Biotechnology and Bioprocess Engineering 18: 567-574 (2013) DOI 10.1007/s12257-012-0398-2

RESEARCH PAPER

Biosequestration of Carbon Dioxide Using a Silicified Carbonic Anhydrase Catalyst Liang-Jung Chien, Manthiriyappan Sureshkumar, Hsiao-Hsin Hsieh, and Jui-Lung Wang

Received: 11 June 2012 / Revised: 1 October 2012 / Accepted: 7 October 2012 © The Korean Society for Biotechnology and Bioengineering and Springer 2013

Abstract Using recombinant DNA technology, we constructed a dual fusion gene expression plasmid, pRCAH-30, encoding carbonic anhydrase (CA) from the cyanobacterium Synechocystis sp. PCC6803, an R5 peptide sequence, and an affinity (His)6 tag, to allow the simultaneous purification and immobilization of the encoded fusion enzyme, termed RCAH. The expressed fusion protein was approximately 30 kDa, and could be rapidly purified using affinity resins. To enhance enzyme activity, the R5 peptide facilitated immobilization by means of silicification with tetramethoxysilane; the aggregated particles were approximately 300 nm in diameter. Activity tests revealed that the enzyme functioned optimally between pH 7.0 and 7.5; maximum stability was achieved between 25 and 45oC, at pH 6.0 ~ 8.0. Activity of the fusion enzyme persisted, even after encapsulation by biomimetic silicification. In fact, silicone embedding stabilized the enzyme structure, thereby increasing its stability and reusability rate under different environmental conditions. In addition, the silicified enzyme reduced waste CO2 gas from 800 to 42 ppm, resulting in a gas capture rate of 94.7% after conversion. Thus, the construct developed in this study can be effectively utilized for the sequestration of industrial waste CO2 gas. Keywords: carbonic anhydrase, biomimetic silicification, R5 peptide sequence, enzyme immobilization, carbon dioxide sequestration Liang-Jung Chien*, Hsiao-Hsin Hsieh, Jui-Lung Wang Graduate School of Biochemical Engineering, Ming Chi University of Technology, New Taipei City, Taiwan Tel: +88-62-2908-9899; Fax: +88-62-2908-3072 E-mail: [email protected] Manthiriyappan Sureshkumar Department of Chemistry, The University of Suwon, Hwasung 445-743, Korea

1. Introduction Over the past few decades, the world’s average temperature has increased by approximately 0.6oC, and this increase is expected to continue at a rapid rate. The warming of the Earth is constantly undergoing changes due to a variety of factors, such as increasing population growth, globalization, and rapid industrialization. The most common means of energy generation is combustion of fossil fuels, which causes emission of the greenhouse gas CO2. The concentration of atmospheric carbon dioxide (CO2) has increased as a consequence of anthropogenic activities, and most scientists consider that this increase has caused global warming. According to recent reports, the rapid increase in environmental CO2 levels during recent years is widely being considered as one of the main driving force for a gradual increase in the earth’s average temperature, serious damage to the polar ecosystem, and imbalance in environmental temperatures. The severity of damaging human-induced climate change and the significance of reduction in environmental CO2 concentrations has become an important research topic. Although numerous techniques are being employed to reduce atmospheric CO2, economically feasible new technology and/or methods to achieve this target are essential. CO2 capture and sequestration have been demonstrated by several reports, by methods based on a variety of chemical and biological approaches. CO2 dissolved in water may undergo a number of transformations, such as solubilizing in an aqueous phase, hydration by water, ionization, or carbonate formation. Hydration of CO2 is the slowest of these steps [1]. Attempts have been made to accelerate this step, in order to enhance the subsequent fixation of CO2 into stable mineral carbonates in the presence of a biocatalyst, carbonic anhydrase (CA) [2-5]. CA (EC 4.2.1.1) is a metallo enzyme, containing a zinc ion in its active site. The primary

