WO1990006996A1 - Procede et appareil de confinement de catalyseurs dans des systemes de reactions multi-phase par membranes - Google Patents

Procede et appareil de confinement de catalyseurs dans des systemes de reactions multi-phase par membranes Download PDF

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WO1990006996A1
WO1990006996A1 PCT/US1989/005671 US8905671W WO9006996A1 WO 1990006996 A1 WO1990006996 A1 WO 1990006996A1 US 8905671 W US8905671 W US 8905671W WO 9006996 A1 WO9006996 A1 WO 9006996A1
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catalyst
membrane
asymmetric membrane
liquid
attached
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PCT/US1989/005671
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English (en)
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Jorge L. Lopez
Stephen L. Matson
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Sepracor, Inc.
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/10Hollow fibers or tubes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/16Hollow fibers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate

Definitions

  • the present invention relates to novel apparatus in which enzymes and other catalysts are confined within membranes for use as membrane reactors in multiphase reaction systems.
  • the invention also relates to a variety i * . of membranes having different solvent-wetting character and Q configurations, and to methods for charging such membranes with catalysts and for regenerating the membrane reactors once the catalysts therein confined have become inactivated through use.
  • a method and apparatus are disclosed for the Q confinement or containment of a catalyst either in an asymmetric membrane or in a composite membrane structure, which is subsequently used to conduct a chemical or biochemical reaction in which multiple phases (e.g. organic and aqueous) are involved.
  • 5 "Immobilization" on solid-phase supports of otherwise homogeneous catalysts is useful because immobilization simplifies the separation of reaction products from catalyst and it facilitates the recovery and reuse of catalyst, which frequently is too expensive for one-time use.
  • catalyst immobilization is often accomplished by covalently attaching the catalyst to the support, generally via irreversible, covalent linking chemistry.
  • Typical supports are membrane structures and partic ⁇ late media such as microporous and gel-type beads.
  • Membrane supports are attractive because membrane reactors have a number of performance advantages relative to packed- bed reactors employing catalysts bound to partic ⁇ late support media.
  • they have the significant disadvantage that " membrane supports are expensive relative to particulate media. Accordingly, the costs associated with periodically replacing membrane-supported " catalysts can be significantly higher than is the case with particulate supports.
  • a significant improvement in membrane bioreactor economics would result from the localization of catalysts in a membrane structure in such a way as to (1) provide effective containment of the catalyst in the membrane, (2) permit high effective catalyst loadings to be realized, and (3) make possible simple catalyst replacement by avoiding the covalent attachment of catalyst to the membrane surface.
  • Such a technology would significantly reduce the cost of catalyst replacement in membrane reactors. Additionally, it could have secondary benefits of avoiding the use of immobilization chemistries that can be expensive and dif icult to control and that sometimes can result in disappointing yields and/or activities of immobilized catalyst.
  • Enzymes have been immobilized in membranes (as opposed to particles) in several different fashions. They have been covalently bound or crosslinked within porous membranes (Thomas, D. , "Artificial enzyme membranes: transport, memory, and oscillatory phenomena," pp. 115-150 in Analysis and Control of Immobilized Enzyme Systems, D. Thomas and J. P. Kernevez, eds., American Elsevier, N.Y.
  • microcapsules and nearly all membrane types — porous and nonporous, electrically charged and neutral — ave been considered in connection with enzyme immobilization.
  • the present invention operates by trapping a catalyst between two catalyst-impermeable boundaries that it cannot cross under normal membrane
  • water-soluble enzymes used in two-phase and/or extractive membrane bioreactors could readily be contained in asymmetric, ultrafiltration-type membranes prepared from suitably hydrophilic polymers/"where 0 the membrane skin and the aqueous/organic phase boundary at opposite surfaces of the membrane would serve to confine the biocatalyst to the interior region of the water-wet porous membrane.
  • a two-layer composite structure consisting of a gel-type diffusion membrane (as used, for 5 example, in dialysis) atop a microporous membrane support could also be employed for catalyst containment.
