Hollow fiber enzyme reactors

Trends in Biotechnology, Vol. 2, No. 1, 1884

Compiled by Anaerobic Energy Systems, sents 3% of the total US energy supply, away; and, third, existing energy Inc., Bartow, Florida a figure similar to those obtained for resources remain sufficient. When any 2 Hashimoto,A. G. (1983) The United States. hydro-electric power or nuclear energy. or all of these factors change in the Proc. lllrd Int. Symp. Anaerobic Digestion, The share of biomethanation remains USA, as they have elsewhere in the Boston, in press. small and there are three main reasons world, we can anticipate a renewed 3 Department of Energy (USA) (1982) Biomass Energy Technology Programme why biomethanation has not yet taken interest in this area ofbiotechnology. Summary. Meridian Corporation/PRC off. First, there are insufficient in- References Systems Service, Falls Church, Virginia centives to invest in biomethanation; 1 Department of Energy (USA) (1980) 4 Altman, L. (1982) Biomass as an Energy second, wastes can still be thrown Anaerobic Digesters in United States. Source, Quarterly review, DOE ed. the entrapped enzyme. The product then diffuses back through the membrane to the substrate solution, from whence it is recovered. (2) Enzyme solution is ultrafdtered into the porous region of an asymmetric hollow fiber membrane or, in a simpler case, the hollow fiber is dipped into the enzyme solution so that enzyme moleHiromi Kitano and Norio Ise cules are absorbed into the spongy Recently there have been some exciting developments in techniques to lumen. Substrate is pumped into the reencapsulate enzymes into hollow fiber membranes. This entrapment actor through the same entry as preprotects enzymes from proteolytic and immunochemical attacks and viously used for the enzyme solution; makes possible industrial and medical applications of the immobilized after the reaction, the product permeates through the thin membrane enzymes. into the membrane lumen and is Various methods for immobilizing Immobilization procedures collected. enzyme molecules have been examEnzyme immobilization methods Characteristics oflmmobllized ined. Physical adsorption and chemical using a hollow fiber dialyser can be clasenzymes in hollow fiber reactors bonding of enzyme molecules to solid sifted into two categories: Since the enzyme molecule entrapsurfaces have been studied extensively, (1) The enzyme solution may be in- ped is not chemically modified, its kinbut these techniques often cause introduced to the outside of the hollow etic behavior in the hollow fiber enactivation of the enzyme and, in fiber or to the inner lumen; the zyme reactor could be expected to be extreme cases, denaturation. Encapsubstrate solution flows on the opposite the same as that of the free enzyme. sulation without chemical reactions side of the membrane to the enzyme However, with hollow fiber reactors in would seem to be the best way to avoid (Fig. 1). The substrate molecules the first category the permeation of these problems. Entrapment of enzyme permeate the membrane and react with substrate into the membrane is often molecules in hollow fibers is easy to achieve by introducing an enzyme soludution tion into the hollow fibre core and then sealing both ends. This method avoids chemical treatment, and prevents access of microbes, proteolytic enzymAnalyser es and antibodies to the enzymes and, as or a result, promotes their operational fraction stability and prolongs their activity. collector The idea of a hollow fiber enzyme reactor was first suggested by Rony in Substrate 1971 t and later he demonstrated its usesolution )llow fulness 2. Since then many researchers ,er have examined the applications of this enzyme immobilization methodL As a result of the rapid technical development by the polymer industries of hollow fiber dialysers for hemodialysis, a variety of hollow fiber dialysers for the immobilization of enzymes are now Thermostated available.

Hollow fiber enzyme reactors

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Hiromi Kitano and Norio he are at the Department of Polymer Chemistry, Fig. I. Schematic drawing of hollow fiber enzyme reactor (from Re(. 4). Kyoto University, Kyoto,Japan. © 1984, Elsevier Science Publishers B.V., Amsterdam 0166 9430/84/$02 043

