Immobilization of biocatalysts with poly(vinyl alcohol) supports

Journal of Biotechnology, 14 (1990) 169-178

169

Elsevier BIOTEC 00511

Immobilization of biocatalysts with poly(vinyl alcohol) supports H i s a o Ichijo, Jun'ichi N a g a s a w a a n d Aizo Y a m a u c h i Research Institute for Polymer5 and Textiles, Ibaraki, Japan

(Accepted 4 December 1989)

Summa~ Two polymer materials, poly(vinyl alcohol) (PVA) superfine fibers and photocrosslinkable PVA bearing styrylpyridinium groups, have been developed to immobilize biocatalysts. The former has a large surface consisting of relatively large-size pores and the fibers can immobilize a large amount of biocatalyst on their surface by ionic interaction. The latter entraps many kinds of biocatalysts by cyclodimerization caused by visible light irradiation. The biocatalysts o n / i n these supports maintain high activity and thermal stability. These materials can easily be formed into various shapes suitable for various applications. A new bioreactor system was constructed for evaluating a variety of biocatalysts and supports. Poly(vinyl alcohol), PVA; Superfine fiber; Photocrosslinkable; Thermostability; DSC; Bioreactor

Introduction A large number of methods and materials for immobilizing biocatalysts have been proposed and tested. However, little attention has been paid to fibrous supports. Dinelli et al. (1976) immobilized various biocatalysts in the pores of fibers by spinning an emulsion consisting of fiber-forming polymers and biocatalysts. Several enzymes were enCorrespondence to: H. Ichijo, Research Institute for Polymers and Textiles, 1-1-4 Higashi, Tsukuba,

Ibaraki 305, Japan. 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

170

trapped in the fibers formed by spinning a mixture of sodium alginate and biocatalysts (Kobayashi et al., 1987). Biocatalysts in intact form were confined in one of the spaces separated by hollow fiber membranes without any change in the properties of the biocatalysts (Engasser et al., 1980). 6-Aminopenicillanic acid has been produced on an industrial scale with the penicillin acylase covalently bound on reduced polyacrylonitrile fibers with glutaraldehyde (Matsumoto, 1984). Photoinduced gelation has also been attracting interest for entrapping bioactive materials including enzymes, organelles and microbial cells, because the photochemical technique possesses merits due to the milder network formation of the polymeric matrix without changes in pH and temperature. An enzyme was immobilized in acrylamide gel by photopolymerization to avoid thermal deactivation (Hicks and Updike. 1966). Fukui et al. have published many reports on the entrapment of various bi~xzatalysts with photocrosslinkable resin prepolymer by near ultraviolet irradiation (Fukui and Tanaka, 1984; Fukui et al., 1987). Since many kinds of functional groups are easily incorporated into poly(vinyl alcohol) (PVA) by acetalization a n d / o r acylation, the polymer can be modified in various ways. So far, much research on fibers and photosensitive polymers derived from PVA has been done in our institute. In this paper, we review the characteristics and applications of both PVA superfine fibers (SFF) and photocrosslinkable PVA bearing styrylpyridinium (SbQ: stilbazole quaternized) groups as supports. This work was carried out in collaboration with Drs. T. Suehiro, H. Uedaira, K. lchimura and N. Aisaka in our institute.

Immobilization of biocatalysts with PVA supports Immobilization of biocatalysts on S F F Preparation of S F F The completely saponified PVA solution is mixed with partially saponified PVA or poly(ethylene oxide). When the mixture is extruded, the jets of the spinning solution are dried with a stream of hot air. The PVA fiber is washed with water at room temperature, then partially saponified PVA or poly(ethylene oxide) dissolves in water and only completely saponified PVA exists in a fiber shape. The fiber is hence split into several tens to some hundreds of extremely fine filaments. Functional groups, such as amino or sulfo groups, are then incorporated into the fine fibers. Fig. 1 shows the pore size distribution curves of three supports determined by the methanol adsorption method. The surface area of SFF is larger than 200 m e g ~ and is as large as 10-20% of that of activated charcoal. Besides, relatively large-size pores form a large proportion of the pore size distribution of SFF. Immobilization of enzymes and yeast on S F F SFF immobilize large amounts of invertase (over 800 mg enzyme per g SFF) (Ichijo et al., 1982), while the proteinbinding capacity of other adsorbents is much smaller than that (Goldstein and