  • the present invention makes possible the removal of deactivated catalyst
  • microencapsulation comes closest to the present invention, 3 *5 involving as it does a selective membrane barrier that prevents loss of catalyst from the interior; generally, selectivity is based on the size of the catalyst relative to the .diameter of the pores in the microcapsule wall.
  • microcapsules have but a single interface with the process stream (i.e., the microcapsule wall), and as a result the encapsulated catalyst is in contact with **onl a single process stream. ⁇ - in contrast, it is su*-* urpose of the present invention to immobilize catalysts in membrane structures that permit close contact of the catalyst with multiple (and often immiscible) process streams.
  • Figs. IA, IB, and 1C are schematic representations of commonly used catalyst immobilization techniques, with conventional methods for enzyme and for non-biological catalyst immobilization shown in Fig. IA and Figs. IB and 1C, respectively;
  • Figs. 2A, 2B and 2C are schematic representations of some conventional hollow fiber membrane/enzyme reactors as have been investigated for fully aqueous systems, with enzyme outside a fiber in a shell, enzyme in the porous matrix of a fiber, and enzyme in the lumen of a fiber shown in Figs. 2A, 2B and 2C, respectively;
  • Fig. 3 is a schematic representation of an illustrative embodiment of the invention in which a biocatalyst is contained within an inside-skinned, hydrophilic hollow fiber
  • Fig. 4 is a schematic representation of an illustrative embodiment of the invention in which a non- biological homogeneous catalyst is contained within an outside-skinned, hydrophobic hollow fiber
  • Fig. 5 is a schematic representation of an illustrative embodiment of the invention in which a biocatalyst is contained within a composite, hydrophilic hollow fiber.
  • FIG. 3 shows a preferred embodiment of the
  • Asymmetric membranes suitable"for the practice of this invention are chosen from the group of anisotropic ultrafiltration (UF) and microfiltration (MF) membranes. These are characterized by a more-or-less thin "skin" layer, which in the case of UF membranes is on the order of 0.1-0.2 ⁇ in thickness, supported atop a much thicker (100 200 ⁇ m) and highly porous substrate region.
  • UF anisotropic ultrafiltration
  • MF microfiltration
  • the skin of appropriate asymmetric UF-and MF-type membranes is characterized by sufficiently small pores (10's of Angstroms to perhaps 100 Angstroms in diameter) that macromolecular catalysts such as enzymes and colloidal or particulate catalysts are prevented from diffusing across to be lost to a process stream.
  • macromolecular catalysts such as enzymes and colloidal or particulate catalysts are prevented from diffusing across to be lost to a process stream.
  • the skin or surface region of the asymmetric membrane forms one catalyst- impermeable boundary.
  • the required characteristics of the "skin” layer will, of course, depend strongly on the size and other properties of the catalyst that is to be retained.
  • the pores in the highly porous substrate region underlying the i, skin" region of the membrane support can be and preferably are much larger ( ⁇ .02 ⁇ m to several ⁇ m,s in diameter) than those in th& skin. 1*
  • the dl&meter of these substrate pores is chosen with two constraints in mind: (1) the pores must be large enough to accommodate the catalyst, which, in the case of whole cells, may be several microns in diameter and (2) the pores must be sufficiently small (a few microns at most) that capillary forces within them are significant.
  • the latter consideration is important because the porous membrane substructure must be "wet” or impregnated by the "correct” liquid phase (e.g., usually the aqueous phase in the case of an enzyme-catalyzed conversion) , and hence it is important that the intrusion pressure (the pressure at which the "incorrect” fluid can be forced into the pores of the substrate) not be exceeded during operation of the reactor.
  • the intrusion pressure A P is inversely related to pore radius r r by the Young- LaPlace equation:
  • is the interfacial tension between the organic and aqueous phases and ⁇ is the contact angle between the membrane material and the liquid phase contained therein.
  • substrate pore sizes will be chosen such that the intrusion pressure is at least several psi, to ensure stable operation of the membrane contained catalyst.
  • the second catalyst-impermeable boundary that defines the catalyst-containing region is defined by the liquid-liquid interface (typically, an aqueous/organic phase boundary) that is located at the surface of the membrane furthest removed from the "skin" layer.