568

reaction catalyzed by this enzyme is as follows: CO2 + H2O ↔ HCO3− + H+. Its reaction rate can be as high as 104 ~ 106 reactions/s [6]. Hydration of CO2 by the CA enzyme is initiated by a nucleophilic attack on the carbon atom of CO2 by the zinc-bound OH group to produce bicarbonate, which is then displaced from zinc by a water molecule [4]. Although CA demonstrates good activity during the reaction, the free enzyme has low stability, and limited reusability. In addition, recovery of this enzyme from the reaction environment is generally not possible. Therefore, many researchers have sought to increase the structural stability of CA via enzyme immobilization, thereby increasing its activity and reusability [4,7-13]. Silicon dioxide has a high-density porous structure that can interact with medium and low molecular weight substances, to allow the embedding of enzymes, thereby stabilizing enzyme structures. When enzymes are encapsulated in silicon dioxide during the silica sol-gel process, the activity of the embedded enzyme is preserved, making this an effective strategy [1416]. The most common precursors for enzyme encapsulation in this process are tetramethoxysilane (TMOS) and tetraethoxysilane (TEOS), both of which can produce soluble hydroxy derivatives, such as silicate and hydroxysilane, via hydrolysis. Mixing these hydroxyl derivatives with target enzymes can lead to particle aggregation, which embeds and encapsulates the enzymes in hydrolyzed silica [17,18]. The polycationic peptide silaffin, which is phosphorylated at many serine and lysine residues in the diatom Cylindrotheca fusiformis, can promote silicone condensation in a silicate solution at neutral pH, and under standard temperature and pressure conditions. The silaffin polypeptide unit is an unmodified R5 peptide repeat sequence (SSKKSGSYS GSKGSKRRIL); silica-condensing synthetic R5 peptide can induce silica particle precipitation in a neutral pH silicate solution within seconds [19-22]. Encapsulation and immobilization of enzymes by biomimetic silicification can prevent contact between enzymes and proteases or denaturants, and also stabilize the tertiary structure of enzymes [22,23], thereby preserving enzyme activity after immobilization. In this study, we used the cyanobacterium Synechocystis sp. PCC 6803 and recombinant DNA technology to create a dual fusion plasmid, pRCAH-30, encoding CA from Synechocystis sp. PCC 6803, the R5 peptide sequence, and a hexahistidine (His)6 affinity tag. The (His)6 affinity tag was included to facilitate enzyme purification by metal affinity chromatography. The purified enzyme was used for biomimetic silicification, and the stabilities of the immobilized and non-immobilized enzymes at different temperatures and pH values were compared. We evaluated the enzyme reusability rate and the efficiency of waste CO2 gas

Biotechnology and Bioprocess Engineering 18: 567-574 (2013)

conversion, by investigating the conversion of industrial waste CO2 gas, and the CA activity after encapsulation in biomimetic silica.

2. Materials and Methods 2.1. Plasmid construction DNA from Synechocystis sp. PCC6803 was extracted using DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany), and the CA gene was amplified by PCR, using primers designed with Vector NTI software. The resulting PCR product was digested with BglII and XhoI (New England Biolabs, Beverly, MA), and ligated into the pET30b(+) expression vector (Novagen Inc., Madison, WI), to generate the CA-expressing plasmid pCAH-30. The integrity of this construct was confirmed by sequencing (Mission Biotech, Taiwan). To allow immobilization of the enzyme using a biomimetically silicified peptide, the R5 sequence was synthesized using synthetic gene technology [20], and inserted into the plasmid. Two single-stranded nucleotide sequences, i.e. R5-F: 5'-tatgtcctccaagaaatccggatcctactcgggatccaagggttccaa gcgtcgcatcttgcca-3', and R5-R: 5'-gatctggcaagatgcgacgct tggaacccttggatcccgagtaggatccggatttcttggaggaca-3', were synthesized. These oligonucleotides were first separately incubated with 10 mM ATP and T4 polynucleotide kinase at 37oC for 1 h; thereafter, the R5-F and R5-R nucleotides were mixed, and DNA polymerase was added to the mixture. The mixture was placed in boiling water for 2 ~ 5 min. After cooling, the ligated R5 peptide sequence was used as the template for PCR, to generate the R5 sequence. This amplicon was then ligated into the NdeI and BamHI sites of the digested pCAH-30 plasmid, and the integrity of the pRCAH-30 construct generated was verified by sequencing (Mission Biotech) (Fig. 1). 2.2. Protein expression The pRCAH-30 plasmid was first expressed in Escherichia coli BL21(DE3) (Yeastern Biotech Co., Ltd.) cells, which were cultured in a 500-mL flask with 100 mL of LB medium and 30 µg/mL kanamycin at 37oC in a shaker, until an OD600 of approximately 1.0 was reached. Protein expression was induced with 0.05 mM IPTG, at 30oC for 6 h; cells were then collected by centrifugation (5 min, 6,000 rpm, 4oC), resuspended in buffer solution (50 mM Na2HPO4, 0.3 M NaCl, and 30 mM imidazole; pH 8.0), and disrupted, using a sonicator (Ultrasonic Processor, type XL2000). After centrifugation, the supernatant, which contained the soluble target protein, was collected. The enzyme present in the lysate was then purified by immobilized metal affinity chromatography (IMAC), making use