171

A 'c~ 100C ~"

800

~.~

~

600

o~

400

(b)

200

0

1

2

3

Pore radius

4

5

6

(nm)

Fig. 1. Pore size distribution: (a) superfine fiber (217 rn2 g - l ) , (b) activated carbon (1400 m 2 g - l ) . (c) ion exchange resin (410 m 2 g - i ) .

Manecke, 1976). SFF are hence found to be an excellent support for immobilizing large molecules such as enzymes. Acidic enzymes (invertase, glucose oxidase, fl-galactosidase from Escherichia coil) combine with anion exchangers such as dimethylaminated SFF and trimethylaminated SFF, whereas basic enzymes (lipase, fl-galactosidase from jack bean, fl-glucosidase) combine with acidic forms of SFF (Ichijo et al., 1985a, 1986a). The amount of invertase immobilized on aminated SFF increases with the increase in the amino groups on SFF. Baker's yeast is well immobilized on dimethylaminated and trimethylaminated SFF (see Fig. 2) (lchijo et al., 1987), but not on sulfonated SFF. These results suggest that SFF bind biocatalysts by electrostatic interaction. Therefore, SFF are easily regenerated after immobilized biocatalysts lose their activity. The adsorption rate of invertase onto dimethylaminated SFF is very fast. At initial enzyme concentrations below 1 mg m1-1, the observed rates agreed well with the results calculated from simple equations based on the Langmuir adsorption mechanism (lchijo, 1983). It was found that the enzymic reaction by the invertase both in solution and in the immobilized state on SFF followed Michaelis-Menten reaction kinetics.

Immobilization of biocatalysts in PVA-SbQ Properties of PVA-SbQ The SbQ group is incorporated into PVA by acetalization (Ichimura, 1982; Ichimura and Watanabe, 1982). The mixture of a biocatalyst and water-soluble PVA-SbQ is insolubilized on irradiation with visible light by crosslinking through the cyclodimerization of the SbQ. This reaction occurs specifically without free radicals which may attack biocatalysts. Various enzymes or microbial cells are completely and efficiently entrapped in the network of the polymer when the content of the photosensitive group is more than about 1.0 tool% attached to

172

O Fig. 2. Scanning electron micrographs of yeast ceils immobilized on aminated SFF before (a) and after proliferation (b). PVA of P = 1700 (Ichimura, 1984). The size of the network can be controlled by the SbQ content and molecular weight of PVA. The gel can be easily formed into various shapes such as membrane, particle, fiber, coating of support, etc. and has excellent mechanical strength so that PVA-SbQ is used as a printing plate. PVA-SbQ can be modified to be more hydrophobic or more ionic by introducing functional groups at the hydroxyl groups of PVA.

lrnmobilization of hiocatalysts in PVA-ShQ

A suspension of baker's yeast in a PVA-SbQ solution was coated on the inner surface of a tube. After a solution of glucose (18 wt%) and some minerals was circulated through the tube at 30 ° C for 8 d, ethanol was produced at 3 wt% and the yeast proliferated well (Suehiro et al., 1987). Lipase was entrapped in water-containing gel particles by dispersing the solution of lipase and PVA-SbQ in fine droplets into an adequate water-immiscible organic solvent like silicon oil with vigorous stirring under irradiation (Suehiro et al., 1988b). This method produced particles of 20-100 ~tm diameter containing 60-85% water. The immobilized lipase hydrolyzed fat in hydrophobic solvents by consumption of water in gel particles, and the relative activity was approx. 10% of that in emulsions of native lipase. It also catalyzed the interesterification of a triglyceride and a fatty acid in organic solvents (see Table 1; Suehiro et al., 1988a). It was found that only a small amount of water contained in the gel was enough to maintain the

