Membrane reactors immobilized enzyme

In the past many attempts have been done to implement membranes into immobilized enzyme reactors, especially within inunobilized enzymes for food processing (Pitcher,... 

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 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... 

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. 

On the other hand, in nature a continuous uptake of substrate and release of product without loss of biocatalysts is not achieved by carrier fixation but by means of cellular membranes. Efficient immobilized enzyme reactor systems for technical applications can therefore be established replacing cellular membranes by ultrafiltration or reverse osmosis synthetic membranes, and the activated transport through the cellular wall by a forced flow across the membrane.7... 

Enzymatic bioreactor Immobilized microfluidic enzyme reactor (IMER) Immobilized enzyme reactor Immobilized enzyme biosensor Membrane reactor... 

In most cases, hoUow fibers are used as cylindrical membranes that permit selective exchange of materials across their waUs. However, they can also be used as containers to effect the controUed release of a specific material (2), or as reactors to chemically modify a permeate as it diffuses through a chemically activated hoUow-fiber waU, eg, loaded with immobilized enzyme (see Enzyme applications). 

Membrane reactors, where the enzyme is adsorbed or kept in solution on one side of an ultrafHtration membrane, provides a form of immobilized enzyme and the possibiHty of product separation. 

The most effective of these include immobilization [80], lipid coating [81], surfactant coating [82], use of cross-linked enzyme crystals [83], cross-linked enzyme aggregates [84], and membrane reactors [85]. 

Concerning function integration, for example, micro-flow membrane reactors can exhibit similar process intensification, as shown already for their large-scale counterparts [75]. Separation columns for proteomics, immobilizing enzymes, utilize the large surface-to-volume ratios. Surface tension differences can guide and transport liquids selectively. 

Different enzymes - particularly lipases - immobilized in membrane reactors have been studied in the presence of two-liquid phases (Table 5). Organic and aqueous phases containing respectively hydrophobic and hydrophilic reactants are separated by a solid mem-... 

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. 

Biological catalysts in the form of enzymes, cells, organelles, or synzymes that are tethered to a fixed bed, polymer, or other insoluble carrier or entrapped by a semi-impermeable membrane . Immobilization often confers added stability, permits reuse of the biocatalyst, and allows the development of flow reactors. The mode of immobilization may produce distinct populations of biocatalyst, each exhibiting different activities within the same sample. The study of immobilized enzymes can also provide insights into the chemical basis of enzyme latency, a well-known phenomenon characterized by the limited availability of active enzyme as a consequence of immobilization and/or encapsulization. 

Immobilized biocatalysts are enzymes, cells or organelles (or combinations of these) which are in a state that permits their rense (The Working Party of Immobilized Biocatalysts, 1983). Examples are insolnble enzymes, e.g. nsed in a fixed bed reactor or soluble enzymes, e.g. used in a semipermeable membrane reactor. This chapter will describe methods of industrial interest for making biocatalysts insoluble. 

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. 

Reaction engineering helps in characterization and application of chemical and biological catalysts. Both types of catalyst can be retained in membrane reactors, resulting in a significant reduction of the product-specific catalyst consumption. The application of membrane reactors allows the use of non-immobilized biocatalysts with high volumetric productivities. Biocatalysts can also be immobilized in the aqueous phase of an aqueous-organic two-phase system. Here the choice of the enzyme-solvent combination and the process parameters are crucial for a successful application. 

Membrane reactors can be considered passive or active according to whether the membrane plays the role of a simple physical barrier that retains the free enzyme molecules solubilized in the aqueous phase, or it acts as an immobilization matrix binding physically or chemically the enzyme molecules. Polymer- and ceramic-based micro- and ultrafiltration membranes are used, and particular attention has to be paid to the chemical compatibility between the solvent and the polymeric membranes. Careful, fine control of the transmembrane pressure during operation is also required in order to avoid phase breakthrough, a task that may sometimes prove difficult to perform, particularly when surface active materials are present or formed during biotransformahon. Sihcone-based dense-phase membranes have also been evaluated in whole-cell processes [55, 56], but... 

Enzymes, when immobilized in spherical particles or in films made from various polymers and porous materials, are referred to as immobUized enzymes. Enzymes can be immobilized by covalent bonding, electrostatic interaction, crosslinking of the enzymes, and entrapment in a polymer network, among other techniques. In the case of batch reactors, the particles or films of immobilized enzymes can be reused after having been separated from the solution after reaction by physical means, such as sedimentation, centrifugation, and filtration. Immobilized enzymes can also be used in continuous fixed-bed reactors, fluidized reactors, and membrane reactors. 

Initial preparative work with oxynitrilases in neutral aqueous solution [517, 518] was hampered by the fact that under these reaction conditions the enzymatic addition has to compete with a spontaneous chemical reaction which limits enantioselectivity. Major improvements in optical purity of cyanohydrins were achieved by conducting the addition under acidic conditions to suppress the uncatalyzed side reaction [519], or by switching to a water immiscible organic solvent as the reaction medium [520], preferably diisopropyl ether. For the latter case, the enzymes are readily immobilized by physical adsorption onto cellulose. A continuous process has been developed for chiral cyanohydrin synthesis using an enzyme membrane reactor [61]. Acetone cyanhydrin can replace the highly toxic hydrocyanic acid as the cyanide source [521], Inexpensive defatted almond meal has been found to be a convenient substitute for the purified (R)-oxynitrilase without sacrificing enantioselectivity [522-524], Similarly, lyophilized and powered Sorghum bicolor shoots have been successfully tested as an alternative source for the purified (S)-oxynitrilase [525],... 

Immobilized enzyme and enzyme membrane reactors each have advantages and disadvantages (Table 5.1). 

In the preceding section, we analyzed an immobilized enzyme process and calculated some important parameters such as productivity. In this section, we investigate another process configuration for retaining biocatalysts, the membrane reactor. The advantages and disadvantages of immobilization and membrane retention have already been discussed in Chapter 5. As in the case of immobilization, retention of catalyst by a membrane vastly improves biocatalyst productivity, a feature important on a processing scale but usually not on a laboratory scale. 

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. 

A third type of membrane reactor combines the functions of contactor and separator. An example of this combination membrane reactor is shown in Figure 13.16(c), in which the membrane is a multilayer composite. The layer facing the organic feed stream is an immobilized organic liquid membrane the layer facing the aqueous product solution contains an enzyme catalyst for the deesterification reaction... 

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