Biocatalytic Reactors Research Articles

Renewable Dehydrogenase-Based Interfaces for Bioelectronic Applications

Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. However, these interfaces contain labile components, including enzymes and cofactors, which have limited lifetimes and must be replaced periodically to allow long-term operation. Current methods to fabricate bioelectronic interfaces do not allow facile replacement of these components, thus limiting the useful lifetime of the interfaces. In this paper we describe a versatile new fabrication approach that binds the enzymes and cofactors using reversible ionic interactions. This approach allows the interface to be removed via a simple pH change and then replaced to fully regenerate the biocatalytic activity. The positively charged polyelectrolyte poly(ethylenimine) was used to ionically bond a dehydrogenase enzyme and its cofactor to a gold electrode that was functionalized with 3-mercaptopropionic acid and the electron mediator toluidine blue O. By reducing the pH, the surface-bound 3-mercaptopropionic acid was protonated, disrupting the ionic bonds and releasing the enzyme-modified polyelectrolyte. After neutralization, fresh enzyme and cofactor were bound, regenerating the bioelectronic interface. Cyclic voltammetry, chronoamperometry, constant potential amperometry, electrochemical impedance spectroscopy, and Fourier transform infrared spectroscopy analyses were used to characterize the bioelectronic interfaces. For the two enzymes tested (secondary alcohol dehydrogenase and sorbitol dehydrogenase) and their respective cofactors (beta-nicotinamide adenine dinucleotide phosphate and beta-nicotinamide adenine dinucleotide), the reconstituted interface exhibited a surface coverage, an electron-transfer coefficient, and a turnover rate similar to those of the original interface.

Mutation of Tyr-218 to Phe in Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2 degrees ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, beta-nicotinamide adenine dinucleotide phosphate (NADP+), is more expensive and less stable than beta-nicotinamide adenine dinucleotide (NAD+). PCR-based, site-directed mutagenesis was performed on 2 degrees ADH in an attempt to adjust the cofactor specificity toward NAD+ by mutating Tyr218 to Phe (Y218F 2 degrees ADH). This mutation increased the Km(app) for NADP+ 200-fold while decreasing the Km(app) for NAD+ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2 degrees ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD+. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD+ was electrically recycled 6.7 x 10(4) times, suggesting that the Y218F 2 degrees ADH-modified bioelectronic interface is stable.

Monoliths as Biocatalytic Reactors: Smart Gas—Liquid Contacting for Process Intensification

AbstractFor Abstract see ChemInform Abstract in Full Text.

Versatile bioelectronic interfaces based on heterotrifunctional linking molecules

Bioelectronic interfaces that allow dehydrogenase enzymes to communicate with electrodes have potential applications such as biosensors and biocatalytic reactors. A major challenge in creation of such bioelectronic interfaces is to orient the enzyme, its cofactor, and an electron mediator properly with respect to the electrode in order to achieve efficient, multistep electron transfer. This paper describes a versatile, new method that uses cysteine, an inexpensive, branched amino acid having sulfhydryl, amino, and carboxyl functional groups, to achieve such orientation. This approach provides greater flexibility in assembling complex bioelectronic interfaces than previously reported approaches that bind the enzyme, cofactor, and mediator in a linear chain. Cysteine was attached to a gold electrode through the sulfhydryl groups, to the electron mediator toluidine blue O (TBO) through the carboxyl group, and to the cofactor (e.g., NAD(P)+) through the amino group. Cyclic voltammetry, impedance spectroscopy, chronoamperometry and quartz crystal microbalance gravimetry were used to demonstrate the sequential assembly steps and the electrical activity of the resulting bioelectronic interface.

Monoliths as Biocatalytic Reactors: Smart Gas−Liquid Contacting for Process Intensification

In this paper, the advantages and disadvantages of using segmented flow in microchannels to intensify biocatalytic processes are discussed. We consider both suspended cell cultures and immobilized enzymes. Once bubbles are formed in microchannels, they can no longer coalesce and, hence, no energy is required to break up larger bubbles. As a result, the same gas−liquid mass-transfer behavior can be obtained at an order of magnitude lower power input. The gas−liquid−solid mass transfer, i.e., to a catalyst on the channel walls, improves with decreasing energy input, allowing the combined reduction of power input and reactor size. These findings are supported by a scaling analysis based on the pressure drop work of Kreutzer et al.1 and the mass-transfer work of Van Baten and Krishna.2 For the practical application of monoliths for liquid-phase cell cultures, the stability of a downflow monolith reactor with respect to clogging by biofilm was investigated experimentally. The results indicated that, as long as flushing out of the suspended cells was prevented, stable operation was possible.

