Self-powered enzyme-based biosensors.
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Biosensing Techniques↗
Biomedical subjects
A F Bückmann
Publications and source records attributed to A F Bückmann.
Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Reprinted from Biotechnology and Bioengineering, Vol. XXIII, No. 12, Pages 2789-2802 (1981).
Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10(4) [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L-leucine dehydrogenase (LEUDH) was used as catalyst for the reductive amination of alpha-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L-leucine. Formate dehydrogenase (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L-leucine. The kinetic constants V(max) and k(m) of the enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L-leucine was produced continuously out of alpha-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).
Bioreactors↗
An integrated NAD+-dependent enzyme-functionalized field-effect transistor (ENFET) system: development of a lactate biosensor.
An integrated NAD+-dependent enzyme field-effect transistor (ENFET) device for the biosensing of lactate is described. The aminosiloxane-functionalized gate interface is modified with pyrroloquinoline quinone (PQQ) that acts as a catalyst for the oxidation of NADH. Synthetic amino-derivative of NAD+ is covalently linked to the PQQ monolayer. An affinity complex formed between the NAD+/PQQ-assembly and the NAD+-cofactor-dependent lactate dehydrogenase (LDH) is crosslinked and yields an integrated biosensor ENFET-device for the analysis of lactate. Biocatalyzed oxidation of lactate generates NADH that is oxidized by PQQ in the presence of Ca2+-ions. The reduced catalyst, PQQH2, is oxidized by O2 in a process that constantly regenerates PQQ at the gate interface. The biocatalyzed formation of NADH and the O2-stimulated regeneration of PQQ yield a steady-state pH gradient between the gate interface and the bulk solution. The changes in the pH of the solution near the gate interface and, consequently, the gate potential are controlled by the substrate (lactate) concentration in the solution. The device reveals the detection limit of 1 x 10(-4) M for lactate and the sensitivity of 24+/-2 mV dec(-1). The response time of the device is as low as 15 s.
Biosensing Techniques↗
Synthesis of N6-(2-aminoethyl)-FAD, N6-(6-carboxyhexyl)-FAD, and related compounds.
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Flavin-Adenine Dinucleotide↗
Covalent attachment of FAD derivatives to a fusion protein consisting of 6-hydroxy-D-nicotine oxidase and a mitochondrial presequence. Folding, enzyme activity, and import of the modified protein into yeast mitochondria.
Autoflavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO) was successfully employed to modify the protein covalently with FAD derivatives. The model compounds N6-(2-aminoethyl)-FAD and N6-(6-carboxyhexyl)-FAD were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9 (Su9) attached to 6-HDNO. When translated in the rabbit reticulocyte lysate, Su9-6-HDNO was in the trypsin-sensitive apoenzyme form; when translated in the presence of flavins it adopted a trypsin-resistant conformation characteristic of the 6-HDNO holoenzyme. With flavin derivatives, Su9-6-HDNO exhibited approximately 50% of the 6-HDNO activity observed with FAD. The covalently modified Su9-6-HDNO was imported into Saccharomyces cerevisiae mitochondria with an efficiency equal to that of the apoenzyme. Apparently the increase in size and charge of the FAD moiety did not hamper translocation across the mitochondrial membranes. Yeast mutant ssc1-2 mitochondria deficient in mtHsp70 unfoldase activity imported the flavinylated Su9-6-HDNO protein. In mutant ssc1-3 mitochondria deficient in both mtHsp70 unfoldase and translocase activity Su9-6-HDNO was trapped as translocation intermediate; the Su9 presequence was passed to the matrix where it was proteolytically cleaved by the mitochondrial processing peptidase; (MPP); the translocation-arrested 6-HDNO moiety adopted a trypsin-sensitive conformation. Our results indicate that unfolding of the FAD-stabilized flavin-binding domain of 6-HDNO in passage through the mitochondrial general insertion pore does not require the activity of mtHsp70.
Animals↗
Synthesis, characterization and preliminary crystallographic data of N6-(6-carbamoylhexyl)-FAD-D-amino-acid oxidase from pig kidney, a semi-synthetic oxidase.
The FAD analogue, N6-(6-carboxyhexyl)-FAD, carrying a hexanoic acid residue at the N6 position of the adenine moiety was synthesized. A new semi-synthetic oxidase, N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase, was prepared by reacting the succinimido ester of N6-(6-carboxyhexyl)-FAD with apo-D-amino-acid oxidase from pig kidney in the presence of benzoate. Reaction conditions and methods have been developed for preparing pure semi-synthetic and fully active N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase that contains 1 covalently bound FAD analogue/subunit, as verified by redialysis, ultraviolet spectrophotometry, electrospray ionization (ESI)-MS and peptide mapping. Presumably, the N6-(6-carbamoylhexyl)-FAD moiety of this semi-synthetic D-amino-acid oxidase (DAAO), selectively bound to Lys163, has a structurally similar position to that of the non-covalently bound FAD of the native holoenzyme, since both DAAO forms show very similar kinetic properties (semi-synthetic DAAO, Vmax(app) = 17.7 mumol min-1 mg-1; KM(app) = 4.5 mM; native holo-DAAO, Vmax = 12.2 mumol min-1 mg-1; KM = 1.8 mM). Compared with the native holo-D-amino acid oxidase. this new semi-synthetic N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase is a considerably more stable enzyme that shows meso-thermostability and withstands inactivation on dilution. Probably, the lack of dissociation of FAD and, consequently, the absence of the instable apoenzyme are responsible for these phenomena. Preliminary investigations resulted in finding convenient and reproducible crystallization conditions for N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase. The single crystals, obtained by the sitting-drop method using ammonium sulfate as precipitant, belong to the tetragonal space group I422 with cell dimensions a = 16.3 nm, c = 13.6 nm. The crystals diffract to 0.3-nm resolution, with two molecules being present in the asymmetric unit, demonstrating the two-subunit quarternary structure of this semi-synthetic D-amino-acid oxidase.
