Enzymes, inhibition, substrate reactors

When macromolecular substrates are involved in the transformation under study, concentration polarization phenomena affect the EMR performance more severely. Diffusion limitations of macromolecular substrates hamper the use of immobilized enzymes in the hydrolysis of high-molecu-lar-weight substrates. By selecting membranes with an appropriate molecular weight cut-off, both enzyme and substrate are retained in an EMR in touch with each other, and hydrolysis products and/or inhibitors are continuously removed from the system. Soluble enzymes can then act directly on substrate macromolecules without diffusion limitations and steric hindrance imposed by enzyme fixation to a solid support. The stirring features of CST EMRs moreover assures that substrates and/or inhibitors within the reactor vessel are maintained at the lowest possible concentration level. Such reactor configuration is then extremely useful when substrate inhibited reaction patterns are involved, or when inhibiting species are assumed to exist in the feed stream. 

Flushing the substrate solution along the enzymatic gel causes the substrate to be converted to product even in the axial stream. When the enzyme is product inhibited and the effluent from the reactor is recycled, product accumulates in the feed stream thus inhibiting gelled enzymes. High axial flow rates may reduce conversion of substrate to product in the axial stream and enzyme inhibition, while product conversion in the permeate remains unaltered at a given transmembrane pressure.29 34 36-Under such conditions, the axial flow rate needs to be optimized since it plays an opposite role. 

Biocatalysts in nature tend to be optimized to perform best in aqueous environments, at neutral pH, temperatures below 40 °C, and at low osmotic pressure. These conditions are sometimes in conflict with the need of the chemist or process engineer to optimize a reaction with respect to space-time yield or high product concentration in order to facilitate downstream processing. Furthermore, enzymes and whole cells are often inhibited by products or substrates. This might be overcome by the use of continuously operated stirred tank reactors, fed-batch reactors, or reactors with in situ product removal [14, 15]. The addition of organic solvents to increase the solubility of substrates and/or products is a common practice [16]. 

Substrate and product inhibitions analyses involved considerations of competitive, uncompetitive, non-competitive and mixed inhibition models. The kinetic studies of the enantiomeric hydrolysis reaction in the membrane reactor included inhibition effects by substrate (ibuprofen ester) and product (2-ethoxyethanol) while varying substrate concentration (5-50 mmol-I ). The initial reaction rate obtained from experimental data was used in the primary (Hanes-Woolf plot) and secondary plots (1/Vmax versus inhibitor concentration), which gave estimates of substrate inhibition (K[s) and product inhibition constants (A jp). The inhibitor constant (K[s or K[v) is a measure of enzyme-inhibitor affinity. It is the dissociation constant of the enzyme-inhibitor complex. 

The inhibition analyses were examined differently for free lipase in a batch and immobilised lipase in membrane reactor system. Figure 5.14 shows the kinetics plot for substrate inhibition of the free lipase in the batch system, where [5] is the concentration of (S)-ibuprofen ester in isooctane, and v0 is the initial reaction rate for (S)-ester conversion. The data for immobilised lipase are shown in Figure 5.15 that is, the kinetics plot for substrate inhibition for immobilised lipase in the EMR system. The Hanes-Woolf plots in both systems show similar trends for substrate inhibition. The graphical presentation of rate curves for immobilised lipase shows higher values compared with free enzymes. The value for the... 

Independently of enzyme stability an enzyme reaction at constant temperature and pH should be run with the smallest possible contribution by inhibition. For this reason, a CSTR is most suitable in the case of substrate inhibition because the substrate concentration is evened out across the reactor volume and thus minimized. In the case of product inhibition, however, a batch reactor or a PFR is preferred, as the reactor volume required for complete or nearly complete conversion is much smaller than in the case of a CSTR. 

Closed systems may be employed when buffer recycling is possible, that is when the buffer contains high concentrations of all necessary cosubstrates, when complete consumption of injected substrate occurs within the reactor, and when products of the enzymatic reaction do not inhibit the immobilized enzyme. A closed system for immobilized oxidase enzymes is shown in Figure 4.10. 

Unfortunately, most enzymes do not obey simple Michaelis-Menten kinetics. Substrate and product inhibition, presence of more than one substrate and product, or coupled enzyme reactions in multi-enzyme systems require much more complicated rate equations. Gaseous or solid substrates or enzymes bound in immobilized cells need additional transport barriers to be taken into consideration. Instead of porous spherical particles, other geometries of catalyst particles can be apphed in stirred tanks, plug-flow reactors and others which need some modified treatment of diffusional restrictions and reaction technology. 

The suitability of different reactors is demonstrated for two typical enzyme kinetic examples, involving substrate inhibition in one case and product inhibition in the other (Fig. 7-24). 

The ideal reactor to overcome substrate inhibition (Fig. 7-24A) is the continuous stirred tank reactor (possible in form of an Enzyme Membrane Reactor, see below). In spite of a high feed concentration of substrate a high reaction rate occurs, as the steady state substrate concentration within the reactor is low. 

In the MBR of Salzman et al [5.113] the soluble enzyme exists in the shellside, and is recirculated through en external reservoir to ensure its homogeneity. The substrates were fed in the tubeside and diffused through the hollow fibers to the shellside to react with the enzymes found there. The products that formed diffused back to the tubeside and left the reactor by convection. Back-mixing effects were considered only in the tubeside, whereas other potentially negative effects like side reactions or inhibition were not taken into ac-... 

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