Development of an Amperometric Biosensor Platform for the Combined Determination of l-Malic, Fumaric, and l-Aspartic Acid

Three amperometric biosensors have been developed for the detection of l-malic acid, fumaric acid, and l-aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for l-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM−1 (l-malate biosensor) and 0.4 μA mM−1 (fumarate biosensor). The l-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM−1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.

[1]  Joerg M. Buescher,et al.  Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production , 2017, Metabolic engineering communications.

[2]  Michael J. Schöning,et al.  Characterisation of polymeric materials as passivation layer for calorimetric H2O2 gas sensors , 2012 .

[3]  G. Álvaro,et al.  Immobilized l-aspartate ammonia-lyase from Bacillus sp. YM55-1 as biocatalyst for highly concentrated l-aspartate synthesis , 2012, Bioprocess and Biosystems Engineering.

[4]  W. Buckel,et al.  Effect of an Oxygen-Tolerant Bifurcating Butyryl Coenzyme A Dehydrogenase/Electron-Transferring Flavoprotein Complex from Clostridium difficile on Butyrate Production in Escherichia coli , 2013, Journal of bacteriology.

[5]  E. Šturdı́k,et al.  Comparison of biosensors based on gold and nanocomposite electrodes for monitoring of malic acid in wine , 2012 .

[6]  Johnathan E. Holladay,et al.  Top Value Added Chemicals From Biomass. Volume 1 - Results of Screening for Potential Candidates From Sugars and Synthesis Gas , 2004 .

[7]  Isabelle Migneault,et al.  Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. , 2004, BioTechniques.

[8]  Giuseppe Palleschi,et al.  Amperometric aspartate electrode , 1991 .

[9]  J. Magnuson,et al.  Organic Acid Production by Filamentous Fungi , 2004 .

[10]  S. Campuzano,et al.  Automatic bionalyzer using an integrated amperometric biosensor for the determination of L-malic acid in wines. , 2016, Talanta.

[11]  J. Rhee,et al.  Flow injection system for on-line monitoring of fumaric acid in biological processes , 2003 .

[12]  V. Massey.  Studies on fumarase. II. The effects of inorganic anions on fumarase activity. , 1953, The Biochemical journal.

[13]  F. Scheller,et al.  A bienzyme electrode for L-malate based on a novel and general design. , 1998, Journal of biotechnology.

[14]  J. Švitel,et al.  Amperometric biosensors based on solid binding matrices applied in food quality monitoring. , 1998, Biosensors & bioelectronics.

[15]  Aspartate analysis in formulations using a new enzyme sensor. , 1995, Journal of pharmaceutical and biomedical analysis.

[16]  H. Beyenal,et al.  A Fumarate Microbiosensor for Use in Biofilms , 2017 .

[17]  Michael J. Schöning,et al.  Development of a multi‐parameter sensor chip for the simultaneous detection of organic compounds in biogas processes , 2015 .

[18]  A. M. Almuaibed.  Microscale on-line production and determination of malic acid using flow injection analysis, immobilized fumrase and malate dehydrogenase with chemiluminescence detection , 2001 .

[19]  Y. Chao,et al.  Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli. , 2000, Enzyme and microbial technology.

[20]  G. T. Tsao,et al.  Comparison of fumaric acid production by Rhizopus oryzae using different neutralizing agents , 2002, Bioprocess and biosystems engineering.

[21]  H. Fromm,et al.  The purification and properties of aspartase from Escherichia coli. , 1971, Archives of biochemistry and biophysics.

[22]  G. Guilbault,et al.  Enzyme electrode for the determination of aspartate , 1989 .

[23]  G. Palleschi,et al.  Flow monitoring of glutamate and aspartate in foods and pharmaceutical products with immobilized bienzyme electrochemical cells , 1992 .

[24]  Yang-Chun Yong,et al.  A whole-cell electrochemical biosensing system based on bacterial inward electron flow for fumarate quantification. , 2015, Biosensors & bioelectronics.

[25]  W. Chui,et al.  Prolonged retention of cross-linked trypsin in calcium alginate microspheres. , 1997, Journal of microencapsulation.

[26]  I. Chibata,et al.  Studies on the Fermentative Preparation of L-Aspartic Acid from Fumaric Acid , 1960 .

[27]  T. West.  Microbial Production of Malic Acid from Biofuel-Related Coproducts and Biomass , 2017 .

[28]  N. Kawabata,et al.  Continuous production of l-aspartic acid from ammonium fumarate using immobilized cells by capture on the surface of nonwoven cloth coated with a pyridinium-type polymer , 1995 .

[29]  R. Viola,et al.  Mutagenic investigation of conserved functional amino acids in Escherichia coli L-aspartase. , 1994, The Journal of biological chemistry.

[30]  A. Straathof,et al.  Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae. , 2011, Enzyme and microbial technology.

[31]  Greg M. Swain,et al.  Solid Electrode Materials: Pretreatment and Activation , 2007 .

[32]  G. Broun.  Chemically aggregated enzymes. , 1976, Methods in enzymology.

[33]  J. Stefan Rokem,et al.  Organic acids: old metabolites, new themes , 2006 .

[34]  G. Rechnitz,et al.  Regenerable Bacterial Membrane Electrode for L-Aspartate , 1977 .

[35]  Y. Asano,et al.  Alteration of substrate specificity of aspartase by directed evolution. , 2005, Biomolecular engineering.

[36]  T. Tajima,et al.  Efficient aspartic acid production by a psychrophile-based simple biocatalyst , 2015, Journal of Industrial Microbiology & Biotechnology.

[37]  K. Sakka,et al.  Characterization of a Dihydrolipoyl Dehydrogenase Having Diaphorase Activity of Clostridium kluyveri , 2008, Bioscience, biotechnology, and biochemistry.

[38]  P. Vadgama,et al.  Amperometric determination of L-malic acid in a flow injection analysis manifold using packed-bed enzyme reactors , 1996 .

[39]  G. Broun.  [20] Chemically aggregated enzymes , 1976 .