Animals↗
Improving enzyme-electrode contacts by redox modification of cofactors.
Efficient electron transfer of redox proteins to and from their environment is essential for the use of such proteins in biotechnological applications such as amperometric biosensors and photosynthetic biocatalysts. But most redox enzymes lack pathways that can transport an electron from their embedded redox site to an electrode or a diffusing photoexcited species. Electrical communication between redox proteins and electrode surfaces has been improved by aligning proteins on chemically modified electrodes, by attaching electron-transporting groups and by immobilizing proteins in polymer matrices tethered by redox groups. Generally these methods involve contacting the enzymes at random with electron relay units. Here we report an approach that allows site-specific positioning of electron-mediating units in redox proteins. We strip glucose oxidase of its flavin adenine dinucleotide (FAD) cofactors, modify the latter with redox-active ferrocene-containing groups, and then reconstitute the apoprotein with these modified cofactors. In this way, electrical contact between an electrode and the resulting enzyme in solution is greatly enhanced in a controlled and reproducible way.
Apoproteins↗
Application of a flow injection fibre optic biosensor for the analysis of different amino acids.
The analysis of different amino acids is described using a fibre optic biosensor previously described in its principles. This biosensor works according to the measurement principle of flow injection analysis (FIA) and consists of a fluorosensor and a measurement cell operating under pressure. In the measurement cell, enzymes and molecular weight enlarged coenzyme PEG (MW 20000)-N6-(2-aminoethyl)-NAD(H) are confined behind a solid ultrafiltration membrane. Assays for the analysis of L-phenylalanine and L-alanine were developed. The analysis frequency is in the range of 1 to 2 samples per hour, and the sensor stability was found to be sensitive to the stability of each enzyme system used. This effect was studied in detail. The L-alanine assay was found to be especially reliable, sensitive, specific, and highly selective for the L-enantiomer.
Alanine↗
Structure/activity relationship of adenine-modified NAD derivatives with respect to porcine heart lactate dehydrogenase isozyme H4 simulated with molecular mechanics.
Using a significantly simplified modification procedure, four charged analogues of the coenzyme NAD, N(1)- and N6-(2-hydroxy-3-trimethylammoniumpropyl)-NAD, N(1)- and N6-(3-sulfopropyl)-NAD were prepared. The kinetic parameters of these derivatives and N(1)-(2-aminoethyl)-NAD, N6-(2-aminoethyl)-NAD and tricyclic 1,N6-ethanoadenine-NAD, all with alterations to the adenine moiety, were determined for porcine heart lactate dehydrogenase isoenzyme H4. The coenzyme activity depends on both position and charge of the introduced groups. Modification of the N6-position leads to a 25-250-fold increase of the kcat/Km value compared to the related N(1) derivative. The kcat/Km value for 1,N6-ethanoadenine-NAD is in the range between that of N(1)-(2-aminoethyl)-NAD and N6-(2-aminoethyl)-NAD. In the case of both N(1) and N6 functionalization, the Km values increase from (3-sulfopropyl)-NAD, with a negatively charged substituent at the adenine, over (2-amino-ethyl)-NAD to (2-hydroxy-3-trimethylammoniumpropyl)-NAD with an uncharged and positively charged substituent, respectively, at the adenine. All N6 derivatives are analogues like NAD with respect to Km and/or Vmax and kcat/Km. The conformation of NAD and its derivatives was calculated and their interaction in the active site of lactate dehydrogenase was simulated using the molecular mechanics program AMBER. The significant differences in activity in correlation to porcine heart lactate dehydrogenase isoenzyme H4 could be rationalized by modelling the three-dimensional structure of the NAD site.
Adenine↗
12 alpha-hydroxysteroid dehydrogenase from Clostridium group P, strain C 48-50. Production, purification and characterization.