173 TABLE 1 Interesterification by immobilized lipase a Relative ratio of triglycerides (%)

Reaction time (h)

O-O-O

P-O-O

P-O-P

P-P-P

0 8 24 48

100 64.8 37.3 22.0

0 25.8 38.4 44.4

0 7.4 24.3 33.6

0 ND ND ND

Rhizopus delemar was entrapped with a 9.0% solution of PVA-SbQ (3.5 mol% of SbQ attached to 88% partially saponified PVA of P = 500) in silicon oil. The particles immobilizing lipase (5.1% enzyme per dry weight), which had been dried to contain 4.5% of water, were mixed with triolein, palmitic acid, and hexane (weight ratio 8 : 7 : 15). The mixture was stirred at 37 o C and triglycerides were analyzed by HPLC. O = oleoyl; P = palrnitoyl, ND = not detected. enzyme activity even in organic solvents. This reaction can be used to reform edible fat. Entrapping methods to immobilize enzymes generally have the disadvantage of lowering the enzymatic activity due to the diffusion limitation of substrate molecules. There are two methods to increase the relative activity of entrapped enzymes by increasing the surface area of the gel matrix. One method is mixing poly(ethylene glycol) with P V A - S b Q and then removing the poly(ethylene glycol) by washing after photocrosslinking (Ichimura, 1987). This gives a porous gel matrix, and the relative activity of invertase entrapped in such a m e m b r a n e is higher than of that entrapped without poly(ethylene glycol). The other method is spraying and air-drying under irradiation an enzyme dissolved in P V A - S b Q solution to give very small particles (0.1-10 ~ m diameter) immobilizing the enzyme (Suehiro and Ichimura, 1988). The relative activity of the enzyme entrapped in these particles is very high. The hydrophilicity of PVA can be controlled by substitution of h y d r o p h o b i c groups such as acetyl. Nakajima et al. (1986) reported the effect of hydrophilicity of the polymer matrix on the production of pigments by Laoandula oera cells entrapped with PVA-SbQ. Furthermore, P V A - S b Q is applied to membranes for biosensors ( M a t s u m o t o et al., 1984; Mizutani et al., 1985; Miyahara et al., 1985; Mizutani and Asai, 1988).

Thermal stability of immobilized enzymes

Comparison of enzyme stability measured by calorimetry and by activity assay Free and immobilized enzymes were heated from 25 ° C to various temperatures at a constant heating rate. The thermal stability of enzymes with or without heat treatment was studied by activity assay and calorimetry. When the free enzyme was heated to over 6 0 ° C , enzyme activity decreased rapidly with the increase in temperature. Immobilized enzyme heated to 75 ° C and

174

100 JE C

50

._>

rr

4ZO

5=0

610

Temperature

708'o-g~0 ` 9O

(*C)

Fig. 3. Thermal stability of free and immobilized invertase. Relative activity: free (~), immobilized (zx). Relative denaturation enthalpy: free (e), immobilized(A).

77 ° C maintained 50% and 25% of its original activity, respectively. These data show that invertase immobilized on SFF is more thermostable than in solution. Furthermore, the results of thermal analysis agree well with those of the activity assay (see Fig. 3) (Ichijo et al., 1985b, 1989). Glucose oxidase bound on SFF is also more thermostable than the free form. For glucose oxidase too, the results of thermal analysis agree well with those of the activity assay (Ichijo et al., 1989).

