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Development and Characterization of a Silica Monolith Immobilized Enzyme Micro-bioreactor Koei Kawakami,* Yoshihide Sera, Shinji Sakai, Tsutomu Ono, and Hiroyuki Ijima Department of Chemical Engineering, Faculty of Engineering, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
Several 10-cm-long capillary tubes [made of poly(ether ether ketone) (PEEK)] with inside diameters of 0.1-2.0 mm were filled with silica monolith-immobilized protease derived by in situ sol-gel transition from a 1:4 mixture of tetramethoxysilane and methyltrimethoxysilane. Transesterification between 20 mM (S)-(-)-glycidol and 0.4 M vinyl n-butyrate in an organic solvent was used as the test reaction. The substrate solution flowed through the column at a flow rate of 0.0004-5.0 mL‚min-1. The conversion in the micro-bioreactor was higher than that in the batch reactor at a high liquid flow rate. When three tubes were connected in series, the conversion at a fixed ratio of the mass of the enzyme to the liquid flow rate was increased by approximately 50%, because of the tripling of the flow rate as compared to the case with a single tube. Changes in the tube diameter had no influence on the conversion at a fixed superficial liquid velocity. Further, the conversion increased with a decrease in the enzyme content. These results were ascribed to the apparent effect of liquid-solid mass transfer and were analyzed quantitatively using a simple mathematical model. Introduction Packed-bed bioreactors are the most popular choice for bioconversions involving particulate immobilized biocatalysts because they permit a higher volumetric productivity than any other type of bioreactor.1 In most cases, they are used for relatively large-scale continuous processing, such as for isomerization of glucose to fructose.2 However, in pharmaceutical applications such as the biocatalytic synthesis of chiral drug substances,3 only a small amount of product might be required in a process that uses expensive starting materials and expensive enzymes. For these purposes, it is desirable to downsize the conventional packed-bed bioreactor or to design a novel micro-bioreactor.4 This type of minior micro-bioreactor would be effective for biocatalytic reactions using expensive enzymes and allow high throughput and on-site production of expensive bioproducts at the point of demand. Xu et al.5 have reported a packed-bed enzyme minireactor for the production of structured lipids using nonimmobilized lipases. Xie et al.6 have developed a high-throughput bioreactor using a hydrophilic poly(vinyldimethylazlactone-co-acrylamideco-ethylene dimethacrylate) monolith as a support for immobilization of trypsin. This type of monolithic bioreactor exhibits very high activity, even at a flow velocity that substantially exceeds those used for packed-bed reactors. A microcapillary reactor with immobilized enzymes on the inner wall7,8 and a chip-based microreactor packed with immobilized enzyme microbeads9 have also been developed. Sol-gel entrapment of enzymes and microorganisms has attracted much attention over the past decade. In particular, for lipases and proteases in organic solvents, entrapment into alkyl-substituted organic silicates results in a significant enhancement of esterification * To whom correspondence should be addressed. Tel. & Fax: (81)-92-642-4127. E-mail:
[email protected].
activity and thermal stability.10,11 This is probably due to the hydrophobic interaction between alkyl groups in the silicate matrix and some hydrophobic amino acid residues closely associated with the conformations of the active sites of enzyme.11 The organic silicates derived from an optimum composition of the silane precursors that provide the entrapped enzymes with beneficial microenvironments consist of an aggregate of fine spherical particles with diameters of a few microns because of microscopic phase separation during the solgel transition. Consequently, these supports are coarse, porous materials with interparticle gaps of a few microns.10 The purpose of this study was to develop and characterize a silica monolith immobilized enzyme microchannel