  • Capillarity acts to confine the desired liquid phase to the highly porous membrane matrix, and to exclude the other, immiscible liquid phase.
  • Shown in Fig. 3 is the situation wherein the catalyst is water-soluble £>r hydrophilic (i.e., preferentially wet by water) , ⁇ -andr' ⁇ he. membrane material is chosen also to be hydrophilic.
  • the aqueous/organic phase boundary will reside essentially at the.iOuter, nskinned surface of the membrane, assuming that the pressure difference across the membrane is not in the direction so as to cause ultrafiltration of aqueous solution across the membrane or not so large as to cause intrusion of organic solvent into it.
  • a water-soluble or hydrophilic catalyst will be confined to the aqueous interior of the membrane by virtue of its inability to partition into the organic solvent phase and subsequently be carried out of the reactor with it.
  • the relatively thick microporous substrate region that underlies the relatively thin skin layer of integrally skinned asymmetric membranes will often contain so-called macrovoids or "fingers” that are characterized by dimensions an order of magnitude or more larger than the diameter of the more prevalent micropores comprising the bulk of the substrate.
  • macrovoid-containing asymmetric membranes are also within the scope of the present invention.
  • the small micropores will be filled with aqueous catalyst-containing solution — retained in the micropores by capillary action — while the much larger macrovoids extending through the substrate will be filled with organic solvent.
  • Intrusion of organic solvent into the macrovoids can be made to occur by applying a small amount of positive pressure to the organic phase sufficient to overcome the relatively small intrusion pressure associated with the larger diameter macrovoids (see Equation I) .
  • the area of aqueous/organic interface can be made larger than the superficial geometric area of the outer envelope of the membrane.
  • such area of the membrane where the aqueous/organic interface is located will be referred to as one of the surfaces of the membrane.
  • Hydrophobic asymmetric membranes containing macrovoids can also be employed for the containment of organic-soluble catalyst in the microporous region of the substrate, the macrovoid being filled with aqueous solution in this case;
  • the geometry of the asymmetric membrane is largely irrelevant to the present invention, whether the membrane be in the form of flat sheets, tubes, or hollow fibers (although the latter will usually be preferred from the points of vievrof mani ⁇ facturability and cost) .
  • both inside-skinned (as shown in Fig. 3) and outside-skinned hollow fibers, sheets or tubes may be employed, and the membrane material may be either hydrophilic (i.e., water-wet) or hydrophobic (i.e., preferentially wet by organic solvents) .
  • hydrophilic i.e., water-wet
  • hydrophobic i.e., preferentially wet by organic solvents
  • the outside-skinned and hydrophobic hollow-fiber of Fig. 4 may have utility in the conduct of phase-transfer catalyzed reactions, where the catalytic species is present predominantly in the organic phase.
  • a acromolecular tail on the catalyst would assist in retaining it in the membrane matrix.
  • the macromolecular tail may be comprised of, generally speaking, a macromolecule such as a polysaccharide, a protein, water soluble polymer (with hydrophilic membrane) or other polymer (i.e. polyethelene glycol).
  • the macromolecular tails may be bonded or otherwise attached to the catalyst by standard methods,known in tl ⁇ e art such : ⁇ as . ⁇ gvalent r attachment using cyanogen bromide or glutaraldehyde.
  • conjugation of. enzymes to peptides, polysaccharides .and polyfunctional, molecules is to increase the effective size of the enzyme and thus its retention by containment in an asymmetric membrane.
  • Conjugation can be performed through a variety of techniques. One of the most used techniques relies on the use.of bifunctional reagents such , as glutaraldehyde [Broun G.B., Methods in Enzymology, 4 263 (1796)].
  • Glutaraldehyde is believed to react with lysine residues in a protein. It can also be used to bind a protein to an inert matrix possessing free amino groups or to another protein.
  • the conjugation chemistry using glutaraldehyde depends strongly on the nature of the protein being conjugated, the type of the inert matrix or protein (if is used) the concentrations of protein, glutaraldehyde, and inert matrix, and pH.