Effect of enzyme loading and pH on thermal stabifiO, of enzymes A marked increase in denaturation temperature (Td) was observed with increasing concentrations of free invertase. The TO values of invertase and glucose oxidase bound on SFF also increased with increasing amounts of enzyme loading (Uedaira et al., 1984, 1988). However, urease entrapped in PVA-SbQ was not stabilized by the increase in enzyme loading. The thermal stability of invertase bound on SFF depends more on enzyme loading than on pH. On the other hand, the thermostability of glucose oxidase immobilized on SFF is influenced by p H much more than by enzyme loading. The Tj values of all the samples tested (invertase and glucose oxidase in solution, invertase and glucose oxidase bound on SFF, invertase and urease entrapped in PVA-SbQ gel) increased when the pH approached their isoelectric points (Uedaira et al., 1984, 1988; Ichijo et al., 1989). The differential scanning calorimetry (DSC) curves for invertase in solution and that immobilized on SFF at the same p H show that the immobilized enzyme is more thermostable than the free form. The other enzymes immobilized on SFF or in PVA-SbQ are also more thermostable than those in solution.

175 Formation of a fiber assembly into an appropriate shape Filter paper composed of short cut S F F (SFP) A filter paper was made of short cut SFF and was inserted into a membrane holder to construct a smallsize bioreactor (lchijo et al., 1986b). A substrate solution was passed through SFP. The glucose produced per unit of time (productivity) increased with the flow rate. The productivity of small amounts of enzyme immobilized on SFP approached a constant value at lower flow rates. It is expected that productivity would increase further with increasing amounts of immobilized enzyme. The space velocity of the reactor with SFP is estimated to be about 36 h - 1 where the conversion is almost 100%. Even at a high space velocity of 1000 h conversion is as high as 40%. More bound enzyme and a higher flow rate would make the reactor more effective. Knitted S F F Although SFP showed a high performance, the hydrodynamic resistance of the SFP reactor increased when a viscous solution was supplied to it for a long period. A reactor with knitted SFF fabric was therefore designed to pass a substrate solution parallel to the immobilization support (Ichijo et al., 1985c). No reduction in flow rate or significant decrease in conversion was observed. The half life was estimated to be around 60-100 d. The efficiency of the reaction increased with the decrease in thickness of the reactor, suggesting that the increase in linear velocity facilitated the diffusion of the substrate and the contact between substrate and bound enzyme. Development o.f a new bioreactor system with a braided S F F module The support module to be used in bench plants should be free from pressure drop increases. A new SFF module was developed by making use of various technologies employed in conventional chemical and textile industries. The braid made of SFF threads was spirally wound around a perforated tube in such a way that the braid bed had enough vacant space for liquids to pass through (Suehiro et al., 1988c). We developed a new bioreactor system consisting of a reactor, two p u m p s for feeding and circulation, a temperature controller, p H sensors, a pressure transducer, a degaser, a flow meter, etc. (see Fig. 4). This system was constructed to evaluate a variety of support materials and to collect data in various operational modes. Alcohol production by yeast and sucrose hydrolysis by invertase were conducted using the braided SFF module and the system described above. In spite of the proliferation of yeast cells, no increase in hydrodynamic resistance was observed. When a honey solution was fed to the system at 40 ml m i n - t and circulated through the reactor containing braided SFF at 2 1 min-1, an ethanol concentration in the effluent of about 6% was maintained. In the case of sucrose hydrolysis, conversion was kept almost constant for 20 repeats. The module is free of pressure drop increases and the biocatalysts immobilized on the module keep their original activity. The system is easily operated and can evaluate various biocatalysts and supports.

176

b

b

o90

fi

Fig. 4. New bioreactor system: (a) reactor, (b) pump, (c) controller and recorder unit. (d) pressure gauge, (e) heat exchanger. (f) reservoir.