bioreactor in which the gaps serve as a passage for the substrate solution. The focus is on the performance of this micro-bioreactor in comparison to that of a conventional batch slurry bioreactor. Transesterification between 20 mM (S)-(-)-glycidol and 0.4 M vinyl n-butyrate by protease was used as the test reaction. Materials and Methods Materials. Protease originating from Aspergillus melleus (Protease P) was kindly supplied by Amano Pharmaceutical Co. and was used without further purification. (S)-(-)-Glycidol was purchased from Aldrich Chemical Co. Vinyl n-butyrate, tetramethoxysilane (TMOS), methyltrimethoxysilane (MTrMOS), and all other chemicals were of reagent grade and were obtained from Tokyo Chemical Industry Co. Poly(ether ether ketone) (PEEK) tubes with inside diameters of 2.0, 1.6, and 0.5 mm and PEEKSIL tubes (fused-silica capillary tube coated with PEEK) with inside diameters of 0.22 and 0.1 mm were purchased from GL Sciences Inc. Preparation of a Silica Monolith Immobilized Enzyme. A mixture of 6.1 mmol of TMOS, 24.2 mmol of MTrMOS, 1.05 mL of distilled water, and 10 µL of
10.1021/ie049354f CCC: $30.25 © 2005 American Chemical Society Published on Web 12/08/2004
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40 mM HCl was mixed in a test tube at room temperature to form a homogeneous sol. After the mixture was cooled to 4 °C, 6.8 mL of 100 mM phosphate buffer (pH 7.5) was added, and then 3 mL of an enzyme solution (pH 7.5) containing 320-900 mg of protease was mixed with the buffered sol. The crude protease sample contained 51% dextrin. During the course of the experiments, it was shown that the dextrin prevented shrinkage of the gel during lyophilization. Optimization of the dextrin content for minimizing shrinkage resulted in the addition of extra dextrin so that a total dextrin content of 455 mg was obtained. A PEEK or PEEKSIL tube of 10 cm in length was placed in a test tube, and it was filled with the enzymecontaining sol mixture. Gelation was allowed to proceed at room temperature for 1 day, and the hydrogels formed were dried in vacuo for 1 day. The PEEK tube containing the silica monolith immobilized enzyme was retrieved, and all of the gel outside the PEEK tube (inside the test tube) was collected and crushed in a mortar. The mass of the gel inside and outside the PEEK tube was measured, and the mass of protease inside and outside the PEEK tube was calculated on the assumption that it had a uniform distribution in the gel. The mass of silica monolith was 60-65 mg for the 100 × 2 mm i.d. tube and 0.64-0.83 mg for the 100 × 0.22 mm i.d. tube. The silica monolith and a prescribed amount of the crushed gels were equilibrated for 1 day in a desiccator containing a saturated aqueous KCl solution (water activity, aw ) 0.84) at 30 °C. The water content in the sample was approximately 9% after this hydration treatment. The silica monolith was used for the flow reactor experiments (micro-bioreactor, abbreviated as µ-BR), and the crushed silica particles were used for the batchwise experiments (batch slurry reactor, abbreviated as BSR). Evaluation of the Enzymatic Activity. Transesterification of 20 mM (S)-(-)-glycidol with 0.4 M vinyl n-butyrate by protease in i-octane or n-decane as the organic solvent was used as a test reaction. End fittings were placed on a 10-cm-long PEEK or PEEKSIL tube loaded with the silica monolith immobilized protease, and the tube was attached to a highperformance liquid chromatograph (HPLC) semi-micropump (PU610, GL Sciences Inc.) or a micropump (MP710, GL Sciences Inc.) and immersed in a constanttemperature bath maintained at 35 °C. In experiments to investigate the effect of the tube length, two or three PEEK tubes, 10 cm in length with a 2-mm i.d., were connected in series. In almost all of the experiments, the 10-cm tube contained 0.14-0.16 mg of crude enzyme/ mg of the monolith. When the enzyme content was increased by 1.5- and 2.25-fold in the PEEKSIL tube of 10 cm in length with a 0.22-mm i.d., the tube was cut to 6.67- and 4.44-cm lengths, respectively, to keep the amount of crude enzyme constant. The substrate solution was fed at a flow rate of 0.15.0 mL‚min-1 in 2.0- and 1.6-mm-i.d. tubes by the HPLC semi-micropump and at a flow rate of 0.0004-0.2 mL‚min-1 in 0.5-, 0.22-, and 0.1-mm-i.d. tubes by the micropump. Steady state for each flow rate was confirmed when the exit concentration of the product became constant, independent of the process time. No inactivation of the enzyme was observed within the range of process times measured. The batch reaction experiments were carried out at 35 °C in 100-mL Erlenmeyer flasks containing both of
