George Bell

Biocatalyst behaviour in low-water systems

An improving understanding of the parameters that affect biocatalyst activity, specificity and stability in low-water non-aqueous media make reliable predictions about the behaviour of such systems increasingly feasible. Here, we discuss some of the key factors, such as control of water activity, and the effects of solvent on K m and protein-ionization state, that must be addressed in order to obtain predictable results.

Water activity fails to predict critical hydration level for enzyme activity in polar organic solvents: Interconversion of water concentrations and activities

Abstract When studying biocatalysts in organic media, it is useful to be able to convert between water concentration and thermodynamic water activity ( a w ). We have obtained relationships for this purpose based on published vapor-liquid equilibrium data for a range of commonly used polar solvents. Their use is illustrated by the reanalysis of literature data for the activity of an immobilized laccase. Kinetic constants measured as a function of water concentration can thus be related to a w . We show that the critical water content for significant V m values is characterized by a similar a w (about 0.4) in five different alcohols; however, the critical a w becomes progressively higher in other water-miscible solvents in the order: dioxane, acetonitrile, tetrahydrofuran, acetone (critical a w about 0.8). This indicates that such polar solvents probably have important direct effects on the enzyme.

The rheology of fibre spinning and the properties of hollow-fibre membranes for gas separation

Abstract The effect of spinning rheology on gas-separation hollow-fibre membranes has been investigated. For polysulfone fibres, permeability decreased with increasing dope concentration. The lowest fibre selectivities occurred with the medium spinning-dope concentration, suggesting that both surface pore area and active layer thickness were high. Both permeability and selectivity increased with increasing dope extrusion rate, possibly due to enhanced orientation of the membrane skin. Permeability increased and selectivity decreased with increasing jet stretch. Polyacrylonitrile membranes, because of the low polymer permeability, proved insensitive to spinning conditions, showing no separation capability due to the dominating effect of surface pores.

Synthesis of glycerides and esters by fungal cell-bound enzymes in continuous reactor systems

SummaryThe synthesis of glycerides and fatty esters using the cell-bound lipolytic enzymes of fungal mycelia is described. Use of organic solvents for substrate solution and a solid-phase enzyme system enable high conversions to be obtained continuously in packed bed and stirred tank reactors.

The deduction of fine structural details of gas separation hollow fibre membranes using resistance modelling of gas permeation

Gas transfer through asymmetric polysulfone hollow fibre membranes has been modelled, allowing fine details of fibre structure to be deduced from gas permeation characteristics. The structural information is used to interpret the relationship between spinning conditions and fibre properties. Dope concentration determines the general morphology of the fibre, such as the porosity (voidage fraction), thickness of the active layer and order of magnitude of surface porosity (fraction of surface area that is pores), and thus it sets the permeability and level of selectivity that are likely to be achieved on coating. The selectivity of the solid polymer (the maximum selectivity achievable by any membrane if coating is highly effective or if no surface pores are present) was found to increase with increasing dope extrusion rate. The elevated levels of shear in the spinneret may enhance the orientation of polymer molecules. Increasing the jet stretch ratio during spinning had a detrimental effect on solid polymer selectivity. Increased elongational strain possibly results in an unfavourable polymer structure.

Organic solvent functional group effect on enzyme inactivation by the interfacial mechanism

Abstract We have used a bubble column apparatus to study interfacial inactivation of enzymes. The amount of enzyme inactivated was proportional to the area of organic solvent exposed, as is characteristic of the interfacial mechanism. Tests were made with a series of 12 solvents of log P close to 4.0, but with different functional groups. With α- and β-chymotrypsin, inactivation was much less severe with amphiphilic molecules like decyl alcohol, than with less polar compounds (heptane as the extreme case). This corresponds to a correlation with aqueous–organic interfacial tension, and presumably reflects a more polar interface as seen by the enzyme adsorbing from the aqueous phase. A 50% mixture of decyl alcohol and heptane behaved similarly to pure decyl alcohol, which would be expected to accumulate at the interface. With pig liver esterase, the correlation was rather weak, however. Accumulated data for interfacial inactivation by alkanes was examined for the above enzymes, and also papain, trypsin, urease and ribonuclease. The differing sensitivities did not show a clear correlation with any enzyme property, although there was some relationship to adiabatic compressibility, thermal denaturation temperature and mean hydrophobicity.

