Enzyme membrane reactors concentration

The separation of homogeneous catalysts by means of membrane filtration has been pioneered by Wandrey and Kragl. Based on the enzyme-membrane-reactor (EMR),[3,4] that Wandrey developed and Degussa nowadays applies for the production of amino acids, they started to use polymer-bound ligands for homogeneous catalysis in a chemical membrane reactor (CMR).[5] For large enzymes, concentration polarization is less of an issue, as the dimension of an enzyme is well above the pore-size of a nanofiltration membrane. 

In an electrochemical enzyme membrane reactor an electrochemical flow-through cell using a carbon-felt anode is combined with an enzyme-membrane reactor. The residence time is adjusted by the flow of the added substrate solution. The off-flow of the enzyme membrane reactor only contains the products p-hydroxy benzaldehyde and p-hydroxy benzylalcohol. By proper adjustment of the residence time and the potential, total turnover of the p-hydroxy toluene, which is introduced into the reactor in 13 mM concentration, can be obtained. In a 10-day run, the enzyme underwent 400000 cycles and the polymer-bound mediator, which was present in a higher concentration than the enzyme, underwent more than 500 cycles. At the end, the system was still active. By proper selection of the residence time, one can either... 

The enzyme p-ethylphenol methylene hydroxylase (EPMH), which is very similar to PCMH, can also be obtained from a special Pseudomonas putida strain. This enzyme catalyzes the oxidation of p-alkylphenols with alkyl chains from C2 to C8 to the optically active p-hydroxybenzylic alcohols. We used this enzyme in the same way as PCMH for continuous electroenzymatie oxidation of p-ethylphenol in the electrochemical enzyme membrane reactor with PEG-ferrocene 3 (MW 20 000) as high molecular weight water soluble mediator. During a five day experiment using a 16 mM concentration of p-ethylphenol, we obtained a turnover of the starting material of more than 90% to yield the (f )-l-(4 -hydroxyphenyl)ethanol with 93% optical purity and 99% enantiomeric excess (glc at a j -CD-phase) (Figure 14). The (S)-enantiomer was obtained by electroenzymatie oxidation using PCMH as production enzyme. 

Fig. 14. Concentration profiles during the continuous indirect electrochemical oxidation of 4-ethylphenol catalyzed by the enzyme EPMH in the electrochemical enzyme membrane reactor...

This system fulfills the four above-mentioned conditions, as the active species is a rhodium hydride which acts as efficient hydride transfer agent towards NAD+ and also NADP+. The regioselectivity of the NAD(P)+ reduction by these rhodium-hydride complexes to form almost exclusively the enzymatically active, 1,4-isomer has been explained in the case of the [Rh(III)H(terpy)2]2+ system by a complex formation with the cofactor[65]. The reduction potentials of the complexes mentioned here are less negative than - 900 mV vs SCE. The hydride transfer directly to the carbonyl compounds acting as substrates for the enzymes is always much slower than the transfer to the oxidized cofactors. Therefore, by proper selection of the concentrations of the mediator, the cofactor, the substrate, and the enzyme it is usually no problem to transfer the hydride to the cofactor selectively when the substrate is also present [66]. This is especially the case when the work is performed in the electrochemical enzyme membrane reactor. 

A solution to this problem is the enzyme membrane reactor (Figure 10.8). This is a kind of CSTR (continuous stirred tank reactor), with retains the enzyme and the cofactor using an ultrafiltration membrane. This membrane has a cut-off of about 10000. Enzymes usually have a molecular mass of 25000-250000, but the molecular mass of NAD(H) is much too low for retention. Therefore it is first derivatized with polyethylene glycol (PEG 20000). The reactivity of NAD(H) is hardly affected by the derivatization with this soluble polymer. Alanine can now be produced continuously by high concentrations of both enzymes and of NAD (H) in this reactor. 

Similarly, enantiomerically pure hydrophobic (S)-l-phenyl-2-propanol, (S)-4-phe-nyl-2-butanol, and (S)-6-methylhept-5-en-2-ol (sulcatol) were obtained with high purities in a dual-loop enzyme membrane reactor unit with separate membrane extraction unit (Kruse, 1996). Whereas substrate concentrations were low at 9-12 mM, space-time yields higher than 100 g (L d) 1 as well as concentrated product solutions were obtained. 

The basic hydrodynamic equations are the Navier-Stokes equations [51]. These equations are listed in their general form in Appendix C. The combination of these equations, for example, with Darcy s law, the fluid flow in crossflow filtration in tubular or capillary membranes can be described [52]. In most cases of enzyme or microbial membrane reactors where enzymes are immobilized within the membrane matrix or in a thin layer at the matrix/shell interface or the live cells are inoculated into the shell, a cake layer is not formed on the membrane surface. The concentration-polarization layer can exist but this layer does not alter the value of the convective velocity. Several studies have modeled the convective-flow profiles in a hollow-fiber and/or flat-sheet membranes [11, 35, 44, 53-56]. Bruining [44] gives a general description of flows and pressures for enzyme membrane reactor. Three main modes... 