Teetering

Before 1940, when the term “fluidization” was coined to describe a gas–solids contacting process, liquid-fluidized beds of granular solids had long been used for classification of minerals by size (sizing) or density (sorting), and were referred to in the ore dressing literature as “teeter columns” or as being in the “teeter condition”. Liquid fluidization or “teetering” has subsequently achieved several other applications, in recent years most notably as extended electrodes for electrowinning metals from dilute solutions, as self-cleaning heat exchangers and as bio-catalytic reactors. Since several important applications, in addition to classification per se, involve particle stratification by size and/or density, the main focus of this paper is on the current understanding of particle segregation and mixing in liquid-fluidized beds.

Scale-down studies on the hydrodynamics of two-liquid phase biocatalytic reactors.

The maintenance of constant interfacial area per unit volume is a key parameter for the successful scale-up of two-liquid phase bioconversion processes. To date, however, there is little published information on the hydrodynamics of such systems and a suitable basis for scale-up has yet to be defined and verified. Here we report power input and hydrodynamic data for a whole-cell bioconversion process using resting cells of Rhodococcus R312 to catalyse the hydration of a poorly water-soluble substrate 1,3-dicyanobenzene (1,3-DCB). Experiments were performed in geometrically similar 3-L and 75-L reactors, each fitted with a three-stage Rushton turbine impeller system. The two-phase system used comprised of 20% v/v toluene dispersed in 0.1 M aqueous phosphate buffer containing up to 10 g(ww) x L(-1) of resuspended biocatalyst and 20 g x L(-1) 1,3-DCB. The power input to the 3-L reactor was first determined using an air-bearing technique for both single-phase and two-phase mixing. In both cases, the power number attained a constant value of 11 at Re>10,000, while the measured power inputs were in the range 0.15-3.25 kW x m(-3). Drop size distributions and Sauter mean drop diameters (d(32)) were subsequently measured on-line in both reactors, using an in-situ light-backscattering technique, for scale-up on the basis of either constant power input per unit volume or constant tip speed. At both scales d(32) decreased with increasing agitation rate, while the drop size distributions obtained were log-normal. All the measured d(32) values were in the range of 30-50 microm, with the lowest values being obtained in systems with biocatalyst present. In all cases, constant power input per unit volume was found to be the most suitable basis for scale-up. This gave virtually identical d(32) values and drop size distributions at both scales. A number of correlations were also identified that would allow reasonable prediction of d(32) values for various agitation rates at each scale. While the results obtained are for a particular phase system, the scale-down methodology presented here would allow the rapid evaluation of other bioconversion processes in the 3-L reactor with a 25-fold reduction in scale. In this way, potential problems that might be encountered at the larger scale, such as the carryover of antifoam from the fermentation stage, could be quickly and efficiently identified.

Comparison of efficiencies of hollow tubes and hollow zones biocatalytic reactors for the enzymatic hydrolysis of benzylpenicillin to 6-aminopenicillanic acid

The comparative efficiencies of two special biocatalytic reactors designed to handle gel-like immobilized biocatalysts, the hollow tubes and the hollow zones reactors, have been assessed for the performance of the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid and phenyl acetic acid by an immobilized penicillin amidase preparation.

Comparison of efficiencies of hollow tubes and hollow zones biocatalytic reactors for the enzymatic hydrolysis of benzylpenicillin to 6-aminopenicillanic acid

Comparison of efficiencies of hollow tubes and hollow zones biocatalytic reactors for the enzymatic hydrolysis of benzylpenicillin to 6-aminopenicillanic acid

Horseradish-Root Tissue Tubular Reactor for Flow Injection Detection of Hydrogen Peroxide

Abstract The use of a horseradish-root tissue as a on-line reactor in connection with flow injection measurements of hydrogen peroxide is described. Such tissue reactor is very suitable for flow operation, as it couples the advantages of very high enzyme loading and good durability, with extreme simplicity and economy. The high conversion efficiency is evaluated under various experimental conditions (reactor dimensions, flow rate, etc.). Response characteristics, such as linearity, reproducibility or response time, are reported. The incorporation of glucose oxidase in the carrier solution allows convenient, low potential, monitoring of glucose (through the liberation and conversion of hydrogen peroxide). Analogous measurements of dopamine, based on an open-tubular apple reactor, are reported. Tissue biocatalytic reactors thus offer a very attractive alternative to enzyme reactors used for flow analysis.