  • Cantarella et al. [Biochem J., 179, 15, (1979)] have described a method for the preparation of soluble polymers consisting of acid phosphatase conjugated with human serum albumin. The report suggests that the molecular weight of this conjugate is in excess of 10 .
  • Peptides can also be conjugated to polysaccharides.
  • Molteni ⁇ Methods in Enzymology, 112, 2 * 85 (1985) describes a method for activating dextrans with CNBr and subsequently coupling the activated dextrans,with primary »amino groups of drugs.,.
  • Axen efc al * [Biopolypers, 9, 410 (1970) describes another method to produce soluble enzyme conjugates with polysaccharides. This method describes the use of conventional CNBr activation and chemistry for coupling the activated enzyme to a solid support, followed by digestion of the solid polysaccharide support with the enzyme dextranese. Examples 10 and 11 which follow, demonstrate these polysaccharide conjugation techniques being applied to the apparatus invention described herein.
  • the present invention can be further categorized according to the nature of the catalysts and reactions involved.
  • the present invention is useful both for localizing enzymes that are dissolved in the aqueous phase, as well as those that operate at aqueous/organic- phase boundaries, such as certain of the lipases.
  • the utility of the invention is not limited to "bioconversions" such as those catalyzed by enzymes and viable or non-living 'whole cells.
  • Various catalytic reactions of synthetic organic chemistry involve multiple phases (e.g., phase-transfer catalyzed reactions), and the present invention is equally useful in these cases.
  • both soluble (typically, macromolecular) and particulate catalysts can be localized according to the method of the present invention.
  • the membrane structure of the present invention is operated in a diffusive mode, i.e., with diffusive transport of reactants into and products out of the catalytic region of the membrane; convective flow through the membrane is to be avoided.
  • reactants diffuse in on one side of the structure, and products diffuse out on the other, so that a separation and/or purification is accomplished simultaneously with the catalytic conversion.
  • the present invention is particularly useful:in the-conduct- ⁇ f catalyzed or "extractive" membrane reactors, where two process streams — one aqueous and one organic — ⁇ ⁇ ⁇ are located on opposite surfaces of the catalyst-containing membrane and serve the purpose of supplying reactant or removing product.
  • the' ⁇ -reactant may be red to the reactor via a stream of organic solution directed past/one surface ofr ⁇ the membrane of the present invention, while the water-soluble product may be withdrawn from the opposite surface of the membrane via a second aqueous process stream.
  • a water-soluble reactant may be supplied via an aqueous stream directed past one surface of the membrane of the present invention while the product is made to partition and is thereby removed into a stream of organic solvent flowing past the other surface of the membrane.
  • aqueous enzyme solution is charged to the shell (or outer) side of the hollow-fiber module and passed though the fiber wall in an ultrafiltration process under a modest pressure difference (i.e., a pressure insufficient to cause disruption or loss of integrity of the skin under "back-flush” conditions) .
  • aqueous enzyme solution is displaced from the shell side of the fiber bundle by flushing it with an immiscible fluid such as air or the organic process solvent. If air or another gas is used in this step, the shell is filled with the organic solvent in a subsequent step.
  • the module is then operated with the organic solvent on. the shell side and an aqueous solution in the lumen of the fiber ⁇ with a slight.excess pressure on the shell side. This pressure difference is too smalil- to1cause intrusioftn of the org - ⁇ .ani*c p**hi Calse into. - therac substrate region of the fiber on the qne hand and is in the wrong direction to cause ultrafiltration of aqueous solution on the other hand.
  • a composite membrane structure is employed in place of the asymmetric, integrally skinned membranes contemplated above.
  • this composite consists of a thin, permselective surface layer of one material supported on a highly porous and much thicker nonselective substrate membrane, generally fashioned from a different material.
  • Techniques for the fabrication of multilayer composite, laminated and coated membrane structures are well known in the art and are the subject of published review articles. Matson, S. L. , Lopez, J. and J. A. Quinn, Chem. Enq. Scl., 38, 503 (1983); Lonsdale, H. K., J. Memb. Sci. , 10, 81 (1982).