NADP(H)-dependent 12 alpha-hydroxysteroid dehydrogenase (HSDH) from Clostridium group P, strain C 48-50, is still expressed at unusual high level (approximately 1% of total protein) under cultivation conditions where the usual expensive brain/heart infusion complex medium is replaced by inexpensive technical grade yeast autolysate. An inexpensive anaerobic bioprocess for the production of HSDH was developed provisionally up to 900-1 scale (9000 U/l, 7 g HSDH, specific activity 1.0 U/mg crude protein, 55 U/g wet cells). By a simple two-step affinity chromatography procedure, easily adaptable to a large-scale operation, using columns of small dimensions of Sephacryl-S-400-Procion-orange-P-2R (5 cm x 28 cm) and Sephacryl-S-400-Procion-red-HE-7B (2.6 cm x 14 cm) approximately 140 mg (1.8 x 10(4) U), HSDH was purified to apparent homogeneity and concentrated directly from a crude cell extract (overall yield 53%, specific activity 128 U/mg). As confirmed by fast native and SDS/PAGE, isoelectric focussing and electron microscopy, HSDH has a molecular mass of approximately 105 kDa and consists of four flattened tetrahedrically arranged identical subunits (26 kDa). The enzyme exhibits a rather low isoelectric point of 4.6, a pH optimum of 8.5-9.5 and a temperature optimum of approximately 55 C for the oxidation of cholic acid. Inhibition by SH reagents and pyridoxal 5'-phosphate has been observed. Chelating agents have no inhibitory effect. The presence of NADP increases considerably the thermostability (t 1/2 4-10 d, 25 C; 2-5 d, 37 C). Steady-state kinetic analysis for both reaction directions indicated that the reaction proceeds through an ordered bi bi mechanism with NADP(H) binding first to the free enzyme. Km, Vmax [forward (Vf) and reverse reactions (Vr)] and the dissociation constants Kd for the binary complexes with NADP and NADPH were as follows. NADP, Km = 35 microns, Kd = 35 microns; cholic acid, Km = 72 microns, deoxycholic acid, Km = 45 microns, Vf = 160 U mg; NAPDH, Kd = 16 microns; 12-oxochenodeoxylic acid, Km = 12 microns, 66 U/mg (conditions, 0.1 M potassium phosphate, pH 8.0, 25 degrees C). N6-functionalized NADP derivatives, e.g. N6-(2-aminoethyl)NADP (Km = 4.5 mM) are poorly accepted as coenzyme by HSDH.
Amino Acid Sequence↗
Coenzymatic properties of low molecular-weight and macromolecular N6-derivatives of NAD+ and NADP+ with dehydrogenases of interest for organic synthesis.
The catalytic activity, expressed as Km and Vmax values, of 16 enzymes of practical interest with the macromolecular coenzymes poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ and their low molecular weight precursors N6-(2-aminoethyl)-NAD+ and N6-(2-aminoethyl)-NADP+, was investigated. The enzymes examined are of direct interest for organic synthesis (i.e. alcohol dehydrogenase from yeast, horse liver, or Thermoanaerobium brockii, lactic dehydrogenase, and several hydroxysteroid dehydrogenases) or are used for the regeneration of NAD+, NADP+, NADH, or NADPH (i.e. glutamate dehydrogenase from liver or Proteus, formate dehydrogenase, glucose dehydrogenase, and malic enzyme). The cycling efficiency of poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ was examined with coupled-enzymes or coupled-substrates systems. Poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and, even more so, poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ were excellent coenzymes with several dehydrogenases. In addition, the coenzymatic properties of N6-(3-sulfonatopropyl)-NAD+, an NAD+ derivative carrying a strong anionic group, were compared with those of the newly synthesized N6-(2-hydroxy-3-trimethylammonium propyl)-NAD+, an NAD+ derivative carrying a strong cationic group. It was expected that the presence of the sulfonic or quaternary ammonium group would enhance the residence time of the coenzyme inside continuous-flow reactors if membranes with anionic or cationic groups, respectively, were used.
Indicators and Reagents↗
Synthesis and application of water-soluble macromolecular derivatives of the redox coenzymes NAD(H), NADP(H) and FAD.
During the past 15 years, the development of strategies to apply the catalytic potential of redox coenzyme-requiring enzymes has been a subject of intensive study; the main purpose of which has been to cut the cost of coenzyme to an economically acceptable level. One approach has been the utilization of isolated coenzyme-dependent enzyme systems with simultaneous enzymatic coenzyme regeneration (recycling). This has been used in conjugation with ultrafiltration reactor technology (enzyme membrane reactor), with coenzyme concentration being kept at a catalytic level. The concept implies confinement (immobilization) and practically 100% retention of both enzymes and coenzymes being dissolved in homogeneous solution within the reactor space that is closed off by an ultrafiltration membrane through which low-molecular-weight reactants (substrates and products) can freely pass. Since the problem of retaining nearly 100% native coenzymes of relatively low molecular weight by ultrafiltration membranes has not been satisfactorily solved, active macromolecular coenzyme derivatives are required. In this review, the syntheses, properties and merits of water-soluble macromolecular derivatives of NAD(H), NADP(H) and FAD are considered with respect to their biotechnological application.
Chemical Phenomena↗
Large-scale isolation of enzymes.
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Aldehyde Oxidoreductases↗
Novel applications of fluorescence sensors.
Typically, NAD(P)H-sensitive culture probes have been used to estimate biomass concentrations in suspended-cell cultivations, but these sensors have other uses as well. A number of applications, ranging from biosensors to immobilized-cell metabolic studies, are presented.
Alanine Transaminase↗