Membrane reactors substrates

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). 

In this case study, an enzymatic hydrolysis reaction, the racemic ibuprofen ester, i.e. (R)-and (S)-ibuprofen esters in equimolar mixture, undergoes a kinetic resolution in a biphasic enzymatic membrane reactor (EMR). In kinetic resolution, the two enantiomers react at different rates lipase originated from Candida rugosa shows a greater stereopreference towards the (S)-enantiomer. The membrane module consisted of multiple bundles of polymeric hydrophilic hollow fibre. The membrane separated the two immiscible phases, i.e. organic in the shell side and aqueous in the lumen. Racemic substrate in the organic phase reacted with immobilised enzyme on the membrane where the hydrolysis reaction took place, and the product (S)-ibuprofen acid was extracted into the aqueous phase. 

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... 

Since in continuous degradation processes it is expected to reach a molecular weight distribution of the products, which is optimal for their further use, the investigation was devoted to test the effect of a key parameter such as the enzyme to substrate ratio (E/S). For a fixed mean retention time in the UF-membrane reactor, the following behaviour can be... 

Enzymatic degradation of pectin can be satisfactory performed in UF-membrane reactors which have been proved to be helpful tool for laboratory scale investigations. Reaction products can be continuously recovered in a sequence of filtration stages. The obtained product distribution depends on the enzyme to substrate ratio, which affects particularly the... 

Moreover, the membrane could be mounted as an interface between the apolar substrate and the polar oxidant in a membrane reactor, avoiding the use of any solvent. Dilution of the reagents by solvent and competition between solvent and reagents on the active sites can thus be avoided. In the countercurrent membrane reactor, the substrate and the oxidant are circulated at each side of the membrane and alkanes can be oxidized with peroxides without solvents. Of course, the system carries all of the other advantages of membrane reactors continuous operation and easy separation. 

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. 

After preformation, the substrates and carbon dioxide were supplied continuously. The membrane reactor was pressurized at the feed side up to 20 MPa with the reaction mixture. A trans-membrane pressure was created by opening a needle valve on the permeate side after which the continuous process started. 

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... 

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. 

The research group of Van Leeuwen reported the use of carbosilane de-ndrimers appended with peripherial diphenylphosphino end groups (i.e. 25, Scheme 26) [37]. After in situ complexation with allylpalladium chloride, the resultant metallodendrimer 25 was used as catalyst in the allylic alkylation of sodium diethyl malonate with allyl trifluoroacetate in a continuous flow reactor. Unlike in the batch reaction, in which a very high activity of the dendrimer catalyst and quantitative conversion of the substrate was observed, a rapid decrease in space time yield of the product was noted inside the membrane reactor. The authors concluded that this can most probably be ascribed to catalyst decomposition. The product flow (i.e. outside the membrane reactor)... 

Figure 11.4 Schematic diagram of membrane reactor. The reaction is designed so that reagent X may pass through the membrane and is therefore available for reaction. The substrate and product cannot pass through and the phases remain separate...

Pharmaceutical production generally uses multipurpose equipment, and so entrapment behind a membrane would require significant capital expenditure on specialized equipment. In spite of this, the use of membrane reactors in biocatalysis represents an efficient method of enzyme immobilization, given the large molecular weight difference between enzymes (10-150 kDa) and most substrates (300-500 Da). The reader is referred to some recent reviews on the topic. 

Fig. 2. Schematic presentation of a membrane reactor (left) and theoretical relative concentrations (Cfl of the dendritic species versus the substrate flow (in residence times Nfl calculated for various retention factors.

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. 

Figure 9.2-4. High-pressure continuous stirred tank membrane reactor S, substrates 1, reactor 2, separator 3, magnetic stirrer P, high pressure pump TIR, temperature regulator and indicator PI, pressure indicator.