  • composite membrane structures suitable for the localization of various biocatalysts might be manufactured based on the use of thin surface coatings of regenerated cellulose dialysis-type membrane supported on microporous membranes, particularly microporous hollow fibers.
  • a suitably microporous layer might be deposited upon or within a regenerated cellulose hollow fiber.
  • Hydrophilic polyacrylonitrile-based copolymer membranes also appear to be well suited to construction of such types of composite membrane structures. ;
  • An enzyme solution was prepared by dissolving 50 grams of Candida lipase (Mol. Wt. 100,000; Sigma Chemical 5 Co. Cat # L 1754) in 1.25 liters of water and then filtering this solution to remove the insoluble material.
  • This inter-facially acting enzyme is known to hydrolyze a large number of organic esters, among them, phenoxyacetate methyl ester and amyl acetate.
  • the enzyme was loaded into a i 2 c*ustom-made solvent-resistant membrane module fabricated with aniso- tropic polyacrylonitrile (PAN) hollow fibers taken from a PAN-200 hemofilter (ASAHI Medical Co.).
  • PAN aniso- tropic polyacrylonitrile
  • ASAHI Medical Co. ASAHI Medical Co.
  • the enzyme solution was recirculated from the shell side to the lumen side and back to the solution reservoir in an ultrafiltration mode. 0 Throughout the loading process the pressure difference between the shell and lumen compartments was kept to 8 psi by adjusting the ultrafiltration rate (generally between 200 to 20 ml/min) . The procedure was completed in one hour. The initial and final enzyme solution activities are shown 5 below in Table 1.
  • the reaction progress and rate were monitored by following the caustic consumption and observing the organic phase reservoir level.
  • the rate of ester hydrolysis was 3000 ⁇ moles/min and at the end it was 1500 ⁇ moles/min.
  • the phenoxyacetic acid product in the aqueous reservoirs was subsequently recovered by acidifying to a pH of 1.0 with concentrated HC1 and filtering the precipitated solids. After drying, the solids were assayed titrimetrically and found to be 96.3 % acid. A sample of the dried solids was dissolved in a mixture of chloroform and water with a subsequent drying/evaporation of the chloroform phase.
  • the rate of amyl acetate hydrolysis in the reactor was measured in exactly the same manner described above with the exception that 25 mM NaOH was used.
  • the reaction rate was 6.8 ⁇ moles per minute which corresponds to 3% of the initial activity.
  • Example 2 At the conclusion of the membrane regeneration procedure described in Example 2, the module was charged with 20 grams of Candida lipase (the same type and in the same concentration described in Example 1) . Amyl acetate was used as a substrate and the reactor operated in exactly the same manner as described in Example 2. The rate of reaction was measured at 70 ⁇ moles/min.
  • An enzyme ' solution was prepared by dissolving ⁇ 10.8 ml of a pig liver estera ⁇ e preparation (Mol. Wt.
  • the enzyme was loaded into the same membrane module described in Example 1 by recirculating the enzyme solution from the shell side to the lumen side and back to 5 the solution reservoir in an ultrafiltration mode. Throughout the loading process the pressure difference between the shell and lumen compartments was kept to 9.5 psi by adjusting the ultrafiltration rate (generally between 200 to 20 ml/min) . The'procedure was completed in one hour. 0 The initial and final enzyme solution activities are shown below in Table 2.
  • the enzyme was removed from the reactor through the following procedure: - backflushed with 6 liters of distilled water entering on the lumen side and exiting on the shell side at a flow rate of 50 ml/min
  • the rate of ethyl butyrate hydrolysis in the reactor was measured in exactly the same manner described above with the exception that 25 M NaOH was used.
  • the reaction rate was 30 ⁇ moles per minute corresponding to 0.3% of the initial activity in the module.
  • alpha-Chymotrypsin (Mol. Wt. 23,000, Sigma Chemical Co. Cat # C 4129) was prepared by dissolving 0.5 grams of the enzyme in I liter of 0.1 M K.HPO./l.O M NaCl pH 7.8. This solution was recirculated on the shell side of a 1 m 2 ASAHI PAN-150 hemofilter (ASAHI Medical Co.) at a flowrate of 50 ml/min for 1 hour. Because there " was no flow of enzyme solution from the shell to the lumen side, the enzyme was loaded into the membrane solely in a diffusive mode.