Conclusion

PVA superfine fibers and photocrosslinkable PVA have been developed as immobilization supports with excellent characteristics. Various biocatalysts immobilized o n / i n those supports showed high activities and reactive efficiencies in both continuous and repeated enzymic reactions. Several enzymes became more thermostable by the immobilization. The stability measured by activity assay agreed well with that by thermal analysis. These supports can be easily formed into various shapes such as particle, film, filter paper, knitted fabric, braid, etc. In new bioreac-

177 tor systems with braided SFF, the immobilized biocatalysts kept their original a c t i v i t y a n d t h e m o d u l e s w e r e free o f p r e s s u r e d r o p i n c r e a s e s in s p i t e o f t h e proliferation of yeast. It is c o n c l u d e d t h a t t h e s e P V A s u p p o r t s h a v e a d v a n t a g e o u s f e a t u r e s to b e a p p l i e d t o v a r i o u s b i o t e c h n o l o g i c a l fields.

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178 Kobayashi, Y., Matsuno, R., Ohya, T. and Yokoi, N. (1987) Enzyme-entrapping behaviors in alginate fibers and their papers. Biotechnol. Bioeng. 30, 451.-457. Matsumoto, K. (1984) Process for preparing an amino group containing polyacrylonitrile polymer. U.S. Patent 4,486,549, Dec. 4, 1984. Matsumoto, K., Mizoguchi, H. and Ichimura, K. (1984) Application of photocrosslinkable poly(vinyl alcohol) to enzyme membranes for biosensors. Kobunshi Ronbunshu 41,221-228. Miyahara, Y., Moriizumi, T. and lchimura, K. (1985) Integrated enzyme FETs for simultaneous detections of urea and glucose. Sens. Actuators 7, 1-10. Mizutani. F, and Asai, M. (1988) Ferrocene-mediated enzyme electrode for glucose with the use of eonducting polymer support. Bull. Chem. Soc. Jpn. 61, 4458-4460. Mizutani, F., Yamanaka, T., Tanabe, Y. and Tsuda, K. (1985) An enzyme electrode for l,-lactate with a chemically-amplified response. Anal. Chim. Acta 177, 153-166. Nakajima, H., Sonomoto, K., Morikawa, H.. Sato, F.. Ichimura, K., Yamada, Y. and Tanaka. A. (1986) Entrapment of Lat.,andula vera cells with synthetic resin prepolymers and its application to pigment production. Appl. Microbiol. Biotechnol. 24, 266-270. Suehiro, T. and lchimura, K. (1988) Enzyme immobilization in the particles. Application for Jpn. Patent, 63-79676, Mar. 31, 1988. Suehiro, T., Ichijo, H., Nagasawa, J., Ichimura, K. and Yamauchi, A. (1987} Preparation of a tubular enzyme reactor. Jpn. Patent 1,405,591, Oct. 27, 1987. Suehiro, T., lchijo, H., Nagasawa, J., Ichimura. K. and Yamauchi, A. (1988a) Interesterification by immobilized lipase. Jpn. Patent 1,440,132, May 30, 1988. Suehiro, T., Ichijo, H., Nagasawa, J.. Ichimura, K. and Yamauchi, A. (1988b) Enzyme immobilization with poly(vinyl alcohol) by photopolymerization. Jpn. Patent 1,454, 372, Aug. 25, 1988. Suehiro, T., Ichijo, tt.. Nagasawa, J., Uedaira, H. and Yamauchi, A. (1988c) Bioreactor with yeast immobilized on braided PVA fiber support. ('hem. Eng. Syrup. Ser., Vol. 17, Soc. Chem. Eng. Jpn., Tokyo, pp. 83-87. tJedaira, H., Yamauchi, A., Nagasawa, J., lchijo, H., Suehiro, T. and lchimura, K. (1984) The effect of immobilization in photocrosslinked polymer on the thermal stability of invertase. Sen-i Gakkaishi 40, T317-T321. Uedaira, H., Ichijo, H., Nagasawa, J., Suehiro, T. and Yamauchi, A. (1988) Thermal stability of inverlase immobilized on superfine filaments of a poly(vinyl alcohol) derivative. Thermochim Acta 123, 183-190.