the substrates in 20 mL of i-octane or n-decane. The reaction mixture was agitated by a magnetic stirrer bar with its rotating speed set to 180 rpm, to prevent deposition of the gel powder on the flask wall. The reaction was initiated by addition of the immobilized protease (75-100 mg for an enzyme content of 14-15%) into the substrate solution. The organic samples were analyzed using a gas chromatograph (Shimadzu GC14 B) equipped with a FSS ULBON HR-20M capillary column. Comparison of Conversion in a Micro-bioreactor with That in a Batch Slurry Reactor. Although the flow pattern in the microreactor may be characterized by laminar flow, we assumed plug flow for the purpose of simple modeling and comparison. Then, the performance may be described by the following basic equation:
W ) C 0Sb v
dx
S ∫0x -rS,enz S
(1)
in which W [mg] is the mass of enzyme loaded, v [cm3‚min-1] the volumetric flow rate of the substrate solution, C 0sb the feed concentration of the substrate, xS the conversion of the substrate, and -rS,enz [mol‚(mg of enzyme)-1‚min-1] the reaction rate of the substrate, based on the unit mass of the enzyme loaded. On the other hand, the time course of the enzyme reaction in BSR is given by the equation
w t ) C 0Sb V
dx
S ∫0x -rS,enz S
(2)
where w [mg] is the mass of the enzyme, V [cm3] the volume of the reacting mixture, and C 0Sb the initial concentration of the substrate. From a comparison between eqs 1 and 2, it follows that, for the same conversion, the ratio of the mass of crude enzyme to the volumetric flow rate of the substrate solution, W/v, with the continuous µ-BR corresponds to the product of the crude enzyme concentration and the reaction time, (w/V)t, with BSR. Results and Discussion Morphology of a Silica Monolith. Figure 1 shows a scanning electron micrograph of the cross section of the silica monolith formed in the PEEK tube. The monolith consisted of an aggregate of fine spherical particles that were a few microns in diameter and had interparticle gaps a few microns in size that served as a passage for the substrate solution. Performance of a Micro-bioreactor. Figure 2 shows a comparison of the conversion in the continuous µ-BR loaded with the silica monolith with that in the BSR in which the silica particles were suspended. To make a comparison of the results easier, the conversions were plotted against W/v or (w/V)t as the abscissa. The conversion in the one-tube µ-BR was slightly higher than that in the BSR under the high volumetric flow rate condition (W/v < 20), while it was below that in the BSR under the low volumetric flow rate condition (W/v > 20). This might be due to an increase in the resistance to mass transfer of the substrate from the bulk liquid to the solid surface with a decrease in the liquid flow rate. Figure 2 also shows the relationship
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Figure 1. Scanning electron micrographs of the cross section of the protease-immobilizing silica monolith: (a) ×52; bar indicates 200 µm; (b) ×1000; bar indicates 10 µm.
Figure 2. Effect of the number of tubes connected in series on conversion in a comparison of the time courses of conversion in a micro-bioreactor and a batch slurry reactor: i-octane was used as the organic solvent. Inside diameter and length of the tube: 2.0 mm and 10 cm, respectively. Enzyme content: enz ) 0.148. Amount of protease and solid and liquid flow rate: (closed circles) 8.8 and 59.6 mg and 0.20-1.60 mL‚min-1 in a one-tube µ-BR; (closed triangles) 18.3 and 123.5 mg and 0.41-3.30 mL‚min-1 in a two-tube µ-BR; (closed squares) 26.3 and 177.4 mg and 0.604.80 mL‚min-1 in a three-tube µ-BR; (open circles) 11.1 and 74.7 mg and 20 mL (liquid volume) in BSR.
