Enzymatic bioreactors: An electrochemical perspective

Biocatalysts provide a number of advantages such as high selectivity, the ability to operate under mild reaction conditions and availability from renewable resources that are of interest in the development of bioreactors for applications in the pharmaceutical and other sectors. The use of oxidoreductases in biocatalytic reactors is primarily focused on the use of NAD(P)-dependent enzymes, with the recycling of the cofactor occurring via an additional enzymatic system. The use of electrochemically based systems has been limited. This review focuses on the development of electrochemically based biocatalytic reactors. The mechanisms of mediated and direct electron transfer together with methods of immobilising enzymes are briefly reviewed. The use of electrochemically based batch and flow reactors is reviewed in detail with a focus on recent developments in the use of high surface area electrodes, enzyme engineering and enzyme cascades. A future perspective on electrochemically based bioreactors is presented

Study of ALDH from thermus thermophilus–expression, purification and characterisation of the non-substrate specific, thermophilic enzyme displaying both dehydrogenase and esterase activity

Aldehyde dehydrogenases (ALDH), found in all kingdoms of life, form a superfamily of enzymes that primarily catalyse the oxidation of aldehydes to form carboxylic acid products, while utilising the cofactor NAD(P)+. Some superfamily members can also act as esterases using p-nitrophenyl esters as substrates. The ALDHTt from Thermus thermophilus was recombinantly expressed in E. coli and purified to obtain high yields (approximately 15–20 mg/L) and purity utilising an efficient heat treatment step coupled with IMAC and gel filtration chromatography. The use of the heat treatment step proved critical, in its absence decreased yield of 40% was observed. Characterisation of the thermophilic ALDHTt led to optimum enzymatic working conditions of 50 ◦C, and a pH of 8. ALDHTt possesses dual enzymatic activity, with the ability to act as a dehydrogenase and an esterase. ALDHTt possesses broad substrate specificity, displaying activity for a range of aldehydes, most notably hexanal and the synthetic dialdehyde, terephthalaldehyde. Interestingly, para-substituted benzaldehydes could be processed efficiently, but ortho-substitution resulted in no catalytic activity. Similarly, ALDHTt displayed activity for two different esterase substrates, p-nitrophenyl acetate and p-nitrophenyl butyrate, but with activities of 22.9% and 8.9%, respectively, compared to the activity towards hexanal

Use of self-assembled monolayers for the sequential and independent immobilisation of enzymes

The precise spatial control of the immobilisation of enzymes is necessary for the sequential localisation of an enzymatic cascade. In this contribution, the sequential immobilisation of alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FLDH) and formate dehydrogenase (FoDH) on self-assembled monolayer modified electrodes has been demonstrated. A range of thiols were screened and optimal enzymatic activity was obtained with thiols bearing -OH, a positively charged heterocyclic aromatic ring and -COOH for the immobilisation of ADH, FLDH and FoDH, respectively. Scanning of the applied potential was utilised to separately modify individual electrodes with the appropriate thiol and enzyme to deliver a three-enzyme system with catalytically active enzyme. Immobilised ADH, FLDH and FoDH retained 46, 32 and 76% of initial activity, respectively, on storage in aqueous solution at 4°C for a period of two weeks

Use of polymer coatings to enhance the response of redox-polymer-mediated electrodes

The successful use of biosensors requires that the sensor can operate over abroad enough linear range that encompasses the physiological concentration of the substrate of interest. A polymer coating layer functioning as a mass transport barrier is typically used to expand the linear range of biosensors, with however, the concomitant disadvantage of a reduction in the response. Effects of a poly(acrylic acid) (PAA) coating layer on the response of a glassy carbon electrode modified with an Os redox polymer and lactate oxidase (LOx) were evaluated. The coating layer resulted in an expanded linear range from 7 to 15 mM, doubled catalytic response towards the oxidation of 35 mM lactate and improved operational stability. Detailed voltammetry studies revealed that the coating layer can improve the amount of the redox polymer that is available as a mediator, leading to the increased catalytic response at high concentrations of substrate. Similar results were obtained with other polymer layers (polystyrene sulfonate (PSS), poly(diallyldimethyl-ammonium chloride) (PDADMAC) and poly(3,4-ethylenedioxythiophene) (PEDOT)) and with the enzymes, glucose oxidase and bilirubin oxidase demonstrating the general nature of the method

Significant Enhancement of Structural Stability of the Hyperhalophilic ADH from <i>Haloferax volcanii</i> via Entrapment on Metal Organic Framework Support

The use of an in situ immobilization procedure for the immobilization of hyperhalophilic alcohol dehydrogenase in a metal organic framework material is described. The easy and rapid in situ immobilization process enables retention of activity over a broad range of pH and temperature together with a decrease in the halophilicity of the enzyme. The catalytic activity of the immobilized enzyme was studied in nonaqueous solvent mixtures with the highest retention of activity in aqueous solutions of methanol and acetonitrile. The approach demonstrates that this immobilization method can be extended to hyperhalophilic enzymes with enhancements in activity and stability

Characterization of Nanoporous Gold Electrodes for Bioelectrochemical Applications

The high surface areas of nanostructured electrodes can provide for significantly enhanced surface loadings of electroactive materials. The fabrication and characterization of nanoporous gold (np-Au) substrates as electrodes for bioelectrochemical applications is described. Robust np-Au electrodes were prepared by sputtering a gold–silver alloy onto a glass support and subsequent dealloying of the silver component. Alloy layers were prepared with either a uniform or nonuniform distribution of silver and, post dealloying, showed clear differences in morphology on characterization with scanning electron microscopy. Redox reactions under kinetic control, in particular measurement of the charge required to strip a gold oxide layer, provided the most accurate measurements of the total electrochemically addressable electrode surface area, <i>A</i>_real. Values of <i>A</i>_real up to 28 times that of the geometric electrode surface area, <i>A</i>_geo, were obtained. For diffusion-controlled reactions, overlapping diffusion zones between adjacent nanopores established limiting semi-infinite linear diffusion fields where the maximum current density was dependent on <i>A</i>_geo. The importance of measuring the surface area available for the immobilization was determined using the redox protein, <i>cyt</i> c. The area accessible to modification by a biological macromolecule, <i>A</i>_macro, such as <i>cyt</i> c was reduced by up to 40% compared to <i>A</i>_real, demonstrating that the confines of some nanopores were inaccessible to large macromolecules due to steric hindrances. Preliminary studies on the preparation of np-Au electrodes modified with osmium redox polymer hydrogels and Myrothecium verrucaria bilirubin oxidase (<i>Mv</i>BOD) as a biocathode were performed; current densities of 500 μA cm^–2 were obtained in unstirred solutions

Charge pump design.

<p>Overall scheme of the charge pump design divided into different modules connected to electronics for sensing, sampling, and wireless radio transmission of data.</p

Bench-top device test.

<p>Photographs of the set-up for the bench-top device test, showing (A) the oxygen sensitive wireless self-powered biodevice, <i>i.e.</i> an EFC (electrochemical cell containing the anodes, 1, and cathodes, 2) connected to the wireless operational unit (white box, 3) and a control device (voltmeter, 4) and (B) a computer with the developed control software and receiver (CC2530 radio highlighted with the white arrow, 5), placed roughly 4 m from the device.</p

Wireless carbohydrate sensing.

<p>Recorded signal from the carbohydrate sensitive self-contained biodevice in buffers with varying lactose concentrations.</p

Wireless oxygen sensing.

<p>Recorded signal from the self-contained biodevice for oxygen monitoring in buffers with varying oxygen concentrations.</p