Enzymes hollow fiber reactors

Another favorable aspect of stirred batch reactors is the fact that they are compatible with most forms of a biocatalyst. The biocatalyst may be soluble, immobilized, or a whole-cell preparation in the latter case a bioconversion might be performed in the same vessel used to culture the organism. Recovery of the biocatalyst is sometimes possible, typically when the enzyme is immobilized or confined within a semi-permeable membrane. The latter configuration is often referred to as a membrane reactor. An example is the hollow fiber reactor where enzymes or whole cells are partitioned within permeable fibers that allow the passage of substrates and products but retain the catalyst. A hollow-fiber reactor can be operated in conjunction with the stirred tank and operated in batch or... 

Cass, B.J. et al., Production of tomato flavor volatiles from a crude enzyme preparation using a hollow-fiber reactor, Biotechnol. Bioeng., 67, 372, 2000. 

Up to four enzymes involved in the metabolic pathways of purine bases-allantoinase, allantoicase, uricase and catalase-have been immobilized together by means of glutaraldehyde on the outer surface of cellulosic hollow fibers. Reactor performances are depicted in Figure 7.37, when uric acid is fed to the multi-enzyme reactor.71... 

Increasing the molecular weight of the cofactor by con-valent attachment to soluble or Insoluble supports is expensive and usually results in decreased enzyme activity due to sterlc hindrance (13, 14). In contrast it has been demonstrated experimentally that effective cofactor reuse can be achieved without modification of the cofactor (15, lA, JJ). Preliminary calculations based on a 10,000-15,000 cm2 surface area hollow fiber reactor Indicate that a slow infusion of as little as 26 mg/hr of NADP directly into the pS suspension would maintain a satisfactory NADPH concentration, thus assuring maximal rates of drug detoxification. However, in these preliminary studies adequate concentrations of NADP were included in the circulating drug solution. 

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. 

An immobilized-enzyme continuous-flow reactor incorporating a continuous direct electrochemical regeneration of NAD + has been proposed. To retain the low molecular weight cofactor NADH/NAD+ within the reaction system, special hollow fibers (Dow ultrafilter UFb/HFU-1) with a molecular weight cut-off of 200 has been used [32],... 

In a comparable system, (I ,S)-ibuprofen can be separated by a membrane reactor [83], see Fig. 13.10. The technique comprises a stereo-specific hydrolysis by an enzyme. Subsequently, the enantiomeric ester is extracted into the organic phase on the other side of the membrane. In the system developed by Sepracor Inc., (i )-ibuprofen is selectively hydrolyzed by proteases in a hollow-fiber unit and the (S)-ibuprofen ester can be isolated at 100% yield. This configuration also applies for enantioseparation of other acids such as naproxen and 2-chloropropionic acid. 

Malcata, F.X. and Hill Jr., C.G. (1995) Indnstrial ntihzation of a hollow-fiber membrane reactor for the controlled lipolysis of bntterfat. Enzyme EngineeringXII, edited by M.-D. Legoy and D. N.Thomas. Annals of the New York Academy of Science, Vol. 750, 401-407. 

HOLLOW-FIBER MEMBRANES. A hollow-fiher membrane is a capillary having an inside diameter of - inn and an outside diameter < I mm and whose wall functions as a semipermeahlc membrane. The fibers can he employed singly or grouped into a bundle which may contain tens of thousands of fibers and up to several million libers as in reverse osmosis (Fig. 11. In most eases, hollow fibers are used as cylindrical membranes that permit selective exchange of materials across (heir walls. However, they can also he used as containers to effect the controlled release of a specific material, or as reactors to chemically modify a permeate as il diffuses through a chemically activated hollow-liher wall. e g., loaded with immobilized enzyme. 

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. 

The lipase enzyme stereospecifically hydrolyzes the (+) isomer of naproxen ester. The enzyme is immobilized in the wall of an inside-skinned hollow fiber membrane. The racemic d and / naproxen ester mixture, dissolved in methyl isobutyl ketone, is introduced on the shell side of the fiber and an aqueous buffer solution is circulated through the fiber lumen. The lipase enzyme hydrolyzes the d form of naproxen ester, forming ethanol and naproxen d. Naproxen d is a carboxylic acid soluble in aqueous buffer but insoluble in methyl isobutyl ketone. Consequently naproxen d is removed from the reactor with the buffer solution. The naproxen / ester remains in the methyl isobutyl ketone solution. This technique achieves an essentially complete separation of the d and Z forms. In a clever final step... 

In a multiphase membrane reactor, the conversion of benzylpenicillin to 6-aminopenidllinic acid is performed. The type of microstructured reactor used is a fermentation reactor which contains the enzyme penicillin acylase immobilized on the wall of a hollow-fiber tube. The hollow-fiber tube extracts 6-aminopenicillinic acid at the same time selectively. Benzylpenicillin is converted at the outer wall of the hollow fiber into the desired product, which passes into the sweep stream inside the fiber where it can be purified, e.g. by ion exchange. The non-converted benzylpenicillin is recycled back through the reactor [84],... 

