Enzyme Membrane Reactors EMR

During the last ten years enzyme technology has moved mainly towards the development of new immobilization techniques and the improvement of those already existing. In turn, the attention of applied research has been focused on the engineering of systems based on immobilized biocatalysts. Enzymes involved in this development were enzymes catalyzing simple reactions that normally require no cofactors. A number of drawbacks affected the use of immobilized enzymatic preparations. An often dramatic reduction of initial enzyme activity due to the binding process, and the existence of diffusional resistances limits this approach with low activity enzymes, with macromolecular substrates and in general with enzymes whose cata- 

Depending on the flow dynamics of the reaction vessel, it is possible to distinguish between reactors equipped with flat UF membranes or hollow fibers. 

Performance of dead-end units is largely affected by the flow dynamics of the system in fact, mixing of substrates and catalysts is not fully accomplished, and concentration polarization phenomena strongly limit reactor performance. On the one hand, the existence of a polarization layer above the membrane surface significantly reduces permeate flow rates on the other hand, enzymatic conversion is achieved mostly inside a highly concentrated polarization layer giving lower conversions as compared to those obtained with the same amount of enzyme uniformly distributed within the reactor. 

CST reactors have been more widely adopted, partly due to the possibility of concentration polarization control, and partly due to the easy modeling of enzyme kinetic behavior. In the literature, comprehensive mathematical descriptions of the kinetic behavior of enzymes located in CSTR UF units are reported 9 1-0 as far as low-molecular-weight substrates are concerned. 

Assuming complete mixing within the reactor so that enzyme and substrate concentration in the reactor vessel are uniform, and the latter is equal to its value in the permeate, substrate mass balance in molar form generally looks like  

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Two reactions for the production of L-phenylalanine that can be performed particularly well in an enzyme membrane reactor (EMR) are shown in reaction 5 and 6. The recently discovered enzyme phenylalanine dehydrogenase plays an important role. As can be seen, the reactions are coenzyme dependent and the production of L-phenylalanine is by reductive animation of phenylpyruvic add. Electrons can be transported from formic add to phenylpyruvic add so that two substrates have to be used formic add and an a-keto add phenylpyruvic add (reaction 5). Also electrons can be transported from an a-hydroxy add to form phenylpyruvic add which can be aminated so that only one substrate has to be used a-hydroxy acid phenyllactic acid (reaction 6). 

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 analogy to the enzyme membrane reactors (EMRs) [8], a chemzyme membrane reactor (CMR) is used to retain a polymer-enlarged chemical catalyst of this kind. Tremendous progress could be made in the recycling of polymer-enlarged catalysts (Fig. 3.1.3) by employing different types of catalysts for both the enan-tioselective C-C bond formation and redox reactions. 

The enzyme membrane reactor (EMR), if the reactor contents are well-mixed so... 

The enzyme membrane reactor (EMR) is an established mode for running continuous biocatalytic processes, ranging from laboratory units of 3 mL volume via pilot-scale units (0.5-500 L) to full-scale industrial units of several cubic meters volume and production capacities of hundreds of tons per year (Woltinger, 2001 Bommarius, 1996). The analogous chemzyme membrane reactor (CMR) concept, discussed in Chapter 18, Section 18.4.5, is based on the same principles as the EMR but is far less developed yet. 

For the production of chiral hydrophobic alcohols, FDH from C. boidinii was combined with an NAD+-dependent alcohol dehydrogenase (ADH) from Rhodo-coccus erythropolis. As a first example for the production of hydrophobic alcohols, an enzyme membrane reactor (EMR) was used for the synthesis enantiomerically pure (S)-1 -phenylpropan-2-ol and some related structures out of their corresponding ketones [42]. 

Fig. 5 Enzyme membrane reactor (EMR) used for amino acylase resolution of N-acetyl-D,L-ami-no acids.

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). 

As proof of the kinetic model, fitting of initial rate data or time-course data of batch reactions have been introduced in Sect. 7.4. Additionally, a proper fit of continuous reactions in an enzyme membrane reactor (EMR) may serve as confirmation of the kinetic model. For this coupled enzyme system, calculated and measured conversions at different operating conditions (varying [E] and t values, not further specified) are presented in Fig. 7-34. 

L-Phenylalanine has been produced continuously from ACA with the help of ACA acylase in an enzyme membrane reactor (EMR) with a space-time-yield of 277 g L 1 d1 83 . With ACL acylase, L-Ieucine was produced at 123-180 g L-1 d 1 in the same reactor set-up1821. The dehydroamino acid substrates can be prepared conveniently, either from 2-halogen carboxylic acid esters1841, or, specifically in the case of ACA, via the acetamidomalonic ester route by reaction with benzyl halogenides[851. 

Process Technology Cofactor Regeneration and Enzyme Membrane Reactor (EMR)... 

For soluble reactants and products, enzymes are preferentially immobilized in an enzyme-membrane reactor (EMR). To prevent the cofactor from penetrating through the membrane, it can be enlarged with polyethyleneglycol (PEG) 163l... 

The NADH is regenerated simultaneously by reduction with formate catalyzed by formate dehydrogenase (FDH). For continuous operation an enzyme membrane reactor (EMR) is used9. 

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. 

Use of Enzyme Membrane Reactors (EMR) the method of choice for systems with co-factor recycling or for reactions with expensive enzymes. 

Figure 1. Enzyme Membrane Reactor (EMR) for bulk production - schematic representation. 1, ultrafiltration module 2, peristaltic pump 3, septum 4, sterile filter 5, metering pumps 6, reactor loop (ther-mostated) pHI, pH indication TIR, temperature indication and regulation PI, pressure indication.

Figure 2. Schematic representation of the enzyme membrane reactor (EMR) system used for enzymatic synthesis of P-D>glucuronides.

The work-up of batch processes, run in stirred vessels, had often faced the challenge to efficiently separate and recover the enzyme used. Meanwhile, there is abundant know-how available to immobilise enzymes on different carriers, though some issues need always to be considered maintained activity of the enzyme, its stability towards solvents and the operating temperature used in a reaction. Enzyme immobilisation allows for continuous reactions carried out in columns or in a sequence of continuous stirred-tank reactors. Certain advantages are offered by Degussa s enzyme-membrane-reactor (EMR), where the enzyme is surrounded by a hoUow-fibre membrane, that is permeable to substrate and product. 

Enzyme membrane reactors (EMR) with biocatalyst in a homogeneous solution... 

Figure 13.7 Setup for mass specirrmietry-based quantitative real-time analysis. The outlet flow of a cwtinuous stirred enzyme membrane reactor (EMR) is continuously analyzed by multiple reaction monitoring in an electrospray ionization triple quadrupole mass spectrometer after flow reduction (SI), dilution (T) and another flow reduction (S2) (S6j. Reprinted by permission from Macmillan Publishers Ltd Nature Chemical Biology [Bujara, M., SchumperU, M., Pellaux, R., Heinemann, M., Panke, S. (2011) Optimization of a Blueprint f

The enzyme membrane reactor (EMR), a continuously operated laboratory-scale reaction vessel for biocatalytic transformations in which the reaction compartment is separated from the outside through a membrane in order to retain the enzyme, is the chemical-technological analogue of whole cells ... 

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