  • the activity of the module was calculated by measuring the amount of BT acid that was present in the aqueous effluent from the reactor. The module activity was determined to be 80 ⁇ moles/min. The membrane module was then drained of solvent and buffer and backflushed with 10 liters of 0.1 M phosphate buffer. The membrane module activity was measured in the same manner described above with the exception that the BTEE solution was pumped into the reactor at a rate of 53 ml/min. The activity of the membrane module was 4 ⁇ mole/min corresponding to 5% of the initial activity.
  • An enzyme solution was prepared by dissolving 100 mg of the same alpha-Chymotrypsin used in Example 4 in 500 l of 0.1 M phosphate buffer pH 7.0.
  • the enzyme was loaded on a 1 m 2 ASAH1 PAN _ 150 hemofilter identical to the one used in Example 5 by recirculating the enzyme solution from the shell side to the lumen side and back to the solution reservoir in an ultra-filtration mode. The procedure was completed in 2.5 hours.
  • recirculation of 1 liter of 10 mM BTEE in amyl acetate was started on the shell side.
  • the recirculation rate was 10 ml/min and the average pressure on the shell compartment was kept at 6.5 psi by adjusting a throttling valve at the shell side exit.
  • 200 ml of 2 mM phosphate buffer pH 7.0 was recirculated at a rate of 250 ml/min.
  • the pH of the aqueous reservoir was maintained at"7.0 by addition of 1 M NaOH.
  • the initial rate of BTEE hydrolysis was determined to be 45 ⁇ moles/min.
  • the molecular weight of the enzyme was increased by crosslinking with Bovine Serum Albumin (Sigma Chemical Co., Cat # A 4503) using glutaraldehyde and following conventional protocols for such chemistry ' .
  • the measured activity of this solution was 101 5 moles/min-ml per standard BTEE assay.
  • the enzyme was loaded on a 1-m AHAHI PAN-150 hemofilter identical to the one used in Example 4 by ultrafiltering the solution once from the shell side to the lumen side at a flow rate of 20 ml/min. After loading the enzyme to the reactor, 0 recirculation of 500 ml of 40 mM N-benzoyl-L-tyrosine ethyl ester (BTEE) in octanol (Aldrich Co.) was started on the shell side.
  • BTEE N-benzoyl-L-tyrosine ethyl ester
  • the recirculation rate was 500 ml/min and the average pressure on the shell compartment was kept at 6.5 psig by adjusting a throttling valve at the shell side exit. 5
  • 1 liter of 0.1 M K-HPOl MNaCl buffer pH 7.0 was recirculated at a rate of 500 ml/min.
  • the pH of the aqueous reservoir was maintained at 7.0 by addition of 1 M NaOH.
  • the initial rate of BTEE hydrolysis as catalyzed by the membrane-contained enzyme conjugate was determined to be 42,000 ⁇ moles/hr. 5 EXAMPLE 8
  • the proteolytic enzyme derived from Aspergillus oryzae and obtained commercially from Amano Enzymes Inc. (tradename SEAPROSE) has a molecular weight of 18-30,000 0 Daltons. This enzyme is not retained as effectively as desired by the polyacrylonitrile membrane in hemofilters manufactured by ASAHI Inc., which are rated at a molecular weight cutoff of 50,000 Daltons. This enzyme has been demonstrated to hydrolyze the sulfomethyl ester of R(-)
  • the enzyme conjugate was prepared by making 4 liters of a solution containing 2 mg/ml of Seaprose enzyme, 1 mg/ml of polyethyleneimine (MW 70,000; Aceto 0 Corp., Flushing NY), and 0.005 M CaCl 2 .
  • the pH of the solution was adjusted to pH 7.00 with concentrated HC1.
  • the crosslinking was initiated by adding 16 g of a 25 weight % glutaraldehyde solution. After 40 minutes the pH of the solution was readjusted to pH 7.00 by adding 10% NH40H, 5 followed by addition of 35 g of alanine.