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... 

III (10-DAB) was carried out from which baccatin III was produced in an enzyme reactor. The enzyme reactor comprised a hollow-fiber polymeric ultrafiltration membrane, with immobilized acetyl transferase from Taxus species. The process enabled the production of baccatin III without requiring complicated purification steps of the acetyl transferase. The purification of the baccatin III is also made distinctly easier [20]. 

The first published information on the industrial application of a hybrid system with a HF contactor for production of the drug dilthiazem intermediate was reported by Lopez and Matson [23]. An enzymatic resolution of dilthiazem chiral intermediate is realized in an extractive enzymatic membrane reactor. The enzyme is entrapped in the macroporous sponge part of the hydrophilic hollow-fiber membrane made of a... 

The rapid development of biotechnology during the 1980s provided new opportunities for the application of reaction engineering principles. In biochemical systems, reactions are catalyzed by enzymes. These biocatalysts may be dispersed in an aqueous phase or in a reverse micelle, supported on a polymeric carrier, or contained within whole cells. The reactors used are most often stirred tanks, bubble columns, or hollow fibers. If the kinetics for the enzymatic process is known, then the effects of reaction conditions and mass transfer phenomena can be analyzed quite successfully using classical reactor models. Where living cells are present, the growth of the cell mass as well as the kinetics of the desired reaction must be modeled [16, 17]. 

There have been numerous studies exploring the concept of membrane reactors. Many of them, however, are related to biotechnological applications where enzymes are used as catalysts in such reactions as saccharification of celluloses and hydrolysis of proteins at relatively low temperatures. Some applications such as production of monoclonal antibodies in a hollow fiber membrane bioreactor have just begun to be commercialized. 

Immobilization of lipases on hydrophobic supports has the potential to (1) preserve, and in some cases enhance, the activity of lipases over their free counterparts (2) increase their thermal stability (3) avoid contamination of the lipase-modified product with residual activity (4) increase system productivity per unit of lipase employed and (5) permit the development of continuous processes. As the affinity of lipases for hydrophobic interfaces constitutes an essential element of the mechanism by which these enzymes act, a promising reactor configuration for the use of immobilized lipases consists of a bundle of hollow fibers made from a microporous hydrophobic polymer (137). 

Membrane technology is a well-established technology for the immobilization of enzymes [233] since Degussa [234] introduced a continuous acylase process employing an enzyme-membrane reactor for the enantiomeric production of pure L-amino acids in 1981. Polymer membranes configured into hollow-fiber modules are, by far, the most widely used membrane where the enzyme is held back by the low cutoff of the membrane. 

The continuous enzyme membrane reactor (CMR). (1) Temperature-controlled water-bath (2) Feed tanig (3) Stirrer motor for feed tank (4) Feed pump (5) Feed inlet line to the reaction vessel (6) Reaction vessel (7) Magnetic stirring table (8) Prefilter (9) Recycle pum (10) Flowmeter (11) Membrane inlet pressure gauge (12) Hollow fiber membrane cartridge (13) Membrane outlet pressure gaug (1 Pressure adjusbneut valve (15) Retentate recycle line (16) Air bath environment (17) Pemieate (product) line (18) Permeate collection vessel (19) Electronic balance... 

The substrate specificity of acylase is very broad, and a wide range of pro-teinogenic and nonproteinogenic Af-acetyl and A-chloroacetyl amino acids are transformed by the enzyme. The enzyme membrane reactor (Figure 4) is operated continuously as a recycle reactor, and the enzyme is retained by a UF hollow-fiber membrane (MWCO 10000). 

Lipases can hydrolyze triglycerides into fatty acids and glycerol. They have been used extensively to produce optically active alcohols, acids, esters, and lactones by kinetic resolution. Lipases are unique, in that they are usually used in two-phase systems. A classic example is the use of a lipase for the production of (5, / )-2,3-p-methoxyphenylglycyclic acid, an intermediate for diltiazem. In this process, methyl-/7-methoxyphenylglycidate is stereospecifically hydrolyzed by a lipase immobilized in a hollow fiber membrane reactor. The enzyme is located at the interfacial layer between an organic and an aqueous phase. 

Hollow-fiber membrane reactors with immobilized lipases have been used for the continuous hydrolysis of triglycerides188 and in the esterification of fatty acids.189 There was no deactivation of the enzyme in the former case in 16 days. In a comparable run in solution, the enzyme lost 80% of its activity in 2 days of operation. The latter case used dodecanol and decanoic acid in hexane to give the ester in 97% yield. The half-life of the immobilized enzyme was 70 days. The integration of reaction and separation can decrease product inhibition, increase selectivity, shift equilibria, and reduce the number of downstream operations.190... 

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). 

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