Trends in Biotechnology, Vol. 2, No. 1, 198,1

the rate-determining step. Therefore, the reaction rate does not reach a maximum in the same concentration range as for free enzyme, became the actual substrate concentration in the chamber of enzyme solution is always lower than the bulk substrate concentration as a result of the enzyme reaction 4,s. Thus, the apparent Michaelis constant K~(app) is often larger than the K= of the free enzyme. For example, a bacterial alkaline phosphatase entrapped in the outside region of a cellulose hollow fiber had a K=(app) 1 700 times larger than the K= of the free enzyme s. Similarly, the temperature dependence of the catalytic rate of enzyme in the reactor is different from that of the free enzyme. The activation energy (Ea) of free urease is 8.6 kcal tool -1 and the E a of urease entrapped in a cellulose diacetate hollow fiber is 5.4 kcal mol-L E a for the urease reactor decreases with an increase in the initial concentration of the enzyme solution, supporting the view that the permeation control of this reactor increases with increase in enzyme concentration 4. In addition, if the enzyme reaction produces or consumes ionic species, the p H in the vicinity of the entrapped enzyme is different from that of the bulk substrate solution. So the p H dependence of the reactor is inevitably different from that of free ~ e system. For ~2rnple, the optimum pH of free urease is 6-7 whereas that of urease entrapped in the hollow fiber lumen is lower than 5 because of the ammonia produced 4. Applications of hollow fiber e n z y m e reactors The compatibility between hollow fiber membranes and enzyme molecules can often present serious problems, especially in the case of reactors in the second category. Unfavorable attachment of enzymes onto the membrane surface often causes a decrease in enzyme activity; in fact, polysulfon membranes are now considered unsuitable for the immobilization of enzymes for this reason. For example, the half-life of glucose isomerase soaked in the polysulfon P-10 hollow fiber dialyser is only 5 hours, whereas that of glucose isomerase soaked in the poly(vinylchloride-co-acrylonitrile) is 3 days. To solve this problem the hollow fiber membrane is pre-conditioned with an inert

protein solution such as serum albumin to increase the stability of the reactor (the half-life of glucose isomerase entrapped in the P-10 hollow fibre is increased to 3 days in this way)sT. When enzymes are entrapped in the hollow fiber lumen, the substrate molecule can approach the enzyme directly, thus avoiding the reaction controlled by permeation. On the other hand, some advantages of entrapping enzymes in an hollow fiber dialyser (protection from proteolytic and immunochemical attack) do not apply fully in this case. But another technique, chemical bonding of enzymes within the spongy interior of the hollow fiber, does incorporate these advantages. For this purpose, crosslinkingthe enzyme molecules using glutaraldehydeand albumin has proved to be more useful than other binding methods such as the B r C N activationmethod (immobilization yields of L-asparaginaseare 4.2070 and 2.6%, respectively)8. Immobilized enzymes show only a 15-20070decrease in the activityaftercontinuous reaction for 15 days. In all methods, the membrane must be sterilized with diluted formaldehyde solution before and after the entrapment to extend the half-life of the reactors. This sterilization is found to increase the half-life of entrapped alkaline phosphatase by about 100-fold (30 rain ~ 3 080 rain) s. For coenzyme-requiring enzymes, the cost of coenzymes such as NAD +, N A D H and A T P is a seriousproblem. T w o possible solutions have been examined: (1) The use of an hollow fiber membrane which retains the coenzymes in the enzyme solution by only allowing diffusion of molecules with a lower molecular weights; (2) promotion of an effective turn-over of coenzyme (more than 104-fold increase) using multienzyme systems l°. Recently some researchers have become interested in polymerized coenzymes which are attached to water-soluble polymers such as dextran and polyethyleneglycol, and which are not expected to be permeable through hollow fiber membranes 11. Although at present the cost and the catalytic activity of polymerized coenzymes are not more advantageous than those of free coenzyme, intensive research might make them so in the near future. To confer permeation selectivity on to the membrane, Lo et al. examined a liquid membrane hollow fiber enzyme

Table I. Growth of various types of cells on Amicon XM-50 hollow fiber. Types of cells

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a 37oc, growth period 21 days. b Number of final cells/number of initial cells (table simplified from Ref. 16).

reactor 12. By the impregnation of perfluorinated ether into the hollow fiber, the permeation rate of ethanol through the membrane is enormously reduced (2.8°70 of the initial permeation rate) whereas that of acetaldehyde is only reduced to 32070of the initial rate. Using this hollow fiber, Lo et al. tried to immobilize yeast alcohol dehydrogenase and found that ethanol is completely consumed by the selective permeation of the product, acetaldehyde, through the membrane. Although scaling up this reactor is somewhat difficult, it may be possible to use these reactors for the effective operation of immobilized enzymes with smaller equilibrium constant~.

Whole cells can easily be immobilized on the interior surface of an hollow fiber dialyser. Heat-treated Pseudomohas fluorescens (with L-histidine ammonia lyase activity) entrapped in an hollow fiber reactor continuously converts L-histidine into urocanic acid, which is useful as a sun-screening agent in cosmetics and pharmaceuticals 13. However, the authors point out that there is a lack of diffusion control, probably became of the relatively lower activity of the entrapped cells (permeation rate of substrate is 3 000 times larger than the reaction rate). Clinical applications of hollow fiber enzyme reactors are also possible. Pedersen et aL investigated the possibility of chemotherapy ofleukemla using entrapped L-asparaginase and L-phenylalanine ammonia lyase in an hollow fiber lumen or in the porous lining of an hollow fiber with the help of the giutaraldehyde crosslinking technique 14. By using biocompatible (anti-thrombic) hollow fiber membranes, it will surely be possible to cure some diseases enzymatically*. Hollow fiber dialysers are also useful for culturing animal cells on the surface of membranes (e.g. cellulose acetate and polysulfon) by supplying the fresh medium in the membrane lumen. Knazek et al. Is and Ku et aL 16 succeeded in