between the conversion and the number of tubes connected in series. When three tubes were connected, the conversion was increased by approximately 50%, because of a 3-fold increase in the liquid flow rate as compared to the case with one tube. Figure 3 shows the effect of the inside diameter of the PEEK(SIL) tube on the conversion. Changes in the diameter from 0.1 to 2.0 mm had no influence on the time courses of the conversion because of a constant superficial liquid velocity at a fixed value of W/v. Model-Based Analysis of the Micro-bioreactor Performance. Figure 4 shows the effect of the crude enzyme content, enz [0.145, 0.213, and 0.327 (mg of crude enzyme)‚(mg of solid)-1], on the conversion behavior against W/v or (w/V)t. In this experiment, the length of µ-BR was changed so as to keep the amount of crude enzyme constant; i.e., 10 cm for enz ) 0.145, 6.67 cm for enz ) 0.213, and 4.44 cm for enz ) 0.327 (see the Materials and Methods section). Interestingly, the conversion increased with a decrease in the crude enzyme content at all values of W/v and (w/V)t in both µ-BR and BSR. This may also be due to the apparent effect of the liquid-solid mass transfer. Using these data, the following analysis was made on the basis of a simple mathematical model. If the enzymatic reaction is approximated as firstorder kinetics with respect to the limiting substrate glycidol in this system and is influenced by the liquid-
Figure 3. Effect of the inside diameter of the micro-bioreactor on the time courses of conversion: i-octane was used in the experiments with 2.0- and 1.6-mm-i.d. tubes and n-decane in those with 0.5-, 0.22-, and 0.1-mm-i.d. tubes. Length of the tube: 10 cm. Enzyme content: enz ) 0.144-0.158. Mass of protease and solid and liquid flow rate: (closed circles) 9.4 and 65.1 mg and 0.2-1.6 mL‚min-1 in a 2.0-mm-i.d. tube; (closed squares) 5.8 and 40.4 mg and 0.13-1.0 mL‚min-1 in a 1.6-mm-i.d. tube; (closed diamonds) 0.52 and 3.33 mg and 0.011-0.12 mL‚min-1 in a 0.5-mm-i.d. tube; (closed triangles) 0.101 and 0.64 mg and 0.0021-0.02 mL‚min-1 in a 0.22-mm-i.d. tube; (closed inverse triangles) 0.021 and 0.133 mg and 0.00042-0.005 mL‚min-1 in a 0.1-mm-i.d. tube; (open circles) 12.6 and 99.8 mg and 20 mL (liquid volume) in BSR with i-octane; (open triangles) 12.4 and 89.2 mg and 20 mL (liquid volume) in BSR with n-decane.
solid mass transfer, then the apparent reaction rate based on the unit mass of crude enzyme is written as
-rS,enz )
kcap (C -CSs) ) kenzCSs enz Sb
(3)
-rS,enz ) KenzCsb
(4)
enz 1 1 ) + Kenz kcap kenz
(5)
where
In these equations, kc [cm‚min-1] is the liquid-solid mass-transfer coefficient, ap [cm2‚(mg of solid)-1] is the external surface area per unit mass of solid involving both crude enzyme and gel, enz [(mg of enzyme)‚(mg of solid)-1] is the enzyme content per unit mass of solid, kenz and Kenz [(cm3 of liquid)‚(mg of enzyme)-1‚min-1] are the intrinsic and apparent (overall) first-order enzyme kinetics constants, respectively, and CSb and CSs are the concentrations of the substrate glycidol in the bulk liquid and on the external surface of the solid, respectively.
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Figure 4. Effect of the enzyme content in a silica monolith on the time courses of conversion: n-decane was used as the organic solvent. Inside diameter of the tube: 0.22 mm. Liquid flow rate in µ-BR: 0.0025-0.022 mL‚min-1. Liquid volume in BSR: 20 mL. Mass of protease and solid and enz: (closed circles) 0.121 and 0.833 mg and 0.145 in a 10-cm-long µ-BR; (closed triangles) 0.121 and 0.569 mg and 0.213 in a 6.67-cm-long µ-BR; (closed squares) 0.121 and 0.370 mg and 0.327 in a 4.44-cm-long µ-BR; (open circles) 12.5 and 86.1 mg and 0.145 in BSR; (open triangles) 12.6 and 59.3 mg and 0.213 in BSR; (open squares) 12.5 and 38.3 mg and 0.327 in BSR.
Figure 6. Relationship between kcap separated according to eq 5 and superficial liquid velocity, ul, in a micro-bioreactor.
sion data under the different conditions shown in Figure 4 by utilizing eqs 5 and 6. Figure 6 shows the dependence of kcap on the superficial liquid velocity of the substrate solution. kcap increased in proportion with the 0.5 power of the superficial liquid velocity. The model-based analysis described above suggests that the performance of the silica monolith immobilized enzyme µ-BR is significantly hindered by the effect of the liquid-solid mass transfer. An attempt to reduce this mass-transfer resistance is now in progress. Conclusions
Figure 5. Logarithmic plot of the unconverted fraction of substrate (1 - xS) versus (w/V)t for a batch slurry reactor according to eq 7: (closed circles) enz ) 0.145; (closed triangles) enz ) 0.213; (closed squares) enz ) 0.327. The inset shows a plot of the reciprocal of Kenz versus enz according to eq 5.
