Enzyme membrane reactor, electrochemical

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],... 

Figure 22.7 Design of the electrochemical enzyme membrane reactor (EEMR).

Figure 13. Continuous formation of (S )-4-phenyl-2-butanol from 4-phenyl-2-butanone using the electrochemical enzyme membrane reactor under indirect electrochemical NADH regeneration with a high-molecular-weight rhodium catalyst [26,29,30,65].

Within the electrochemical enzyme membrane reactor, the formation of / -hydroxy-benzaldehyde from / -cresol reached a space-time yield of 4.44 mmol/L h = 11.8g/ g L d using 60 U of the enzyme PCMH. At a residence time of 3 h, the turnover ofp-cresol was 90 to 95%. For the formation of (/ )- -(4-hydroxyphenyl)ethanol from -ethyl-phenol using the electrochemical enzyme membrane reactor, the space-time yield was... 

Figure 25. Principle of the electroenzymatic oxidation of / -ethylphenol catalyzed by EPMH to give (/ )- -(4 -hydroxyphenyl)ethanol in an electrochemical enzyme membrane reactor.

A second obvious area of application is in continuous flow analysis or flow injection analysis systems, in which the immobilized molecules form reactors that can be readily inserted and replaced in a flow analysis manifold. The physical form of the enzymes varies widely packed-bed reactors are often used, but open-tube wall reactors and membrane reactors have also been investigated. A principal advantage of all such systems is that they can use all the optical or electrochemical detectors routinely used in flow analysis. However, the problems of producing stable and robust immobilized enzyme reactors have proved more intractable than many researchers hoped, and other advances (e.g.. the use of more sensitive detectors, improved availability of low-cost soluble enzymes) have minimized the advantages of using solid phase enzymes. 

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