Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode

Abstract

An amperometric enzyme electrode was studied based on the wild-type protein trimethylamine dehydrogenase (TMADH), which catalyses the oxidative N-demethylation of trimethylamine to produce dimethylamine and formaldehyde. Ferrocene derivatives were investigated electrochemically, as free diffusing electron acceptors for recycling of the prosthetic groups of the immobilised enzyme. Ferricinium had the highest rates but, inhibited the enzyme, possibly as a result of a conformational change initiated at the Val-344 residue where it binds close to the 4Fe-4S cluster, interrupting the electron transfer between flavin mononucleotide (FMN) and 4Fe-4S by changing the redox potential of one or both of the prosthetic groups. (Dimethylamino)methylene ferrocene (DMAMFe) (k(s) = 0.93 x 10(5) M(-1) s(-1)) did not show inhibition and was used as a comparison for steady-state characterisation. The sensor response was studied over the pH range 6.0-1.0. Plots of kcat/KM revealed two ionisations with pKa values of 7.5 and 10. The pKa of 10 was attributed to the ionisation of the secondary amine in DMAMFe, whereas the pKa of 7.5 was thought to reflect the ionisations of the intramolecular electron pathway. A TMADH/DMAMFe amperometric enzyme electrode was successfully used for the determination of TMA in different fish samples (detection limit: 2 mg TMA-N per lOOg wet fish muscle). The obtained results compared well with a reference method based on picric acid.

Biogenic amines and fish freshness assessment using a multisensor system based on voltammetric electrodes. Comparison between CPE and screen-printed electrodes

“…Compared with enzyme-based sensors previously published [11][12][13][14][15][16][17][18], chemical modified sensors used in this study have a longer lifetime and do not require special conditions of temperature, humidity or light for conservation. In general, the stability of the enzyme-based sensors is lower than CPEs, and unless they are kept under specific conditions, their stability is guarantee only for a week [17].…”

“…Chemically modified electrodes (CME) containing surface-confined chemical functionalities exhibit some important advantages over conventional bare electrodes in terms of catalytic activity, selectivity and protection from fouling effects [10]. Determination of biogenic amines by using biosensors also represents an interesting alternative [11][12][13][14][15][16][17][18][19].…”

A molecular biology approach to protein coupling at a biosensor interface

“…Loechel et al [9] also introduced a cysteine mutation of the residue Y442 in the protein trimethylamine dehydrogenase (TMADH) to couple into the protein's electron transfer pathway to facilitate charge transfer between enzyme and an electrode.…”

“…Tamerler et al have suggested that hydroxyl and amine groups in methionine, lysine, serine, threonine, glutamine are important for improving adhesion in gold binding peptides. [58] This provided a method for modification of Au nanoparticles, that could then be attached to a SPR gold chip at a density of 1x10 9 particles/cm 2 to offer an antibody immobilization surface. With anti-Salmonella antibodies for detection of Salmonella typhimurium, the authors report a 10 fold improvement in detection, compared with classical SPR methodology.…”

Crystal Structure of Histamine Dehydrogenase from Nocardioides simplex

“…Although histamine oxidase from Arthrobacter globiformis (a topaquinone-containing copper amine oxidase) has been used as a histamine sensor, this enzyme was found to be more sensitive for dopamine and tyramine than for histamine (2). Trimethylamine dehydrogenase (TMADH) 5 from Methylophilus methylotrophus (3) and the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans (4) have been used in general amine-sensing electrodes without the complication of O 2 chemistry, but neither are as effective as histamine sensors.…”