When eq 4 is substituted into eqs 1 and 2, the resulting equations can be easily integrated to give the relations
W v
(6)
w ln(1 - xS) ) -Kenz t V
(7)
ln(1 - xS) ) -Kenz
for the performance of µ-BR and BSR, respectively. Figure 5 shows the semilogarithmic plots of the batch reaction data shown in Figure 4 according to eq 7. The linear relationships obtained may indicate that the assumption of first-order kinetics with respect to glycidol is reasonable. The reciprocal values of Kenz obtained from the slopes of the straight lines were plotted against the enzyme content according to eq 5, as shown in the inset of Figure 5. From the slope and intercept of the straight line obtained here, the values of kcap and kenz are determined to be 0.0014 cm3‚(mg of solid)-1‚min-1 and 0.0105 cm3‚(mg of enzyme)-1‚min-1, respectively. Once the intrinsic first-order enzyme kinetic constant kenz is determined, the volumetric mass-transfer coefficients kcap in µ-BR can be calculated from the conver-
A micro-bioreactor consisting of a 0.1-2-mm-i.d. microcapillary tube filled with silica monolith immobilized protease derived using an in situ sol-gel method was developed, and its performance was evaluated using the transesterification of glycidol with vinyl n-butyrate in organic media. The conversion of glycidol was higher in the micro-bioreactor than in the batch slurry reactor used as a control under the high superficial liquid velocity condition. However, it was shown that the rate of the esterification reaction was significantly influenced by the mass-transfer process of glycidol to the solid surface from the bulk liquid. Thus, it is necessary to reduce this mass-transfer resistance in order to improve the reactor performance. Acknowledgment Financial support from the Asahi Glass Foundation is gratefully acknowledged. This work was also supported by Grants-in-Aid for scientific research (Grant 15656212) and for the 21st Century COE Program, “Functional Innovation of Molecular Informatics”, both from the Ministry of Education, Culture, Science, Sports and Technology of Japan. Literature Cited (1) Hartmeier, W. Immobilized Biocatalysts; Springer-Verlag: Berlin, Germany, 1988. (2) Blanch, H. W.; Clark, D. S. Biochemical Engineering; Marcel Dekker, Inc.: New York, 1996. (3) Bommarius, A. S.; Riebel, B. R. Biocatalysis; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2004. (4) Chovan, T.; Guttman, A. Microfabricated devices in biotechnology and biochemical processing. Trends Biotechnol. 2002, 20, 116. (5) Xu, X.; Zhou, D.; Mu, H.; Adler-Nissen, J.; Hφy, C.-E. A packed-bed enzyme mini-reactor for the production of structured lipids using nonimmobilized lipases. J. Am. Oil Chem. Soc. 2002, 79, 205. (6) Xie, S.; Svec, F.; Frechet, J. M. Design of reactive porous polymer supports for high throughput bioreactors. Poly(2-vinyl-
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4,4-dimethylazlactone-co-acrylamide-co-ethylene dimethacrylate) monoliths. Biotechnol. Bioeng. 1999, 62, 30. (7) Miyazaki, M.; Kaneko, J.; Uehara, M.; Fujii, M.; Shimizu, H.; Maeda, H. Simple method for preparation of nanostructure on microchannel surface and its usage for enzyme-immobilization. Chem. Commun. 2003, 648-649. (8) Kaneno, J.; Kohama, R.; Miyazaki, M.; Uehara, M.; Kanno, K.; Fujii, M.; Shimizu, H.; Maeda, H. A simple method for surface modification of microchannels. New J. Chem. 2003, 27, 1765. (9) Seong, G. H.; Heo, J.; Crooks, R. M. Measurement of enzyme kinetics using a continuous-flow microfluidic system. Anal. Chem. 2003, 75, 3161.
(10) Kawakami, K.; Yoshida, S. Thermal stabilization of lipase by sol-gel entrapment in organically modified silicates formed on kieselguhr. J. Ferment. Bioeng. 1996, 82, 239. (11) Furukawa, S.; Ono, T.; Ijima, H.; Kawakami, K. Activation of protease by sol-gel entrapment into organically modified hybrid silicates. Biotechnol. Lett. 2002, 24, 13.
Received for review July 23, 2004 Revised manuscript received November 18, 2004 Accepted November 19, 2004 IE049354F