Extractive biocatalysis: the use of two-liquid phase biocatalytic reactors to assist product recovery

Two-liquid phase biocatalytic processes are currently attracting wide interest as a means of carrying out industrial conversions involving compounds of low water solubility. In this paper we outline experimental techniques to model product transfer and recovery. Such extractive biocatalysis is of particular benefit for reactions exhibiting product inhibition.

A theoretical model for catalyst‐fluid flow reactors with immobilised cells

AbstractA theoretical model of the behaviour of immobilised cells in porous pellets is developed. Particular attention is given to ethanol production with immobilised yeast cells, the deactivating effect of the alcohol upon the cells and the performance of the reactor. It is demonstrated that by introducing an activity parameter, which is a measurable quantity, all of the effects of immobilisation, decreased activity, increased operational stability and increased thermal resistance, yield to a quantitative description. This description is given in terms of parameters that govern the diffusion and reaction phenomena in the pellet and the dynamics of the cell population in free form. The effects of ethanol are accounted for by treating it as a deactivator acting in series with the main reaction. In this way it can be shown that its action manifests itself by increasing a natural decay constant of the cell system. The growth of the cell population inside the pellet is taken into account. Immobilised enzyme reactors may be considered a special case of heterogeneous biocatalytic reactors in which no growth takes place. The model is able, therefore, to describe enzyme systems under analogous conditions of deactivation‐reaction or under natural irreversible decay.

Two-liquid phase biocatalytic reactors

Two-liquid phase biocatalytic reactors in which a large proportion of the reaction volume is occupied by the organic phase have been investigated for steroid transformations and the modification of oils and fats. The organic phase may consist of the reactant alone or the reactant dissolved in a water-immiscible organic solvent. Under appropriate conditions, immobilized cells and enzymes have reasonable operational stabilities in such reactors, and some commercial processes are being developed.

ChemInform Abstract: TWO‐LIQUID‐PHASE BIOCATALYTIC REACTIONS

Two-liquid phase biocatalytic reactors in which a large proportion of the reaction volume is occupied by the organic phase have been investigated for steroid trans- formations and the modification of oils and fats. The organic phase may consist of the reactant alone or the reactant dissolved in a water-immiscible organic solvent. Under appropriate conditions, immobilized cells and enzymes have reasonable operational stabilities in such reactors, and some commercial processes are being developed.

Two‐liquid‐phase biocatalytic reactions

AbstractThe development of biocatalytic reactors for the conversion of high concentrations of water‐insoluble reactants is at the same stage that systems for water‐soluble reactants had reached almost a decade ago. There are still many problems and much research to be done before we fully understand such systems. Nevertheless, we can confidently expect to see new commercial processes with multiliquid biocatalytic reactors during the next decade.

Collagen‐enzyme complex membranes and their performance in biocatalytic modules

AbstractCollagen was used as carrier for the immobilization of invertase, lysozyme, urease, glucose oxidase, penicillin amidase, and glucose isomerase. Immobilization was accomplished by either impregnation of a preswollen collagen membrane with enzyme solution or electrocodeposition of collagen and enzyme from a collagen dispersion containing dissolved enzyme. The collagen‐enzyme complexes prepared are in membrane form.Membranous collagen‐enzyme complexes were used to construct biocatalytic reactors such as the capillaric coil modular reactor. Such biocatalytic reactors were used in a recirculation system for the conversion of substrates. The biocatalytic reactors showed initial decreases of activity to stable limits which are maintained over a large number of reactor volume replacements. The stable limits correspond to approximately 35% of the initial activities for lysozyme and invertase, 25% for urease, 15% for glucose oxidase.The mechanism of complex formation between collagen and enzyme involves multiple salt linkages, hydrogen bonds, and van der Waals interactions. This protein‐protein interaction which leads to stable complexes by both impregnation and electrocodeposition processes is unique among the enzyme immobilization methods currently available.