The continuous high-pressure enzyme membrane reactor [30] is shown in Figure 9.2-4. The membrane with 35 mm diameter is placed between two sintered plates and fitted in the reactor. A certain amount of the catalyst (hydrated enzyme preparation) is put in the reactor which is electrically heated, with a heating jacket, to constant temperature. The substrates and the gas are pumped into the membrane reactor with the high-pressure pump. The products and unreacted reactants are collected in the separator. The catalyst remains in the reactor (behind the membrane). 

Membrane reactors allow a different option for the separation of biocatalysts from substrates and products and for retention in the reactor. Size-specific pores allow the substrate and product molecules, but not the enzyme molecules, to pass the membrane. Membrane reactors can be operated as CSTRs with dead-end filtration (Figure 5.5e) or as loop or recycle reactors (Figure 5.5f) with tangential (crossflow) filtration. 

Lipases (E.C. 3.1.1.3.) catalyze the hydrolysis of lipids at an oil/water interface. In a membrane reactor, the enzymes were immobilized both on the side of the water phase of a hydrophobic membrane as well as on the side of the organic phase of a hydrophilic membrane. In both cases, no other means for stabilization of the emulsion at the membrane were required. The synthesis reaction to n-butyl oleate was achieved with lipase from Mucor miehei, which had been immobilized at the wall of a hollow fiber module. The degree of conversion reached 88%, but the substrate butanol decomposed the membrane before the enzyme was deactivated. 

Commonly, the reaction is conducted in an emulsion stabilized by surfactants and containing the substrate (PC). In a microporous membrane reactor, PLD stability could be increased sevenfold by the addition of ethers to the chamber side. Additionally, no surfactants were required as the product could be separated in simple fashion 20% product was formed, compared with 4% in the simple emulsion system. 

Under certain conditions, scale-up of membrane reactors is straightforward. Provided that (i) the reactor contents are well mixed so that the reactor is operated as a CSTR, and that (ii) the membrane is configured for filtration in the tangential mode, the pertinent design criterion, besides constant residence time T in the reactor, is constant fluidity F of the substrate/product solution through the membrane at all reactor scales. Fluidity is defined by Eq. (19.36) (V = ultrafiltered volume, AP = transmembrane pressure, t = filtration time, and A = membrane area). 

The acylase-catalyzed resolution of N-acetyl-D,L-amino acids to obtain enantiomerically pure i-amino acids (see Chapter 7, Section 7.2.1) has been scaled up to the multi-hundred ton level. For the immobilized-enzyme reactor (Takeda, 1969) as well as the enzyme membrane reactor technology (Degussa, 1980) the acylase process was the first to be scaled up to industrial levels. Commercially acylase has broad substrate specificity and sufficient stability during both storage and operation. The process is fully developed and allowed major market penetration for its products, mainly pharmaceutical-grade L-methionine and L-valine. 

Acylase (acylase I aminoacylase N-acetyl amino acid amidohydrolase E.C. 3.5.1.14), is one of the best-known enzymes as far as substrate specificity (Chenault, 1989) or use in immobilized (Takahashi, 1989) or membrane reactors (Wandrey, 1977, 1979 Leuchtenberger, 1984 Bommarius, 1992a) is concerned however, its exact mechanism or 3D structure is still not known (Gentzen, 1979 1980). Acylase is available in large, process-scale quantities from two sources, porcine kidney and the mold Aspergillus oryzae. 

An enzyme membrane reactor allows continuous transketolase-catalyzed production of L-erythrulose from hydroxypyruvate and glycolaldehyde with high conversion, stable operational points, and good productivity (space-time yield) of 45 g (L d) 1, thus best overcoming transketolase deactivation by substrates (Bongs, 1997). 

Chiral amines, here (R)-l-aminotetralin, were obtained from racemic amine and pyruvate in a 39 mL hollow-fiber membrane reactor with (SJ-cotransaminases (ft>TA) (Shin, 2001). The substrates were recirculated until the e.e. value exceeded 95%. Simulations suggested residence times should be short to minimize product inhibition. 

---