Aqueous—organic membrane bioreactors. Part I. A guide to membrane selection

Abstract The influence of membrane pore structure on the ease with which an aqueous-organic interface can be maintained in the plane of the membrane of a two-phase membrane reactor is discussed. Four factors affecting the pressure required to cause breakthrough of the non-wetting phase have been identified: (i) membrane pore size, (ii) asymmetry of membrane pore structure, (iii) the placement of the wetting liquid when an asymmetric membrane is used, and (iv) a change in the wetting characteristics of the membrane as the reaction progresses. The fourth factor is particularly important since two phase biocatalytic reactions frequently involve surface active reactants and/or products. It is shown that the ideal pattern of surfactant-membrane interactions - which is reflected by the desired direction of change in the angle of contact between the wetting liquid and the membrane - depends on the second and third factors. An experiment is suggested to assess the importance of the various factors and a set of rules of thumb have been presented to assist in the selection of membrane material and type. The importance of correctly identifying the wetting liquid when an amphiphilic membrane polymer is used has been pointed out.

Measurement and control of water activity with an aluminium oxide sensor in organic two-phase reaction mixtures for enzymic catalysis

Abstract An aluminium oxide film sensor (Endress & Hauser type DY20B) gives correct readings for the partial pressure of water dissolved in hexane or in the presence of various organic solvent vapors. This value, together with temperature, may be used to calculate thermodynamic water activity. The sensor must, however, be used with care, due to limitations of stability, sensitivity, and measurement range. Response time and lifetime are adequate. The sensor has been used for continuous monitoring and control of water activity during lipase-catalysed esterification in a hexane medium: water formed as a reaction product was removed by controlled recirculation of the headspace gases through a drying column. The rate of water production measured with the sensor agreed with the rate of ester synthesis determined by chemical analysis.

Aqueous-organic membrane bioreactors part II. Breakthrough pressure measurement

Abstract The effect of membrane type—structure and wettability—on the operation of two-phase, aqueous-organic, membrane bioreactors has been studied. The influence of surfactants on membrane wettability is reported. A simple, but highly sensitive, technique for the measurement of breakthrough pressures is described. Experimental measurements of the variation in break through pressures as the concentration of tenside in the system was changed are reported. On the basis of the results from these measurements it is concluded that: (i) hydrophilic and, highly retentive, amphiphilic ultrafiltration membranes may be used to operate two-phase bioreactors, (ii) amphiphilic microfiltration membranes should never be used in such reactors and (iii) PTFE membranes would always be a poor choice for use in such devices because they always have a low breakthrough resistance in two-liquid systems—breakthrough pressures as low as 100 mbar were observed for the system ethyl laurate-water-PTFE, which contains no surface-active component. It is shown that these results are in general agreement with rules of thumb for the selection of membranes, presented earlier. The influence of membrane history on its wetting behavior—due to effects such as polymer surface restructuring—is highlighted. The limits on the utility of simple breakthrough pressure tests in determining suitable membranes, for use in two-phase bioreactors, owing to possible complications resulting from the exact mechanism of enzyme action is pointed out.

Kinetics of uptake of organic liquid substrates by microbial cells: A method to distinguish interfacial contact and mass-transfer mechanisms

SummaryWe have measured the rate of (-)-menthol production by whole cells of Bacillus subtilis in a stirred aqueous emulsion of menthyl acetate. With excess cells, the rate is proportional to the organic phase volume, but is the same whether this is pure (-)-menthyl acetate substrate or the racemate. This result is expected if substrate uptake is by direct contact with the interface, but not if mass transfer into the aqueous phase is limiting. This suggests a general approach for distinguishing these two mechanisms, which usually give the same steady-state kinetic behaviour. Addition of 2-propanol, which increases the solubility of menthyl acetate, has little effect on the rate, supporting the conclusion that substrate uptake is by interfacial contact.