Biosequestration of Carbon Dioxide using a Silicified Carbonic Anhydrase Catalyst

569

solution was added; once the color appeared, the reaction was terminated by the addition of TE buffer (50 mM Tris– HCl and 10 mM EDTA; pH 8.0). 2.4. Activity analysis by zymography The method used in this study was originally reported by Hou et al. [21]. In this experiment, 5× loading dye (60 mM Tris–HCl, 2% SDS, 25% glycerol, and 0.1% bromophenol blue) was first incubated with the purified enzyme, at 37oC for 30 min, to where indicator bromophenol blur dye changed color from blue to yellow. Next, 7 µL of each diluted sample was used for analysis on a 15% SDS-PAGE gel. After electrophoresis, the residual SDS on the gels was removed, by washing with 10 mM Tris–HCl (pH 7.9) containing 25% isopropanol. The gels were then incubated in 100 mM Tris–HCl (pH 8.5) containing 2.5% bromophenol blue, followed by incubation in 100 mM Tris–HCl (pH 8.5). When the target protein band turned yellow, the reaction was terminated, by adding distilled water.

Fig. 1. Construction scheme of the dual-fusion expression vector pRCAH-30, for expression in Escherichia coli.

of the (His)6 affinity tag. The protein eluates were dissolved in 5× sample buffer (2.5 mL β-mercaptoethanol, 3 mL glycerol, 3.15 mL of 0.5 M Tris–HCl, 0.01 g bromophenol, and 1.5 g SDS), and boiled at 95oC for 5 min. After cooling, 7 µL of each diluted sample was electrophoresed at 120 V on a 12% SDS-PAGE gel. Coomassie Brilliant Blue R-250 was used for staining. 2.3. Immunoblotting analysis After electrophoresis, the proteins on the gel were transferred onto a PVDF membrane (Hybond-P, Amersham Bioscience, USA), at 24 V for approximately 1 h. The membrane was then incubated in blocking solution (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, and 5% non-fat milk; pH 7.3) at 4oC overnight, followed by incubation with the Anti-Penta-His antibody (QIAGEN, Hilden, Germany), at a 10−4 dilution, at 25oC for 1 h. After several washes with wash buffer (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20; pH 7.3), the membrane was incubated with HRPlabeled mouse anti-rabbit antiserum (Sigma, USA), at a 1 × 10−4 dilution, at 25oC for 1 h. After further washing, the developing NBT/BCIP (Roche Diagnostics GmbH, Germany)

