Enzyme membrane reactors

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). 

Degussa AG uses immobilised acylase to produce a variety of L-amino adds, for example L-methionine (80,000 tonnes per annum). The prindples of the process are the same as those of the Tanabe-process, described above. Degussa uses a new type of reactor, an enzyme membrane reactor, on a pilot plant scale to produce L-methionine, L-phenylalanine and L-valine in an amount of 200 tonnes per annum. 

Many procedures have been suggested to achieve efficient cofactor recycling, including enzymatic and non-enzymatic methods. However, the practical problems associated with the commercial application of coenzyme dependent biocatalysts have not yet been generally solved. Figure A8.18 illustrates the continuous production of L-amino adds in a multi-enzyme-membrane-reactor, where the enzymes together with NAD covalently bound to water soluble polyethylene glycol 20,000 (PEG-20,000-NAD) are retained by means of an ultrafiltration membrane. 

Several hundred tons of L-methionine per year are produced by enzymatic conversion in an enzyme membrane reactor. An alternative approach is dynamic resolution, where the unconverted enantiomer is racemized in situ. Starting from racemic /V-acetyl-amino acid, the enantioselective L-acylase is used in combination with an TV-acyl-amino acid racemase to enable nearly total conversion of the substrate. 

Figure 4.18 Enzyme membrane reactor synthesis of L-tert-leucine from trimethylpyruvic acid in an continuously operated enzyme membrane reactor with ultrafiltration followed by a crystallization step...

Wichmann, R., Wandrey, C., Biickmann, A.F. and Kula, M.-R. (1981) Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Biotechnology and Bioengineering, 23, 2789-2796. 

Chemically functional membranes afford yet another intriguing platform upon which process-intensified chemistry can be performed. For example, an enzyme membrane reactor process is used to produce a... 

Enzyme membrane reactor for production of diltiazem intermediate. A solution of the racemic ester in organic solvent enters the port at the bottom of the reactor and flows past the strands of microporous, hollow-fiber membrane that contain an enzyme. The enzyme catalyzes hydrolysis of one enantiomer of the ester that undergoes decarboxylation to 4-methoxyphenylacetaldehyde (which in turn forms a water-soluble bisulfite complex that remains in the aqueous phase). The other enantiomer of the ester remains in the aqueous stream that leaves the reactor via the port at the top. Courtesy of Sepracor, Inc. 

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. 

For application in an electrochemical enzyme membrane reactor, polymer-supported derivatives of 9 have been synthesized, which could be retained by ul-trafiltration membranes and were thus retained within the electroenzymatic reactor [31, 40]. 

A first application using ferroceneboronic acid as mediator [45] was described for the transformation of p-hydroxy toluene to p-hydroxy benzaldehyde which is catalyzed by the enzyme p-cresolmethyl hydroxylase (PCMH) from Pseudomonas putida. This enzyme is a flavocytochrome containing two FAD and two cytochrome c prosthetic groups. To develop a continuous process using ultrafiltration membranes to retain the enzyme and the mediator, water soluble polymer-bound ferrocenes [50] such as compounds 3-7 have been applied as redox catalysts for the application in batch electrolyses (Fig. 12) or in combination with an electrochemical enzyme membrane reactor (Fig. 13) [46, 50] with excellent results. 

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. 

Thus, it was shown that flavo enzymes 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 [46, 50],... 

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. 

There is huge potential in the combination of biocatalysis and electrochemistry through reaction engineering as the linker. An example is a continuous electrochemical enzyme membrane reactor that showed a total turnover number of 260 000 for the enantioselective peroxidase catalyzed oxidation of a thioether into its sulfone by in situ cathodic generated hydrogen peroxide - much higher than achieved by conventional methods [52],... 

W. Berke, H. J. Schuz, C. Wandrey, M. Morr, G. Denda, and M. R. Kula, Continuous regeneration of ATP in enzyme membrane reactor for enzymatic synthesis, BiotechnoL Bioeng., 32, 130-139 (1988). 

Pfaar, U. Gygax, D. Gertsch, W. Winkler, T. Ghisalba, O. (1999) Enzymatic synthesis of p-D-glucuronides in an enzyme membrane reactor. Chimia, 53, 590-3. 

The reaction in a homogeneous solution with a polar organic solvent in which the enzymes and substrates are both soluble, occurs often at the expense of the enzyme stability [4, 5]. Besides immobilised enzymes in organic solvents [6], emulsion reactors, especially enzyme-membrane-reactors coupled with a product separation by membrane based extractive processes [7-9] and two-phase membrane reactors [10-12], are already established on a production scale. 

Bouwer, S.T., Cuperms, F.P. and Derksen, J.T.P. (1997) The performance of enzyme-membrane reactors with immobilized lipase. Enzyme and Microbial Technology, 21, 291-296. 

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. 

There are maty other examples of cofactor regeneration reactions and/or of reactions which may be performed in an enzyme membrane reactor. An important example is the regeneration of NADH by formate dehydrogenase (FDH), starting with formate (Wichmaim et al, 1981). The advantage of this reaction is that it is irreversible because carbon dioxide is hberated, while formate is a relatively cheap electron donor. 

Figure 10.8 Enzyme membrane reactor for production of L-alanine from DL-lactate (Wandrey, 1984).

For ATP regeneration, a similar concept has been used (Berke et al, 1988). ATP can be regenerated from ADP using acetyl phosphate and the enzyme acetate kinase, upon release of acetate. The reaction is irreversible and acetyl phosphate is a relatively cheap phosphate donor. Thus, in an enzyme membrane reactor, PEGderivatized ATP was consumed by a phosphorylase or a synthetase in a reaction leading to a product of interest, and the ATP was regenerated by the acetate kinase (Figure 10.9). 

The gram-scale preparation of rare sugars by E. coli transketolase was demonstrated successfully for (S)-erythrulose from glycolaldehyde and hydroxypyruvate in an enzyme membrane reactor which allowed the continuous production of (S)-erythrulose with high conversion and a space-time yield of 45 g L" d was reached [12]. 

Hauer, M. Breuer, M. Pohl, Studies on the continuous production of (R)-(-)-phenylacetylcarbinol in an enzyme-membrane reactor. J. Mol. 

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. 

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