The overall mass-transfer rates on both sides of the membrane can only be calculated when we know the convective velocity through the membrane layer. For this, Equation 14.2 should be solved. Its solution for constant parameters and for first-order and zero-order reaction have been given by Nagy [68]. The differential equation 14.26 with the boundary conditions (14.28a) to (14.28c) can only be solved numerically. The boundary condition (14.28c) can cause strong nonlinearity because of the space coordinate and/or concentration-dependent diffusion coefficient [40, 57, 58] and transverse convective velocity [11]. In the case of an enzyme membrane reactor, the radial convective velocity can often be neglected. Qin and Cabral [58] and Nagy and Hadik [57] discussed the concentration distribution in the lumen at different mass-transport parameters and at different Dm(c) functions in the case of nL = 0, that is, without transverse convective velocity (not discussed here in detail). 

A model of a biphasic enzyme membrane reactor for the hydrolysis of triglycerides has been formulated according to the bond graph method of network thermodynamics, and the kinetics, the permeabilities of fatty acids and glycerides, the rates of inhibition of the immobilized enzyme, and the concentration of enzyme in a reaction zone are studied. 

In the described experiments, the space-time yields are limited by the rather low enzyme concentration used. Higher concentrations of the enzyme would increase the space-time yields accordingly. Thus, it could be shown that flavoenzymes and comparable systems can be used for synthetic applications in a continuous process under anaerobic reactivation of the prosthetic group in the electrochemical enzyme membrane reactor [26-31,135,136]. 

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. 

For synthesis on a preparative scale, repetitive batch processing has proved to be an effective and easy-to-handle method1128. The repeated use of the enzyme is possible after concentration of the solution by means of commercially available ultrafiltration equipment and adding fresh substrate solution. Some of the advantages given for the Enzyme Membrane Reactor (see below) are also valid for the repetitive batch technique. 

Compared to batch processes, continuous processes often show a higher space-time yield. Reaction conditions may be kept within certain limits more easily. For easier scale-up of some enzyme-catalyzed reactions, the Enzyme Membrane Reactor (EMR) has been developed. The principle is shown in Fig. 7-26 A. The difference in size between a biocatalyst and the reactants enables continuous homogeneous catalysis to be achieved while retaining the catalyst in the vessel. For this purpose, commercially available ultrafiltration membranes are used. When continuously operated, the EMR behaves as a continuous stirred tank reactor (CSTR) with complete backmixing. For large-scale membrane reactors, hollow-fiber membranes or stacked flat membranes are used 129. To prevent concentration polarization on the membrane, the reaction mixture is circulated along the membrane surface by a low-shear recirculation pump (Fig. 7-26 B). 

Biocatalyst consumption per unit weight of product was found to be about 6000 U/ kg at a conversion of 78 %. For production purposes, enzyme membrane reactors with a working volume up to 500 mL were employed for the synthesis of approximately 2 kg of N-acetylneuraminic acid and other derivatives such as keto-desoxynonulosonic acid (KDN)[129, 132L Downstream processing was achieved mainly by anion exchange chromatography on a 30 L column followed by reverse osmosis to concentrate solutions before lyophilization. 

The separation,purification and concentration of a thermosensitive bioactive compound from a lysate has been carried out combining UF, ion exchange and RO with significant cost reduction and productivity increase. Enzyme membrane reactors have been used for triglyceride enzymatic hydrolysis and product separation. Thermophi1ic,thermostable enzyme ultrafiltration membrane have been prepared, and used in high temperature lactose hydrolysis. 

Enzymatic systems in which membranes are simply used as separation media and not as catalyst carriers are traditionally called "enzyme membrane reactors" (EMR). Concentration polarization phenomena severely affect the performance of such reactors so that it is necessary to control the polarization layer onto membrane pressurized side by means of reactor fluid dynamics or design tricks. Fluid dynamic conditions in some of these reactors make them especially suitable for enzymatic systems for which a homogeneous catalyst distribution is particularly important, such as cofactor-requiring mono- and multi-enzyme systems. 

Concentration polarization phenomena, which are the main drawbacks of the aforesaid enzyme membrane reactors, can nevertheless be used to form, either in dynamic or in static conditions, a gel layer of enzyme proteins on a membrane.27 1 It is even possible to establish more than one enzyme layer and no coupling agent is needed to carry out the immobilization. Due to high protein... 

Enzymes can still be absorbed within symmetric macroporous membranes in order to establish high catalyst concentrations, cross-linked to prevent them from elution, or simply covalently or ionically bound either to symmetric or asymmetric membranes. In spite of short residence times, high conversions can be achieved in most kinds of enzyme membrane reactors.70-83... 

Figure 7.9 Concentration of soluble sugars (cellobiose and glucose) in permeate from an enzyme membrane reactor as a function of time for various space velocities (S).11...

Figure 7.21 Hydrolysis of 1.5 mM sucrose by cogelled invertase in an immobilized enzyme membrane reactor (IEMR). Comparison between experimental data in terms of glucose outlet concentration (Cp) vs. reaction time (t) and theoretical predictions (-). Enzyme amount, 0.398 x 10 s g/cm2 reaction temperature, 30°C pH 4.65 flow rate 1.13 x 103 ml/s.30...

Ultrafiltration of an enzyme solution through a UF membrane does not always result in gel layer formation. Unless a gel layer is formed, this immobilization technique cannot be used for flow systems lacking effective enzyme immobilization. In any event, soluble enzyme membrane reactors can be useful in order to perform kinetic analysis at high enzyme concentrations. Once steady state is attained, the theoretical model permits calculation of reaction rates in both regions. Once the layer is formed it behaves like a secondary membrane,34 capable of separating compounds of different molecular weight in the mixture as well as catalyzing a chemical reaction. 

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