Trends in Biotechnology, VoL 2, No. 1, 1984

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fermenting various types of cells (such as choricarcinoma and SV3T3 cells) on an hoUow fiber membrane surface. By using this procedure they could culture ceils easily (Table 1) and obtain low molecular weight products such as hormones. Other researchers are trying to culture the cells of organs such as pancreatic fl-cells (islet of Langerhans) or liver cells on the surface of hollow fiber membranes, and hollow fiber reactors can be used as artificial hybrid organs by allowing blood to flow through the inner membrane lumen 17-19. By introducing such an 'artificial pancreas' into rats as an arteriovenous shunt, the concentration of plasma glucose is observed to be lowered (Fig. 2) 's. The hollow fibers (Amicon XM-50) used in these experiments had a molecular weight cut-offof 50 000 and, therefore, were permeable to insulin but impermeable to antibodies and lymphocytes,

which reject xenografted islet cells immunologically. Here we have reviewed the practical advantages and problems of hollow fiber enzyme reactors, and their applications. For theoretical treatments of the subject please consult Refs 20-25.

15 16 17 18

References 1 Rony, P. R. (1971)Bioeng. Biotechnol. 13, 431-447 2 Rony, P. R. (1972)J, Amer. Chem. Soc. 94, 8247-8248 3 Chambers, R. P., Cohen, W. and Baricos, W. (1976) in Methods in Enzymology, Vol XLIV (Mosbach, K., ed.), pp. 291-317, Academic Press 4 Kitano, H., Yoshijima, S. and Ise, N. (1980) Bioeng. Biotechnol. 22, 2643-2653 5 Davis, J. C. (1974) Bioeng. Biotechnol. 16, 1113-1122 6 Korus, R. A. and Olson, A. C. (1977)J. Food Sd. 42, 258-260 7 Korus, R. A. and Olson, A. C. (1976) in Enzyme Engineering, Vol. 3 (Pye, E. K. and Weetall, H. H., eds), pp. 543-548, Plenum Press

19 20 21 22 23 24 25

Pietta, P. G., Agnellini, D., Mazzola, G., Vecchio, G., Colombi, S. and Bianchi, G. (1976) in Enzyme Engineering, Vol. 3 (Pye, E. K. and Weetall, H. H., eds), pp. 235-237, Plenum Press Chambers, R. P., Ford, J. R., Allender, J. H., Baricos, W. H. and Cohen, W. (1974) in Enzyme Engineering, Vol. 2 (Pye, E. K. and Wingard, Jr., L. B., eds), Plenum Press, pp. 195-202 Fink, G. J. and Rodwell, V. W. (1975) Bioeng. Biotechnol. 17, 1029-1050 Mosbach, K., Larsson, P-O. and Lowe, C. (1976) in Methods in Enzymology, Vol. XLIV (Mosbach, K., ed.) Academic Press, pp. 859-887 Lo, W. K., Putcha, S., Kim, B. M., Griffith, L., Bissel, S. and Rony, P. R. (1976) in Enzyme Engineering, Vol. 3 (Pye, E. K. and Weetall, H. H., eds), pp. 19-28 Plenum Press Kan, J. K. and Shuler, M. L. (1978) Bioeng. Biotechnol. 20, 217-230 Pederson, H., Horvath, V. and Bertino, J. P. (1976) in Enzyme Engineering, Vol. 3 (Pye, E. K. and Weetall, H. H., eds) pp. 397-408, Plenum Press Knazek, R. A., Gulino, P. M., Kohler, P. O. and Kedrk, R. (1972) Science 178, 65-67 Ku, K., Kuo, M. J., Delente, J., Wildi, B. S. and Feder, J. (1981)Bioeng. Biotechno/. 23, 79-95 Chick, W. L., Like, A. A., Lauris, V. (1975) Science 187, 847-849 Chick, W. L , Perna, J. J., Lauris, V., Low, D., Galletti, P. M., Panol, G., Whittemore, A. D., Like, A. A., Colton, C. K. and Lysaght, M. J. (1977) Science 197, 780-782 Wolf, C. F. W. and Munkelt, B. E. (1975) Tram. Amer. Soc. Artif. Int. Organs 21, 16-20 Waterland, L. R., Michaelis, A. S. and Robertson, C. R. (1974)AIChE J. 20, 50-59 Lewis, W. and Middleman, S. (1974) AICheE 3:. 20, 1012-1014 Mashelkar, R. A. and Ramachandran, P. A. (1975),7. Appl. Chem. Biotechnol. 25, 867-880 Webster, I. A. and Shuler, M. L. (1978) Bioeng. Biotechnol. 20, 1541-1546 Webster, I. A. and Shuler, M. L. (1981) Bioeng. Biotechnol. 23, 447-450 Miyawaki, O., Nakamura, K. and Yano, T. (1982)J. Chem. Eng. Jpn. 15, 142-147; 224-228