Ascorbic acid, citric acid, lactic acid, oxalic acid, tartaric acid (Mundaca-Uribe et al, 2017)…”

Section: Compounds Tested For Interference Amentioning

confidence: 99%

“…The use of iodine for its removal has been used previously for a malate biosensor (Mundaca-Uribe et al, 2017), though it is incompatible with the sensor in this current study. The use of ascorbate oxidase, which similarly converts ascorbic acid to dehydroascorbic acid has been shown to be an effective means of pre-treatment two malic acid biosensors (Maines et al, 2000, Manzoli et al, 2004 and may be easier to integrate into this enzyme-centric architecture. At the time of writing (September 2021), ascorbate oxidase from Cucurbita sp.…”

Section: Identifying the Source Of Interferencementioning

confidence: 99%

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An enzyme-focused approach to create malic acid biosensors for winemaking

Matthews¹

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The tart-tasting malic acid is one of the key flavour determinants in wine. In winemaking, malic acid levels within grapes are carefully monitored prior to harvesting, as well as during secondary fermentations that aim to reduce its concentration. Commonly employed methods of measuring malic acid are expensive, consume large amounts of reagents, and require specialist laboratories and personnel to perform. Best exemplified by blood glucose monitors, enzyme-based amperometric biosensors are a solution to each of these problems, being easy to use, low-cost, and able to be integrated into miniature, portable devices. For measuring malate, these sensors use a malate oxidoreductase enzyme, which catalyses the oxidation of malate and concomitant reduction of a co-factor. When paired with an electrode, this electron transfer reaction can be transduced into a measurable electrical current. Approximately 40 enzyme-based, amperometric malate sensors have been described in the peer-reviewed literature since the first described in 1980. Much of the work into improving devices has focused on the use of specialised materials or chemical additives to enhance electron transfer to the electrode. However, in general, little attention has been given to the choice of malate oxidoreductase used, with most devices using one of three commercially available enzymes. I hypothesised that the selection of a bespoke enzyme can be used to obtain desirable performance characteristics and afford novel utility in a biosensor. As the proof of principle, my choice of enzyme aimed to solve an issue that is particularly prevalent in the malate biosensing literature: the linear ranges of devices are poorly suited for the needs of winemakers. As these ranges are often extremely narrow, a winemaker must perform a careful and accurate dilution series to enable measurements. A device with a wider linear range would be more convenient for the end user. Candidate malate oxidoreductases, characterised in the peer-reviewed literature, were selected based on their reported Michaelis-Menten parameters. I specifically focused on the Michaelis constant (KM), a parameter that governs the hyperbolic relationship between increase in substrate concentration and enzyme saturation, theorising that an enzyme with a large KM would yield a device with a wide linear range. Seven candidate enzymes were expressed, purified, and then characterised using spectrophotometric assays. The R181Q mutant of the malic enzyme from Ascaris suum was taken forward for the project. This enzyme has the highest KM for malate of all characterised malate oxidoreductases and a mechanism to modulate the Michaelis-Menten parameters, including KM, by adding different concentrations of ammonium to assays. An amperometric device was built using this enzyme in solution, on a gold screen-printed electrode. Six electron mediators were tested for their ability to increase the rate of electron transfer from the reduced co-factor, NADH, to the electrode surface. The aim was to produce the highest signal with the lowest operating potential, though it was ultimately found that many were incompatible with the sensor assay, being either insoluble, unstable, or the cause of electrode fouling. Conversely, 2,6-dichlorophenolindophenol proved compatible with the other assay components and gave relatively large signals at the relatively low potential of +150 mV. The device created reports on malic acid concentrations up to 200 mM, the maximum concentration seen in grapes, and has the widest linear range of any enzyme-based malate biosensor described to date. The addition of ammonium to assays was used to increase the sensitivity of the device. However, two other small molecules were also found that were capable of modulating the sensor response: Mn2+ and citrate. These three compounds, added in various combinations, were used to generate three different calibration curves. In this way, a novel type of biosensor was created with the previously undescribed utility of a linear range versus sensitivity trade-off: the sensor could be dynamically made more sensitive if required, making it better at resolving small differences in malic acid, though each increase in sensitivity came with the reduction in the linear range. The device holds value in being operationally flexible to accommodate the needs of winemakers seeking to measure both large and small differences in malic acid. The device was tested by applying a range of Pinot Noir and Pinot Gris grape juice taken from different stages of the winemaking process of grape and wine samples, with measurements being compared to two commercial malic acid testing kits. The sensor consistently produced current readings 5- to 6-fold higher than expected, based on the results of the commercial kits. This was shown to be due to grape juice and wine interfering with both the enzyme catalysed reaction and the mediator, resulting in non-specific current. Many of the compounds found within wine were tested for interfering effects, with ascorbic acid being identified as a potent interferent capable of reducing the mediator. Attempts to pre-treat real samples with chemical additives to remove ascorbic acid were unsuccessful, though an approach using an ascorbate oxidase remains to be tested. This work represents a successful proof of the concept that careful selection of a bespoke enzyme can yield tremendous benefits to an amperometric biosensor, both in terms of the sensor performance and the addition of previously unseen utility. The presence of comprehensive databases, coupled with the current convenience of gene synthesis, suggests that the field of malate-sensing has much to gain from the search for new and better enzymes to be incorporated into devices.

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Current Awareness in Phytochemical Analysis

2005

Phytochem. Anal.

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In order to keep subscribers up‐to‐date with the latest developments in their field, John Wiley &Sons are providing a current awareness service in each issue of the journal. The bibliography contains newly published material in the field of phytochemical analysis. Each bibliography is divided into 13 sections: 1 Books, Reviews &Symposia; 2 General; 3 Nucleic Acids; 4 Amino Acids, Proteins &Enzymes; 5 Carbohydrates; 6 Lipids; 7 Secondary Products; 8 Growth Regulators; 9 Industrially‐Important Products; 10 Toxins/Allergens; 11 Pigments; 12 Vitamins; 13 Others. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted.

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Determination of Malic Acid in Real Samples by Using Enzyme Immobilized Reactors and Amperometric Detection

Cited by 7 publications

References 14 publications

Amperometric biosensor for ascorbic acid

Amperometric biosensor for ascorbic acid

An enzyme-focused approach to create malic acid biosensors for winemaking

Current Awareness in Phytochemical Analysis

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