  • the enzyme conjugate was loaded onto a PAN-200 ASAHI hemofilter (ASAHI Medical Co.).
  • This membrane is an asymmetric, hydrophilic, inside-skinned hollow fiber characterized by 90 % rejection of proteins with a molecular weight higher than 50,000.
  • Aspergillus oryzae proteases obtained from Amano Enzymes Inc. were crosslinked for use in the enzyme membrane reactor of this invention using dimethyl adipimidate obtained from the Aldrich Chemical Company.
  • dimethyl adipimidate obtained from the Aldrich Chemical Company.
  • Solutions of_dimethyl adipimidate. were "t-h ., prepared as follows [D.R, Dodd ⁇ , J.B. Jones, "Enzymes in Organic Synthesis 17.", Can. J. Chem., 57,2533 (1977)].
  • adipimidate solution 8 mmols of the adipimidate were dissolved, in approximately 2 mis of water and the pH adjusted to 8.0 by the addition of concentrated sodium hydroxide solution. The final volume was adjusted to 4 is, giving a final concentration of 2 M adipimidate. 10011 aliquots of the dimethyl adipimidate solution were added to one of the Prozyme 6 solutions and one of the Prozyme 10"' solutions every 15 minutes for 1 hour (four additions) , a fresh adipimidate solution being prepared for each addition.
  • Dextran dialdehyde was prepared by periodic acid oxidation [following a method described in Organic Reactions, Vol 2., p 363]. The molecular weight of the Dextran was 5000, and it was obtained from Sigma Chemical co. Dextran dialdehyde was collected and air dried. Two grams (2.0 g) of the prepared dextran dialdehyde were then dissolved in 100 ml of 0.05 M phosphate buffer, pH 6.5 by warming at 50 ⁇ C for 18 hours. Prozyme 10TM (1.0 g) was dissolved in the dextran dialdehyde solution at room temperature. After the addition of enzyme, 0.4 of sodium cyanoborohydride was added and the solution was stirred for 6 hours.
  • the activity of the solution was measured and found to be 141 U/ml (1 U is the amount of enzyme required to hydrolysis l ⁇ mole of substrate per hour) . In the absence of dextran crosslinking the activity is 185 U/ml, thus demonstrating maintenance of enzyme activity during this chemical procedure.
  • the crosslinked enzyme was then ultrafiltered through a PAN membrane characterized by 95 % rejection of molecules with a molecular weight higher than 50,000 Daltons [the native enzyme has a molecular weight of 18-23,000 Daltons] .
  • the filtrate was assayed for activity.
  • the percent of rejected enzyme was 70 %. In the absence of crosslinking, the amount of rejected enzyme is below 50%, thus demonstrating increased molecular weight and retention.
  • Calcium alginate beads were prepared by the dropwise addition of a 2 % (w/w) sodium alginate solution in water into 350 ml of 0.1 M CaC12. The-beads were subsequently activated with cyanogen bromide by adding 50 ml of a 40 mg/ml CNBr solution in water. The pH was maintained at 11 during the activation by the addition of concentrated NaOH. After consumption of NaOH stopped, the beads were washed with water and subsequently filtered and isolated. Cytochrome C (Sigma Chemical Co., 50 mg) was dissolved in water (50 ml) . The alginate beads were added to the bright red Cytochrome C solution.

Abstract

Dans l'appareil décrit, qui sert à produire une catalyse dans des systèmes de réactions multi-phase, des catalyseurs sont confinés à l'intérieur de plusieurs membranes sans utilisation de couplage covalent. Le confinement des catalyseurs est obtenu grâce à la combinaison d'une pellicule micro-poreuse, placée sur une face de la membrane, et d'un solvant placé sur l'autre face de la membrane et dans lequel les catalyseurs ne sont pas solubles de façon notable. Des procédés sont également prévus pour la préparation d'un tel appareil et pour la régénération des membranes par incorporation d'un nouveau catalyseur.
PCT/US1989/005671 1988-12-19 1989-12-18 Procede et appareil de confinement de catalyseurs dans des systemes de reactions multi-phase par membranes WO1990006996A1 (fr)

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