2.5. Biomimetic silicification of the biological enzyme To form biosilicified nanoparticles, 152 µL freshly prepared TMOS was first acid hydrolyzed to silica acid, by incubation with 848 µL of 1 mM HCl, at room temperature for 15 min. After hydrolysis, a 10% (V/V) silicate solution was added to the purified enzyme solution. After rapid and thorough mixing, round silicified particles were formed. Scanning electron microscopy (SEM) was used to confirm the morphology of these particles. 2.6. Detection of enzyme activity The activity of the purified CA was analyzed using pnitrophenyl acetate (pNPA) as the substrate [24]. CA hydrolyzes pNPA to p-nitrophenol (pNP) and acetate, and the concentration of the product formed can be analyzed at OD348. In total, 930 µL of 15 mM Tris–SO4 (pH 7.6), 3 mM pNPA (dissolved in 25 mL of acetone and stored at −20oC), and 50 µL of enzyme sample was added to a l-mL microfuge tube. After thorough mixing, samples were incubated at 25oC and pH 7.5 for 5 min, before production of pNP, and acetate was analyzed spectrophotometrically at OD348. One unit (U) of activity was defined as the amount of enzyme required to release 1 µmol pNP in 1 min, under the given reaction conditions. 2.7. Analysis of enzyme stability To identify the optimal conditions for CA stability, the optimal pH of the non-silicified enzyme was first evaluated. This optimal pH was then used to determine the temperature and pH ranges that permit enzyme stability. The enzyme was reacted under different pH conditions for 20 h and different temperatures for 15 min, and the enzymatic activity

570

Biotechnology and Bioprocess Engineering 18: 567-574 (2013)

waste CO2 gas produced by China Steel Corporation, using a newly designed reactor (Fig. 2). In total, 800 ppm waste CO2 gas, diluted with air, was passed through the reactor from below, at a flow rate of 5 mL/min, using an air flow regulator. CO2 was then passed from the top to the bottom using water, at a flow rate of 10 mL/min, to increase the CO2 retention time. At various time points, samples were collected from the position indicated by the red arrow in Fig. 2, and analyzed by gas chromatography (Porapak Q, 80 ~ 100 mesh). CO2 concentrations were determined, based on the conversion of the integrated areas before and after the reaction. Thus, the CO2 conversion rate was calculated using the following equation: Moles of CO2 Converted CO2 Conversion (%) = -------------------------------------------------Moles of CO2 Initial Fig. 2. Diagram of the reactor used for waste CO2 gas conversion.

3. Results and Discussion was assessed, as described in section 2.6. As a control for biomimetic immobilization of enzymes purified by IMAC, non-immobilized enzymes were not purified with desorption buffer solution; instead, enzymatic activity analysis was directly performed using enzyme-coated resins. Furthermore, to assess enzyme reusability, silicified and immobilized enzymes were continuously reacted for approximately 1 h in pure CO2 (5 mL/min), and the samples were then collected, to detect enzymatic activity. This experimental procedure was repeated 10 times. 2.8. Measuring CO2 waste conversion We next investigated the effect of CA on conversion of

3.1. Protein expression and purification CA, which has a covalently bound Zn2+ ion cofactor, can quickly and reversibly convert CO2 to HCO3. The RCAH fusion enzyme produced from the dual-fusion plasmid constructed in this study contained a C-terminal (His)6 affinity tag and an N-terminal R5 peptide, which we reasoned would allow easy recovery of the enzyme from the crude cell extract, using IMAC particles, and which could be encapsulated in situ with silica, to preserve its stability. Fig. 3A shows the SDS-PAGE analysis of the dual-fusion RCAH expressed in E. coli BL21(DE3), and its purification by IMAC. A prominent band at approximately

Fig. 3. SDS-PAGE and immunoblot analysis of the target protein before, and after, IMAC purification. (A) SDS-PAGE. (B) Immunoassay of enzymes and zymography. M, Marker; S, Crude extract; IS, Insoluble protein; and P, purified protein. Arrow indicates the target protein, CA.

Biosequestration of Carbon Dioxide using a Silicified Carbonic Anhydrase Catalyst

571

Fig. 4. SEM images of biomimetic silicification of the biological enzyme. (A) Image of the biomimetic particles at 20,000 × magnifications. (B) Image of the biomimetic particles at 50,000 × magnification.

30 kDa, corresponding to the predicted size of the dualfusion RCAH, was detected in the crude extract (lane S) and insoluble protein fraction (lane IS). All proteins isolated and purified by IMAC and analyzed by western blot analysis and zymography had the same molecular weight (Fig. 3B). 3.2. Biomimetic silicification of the biological enzyme The dual-fusion RCAH expressed in E. coli was recovered from the crude extract, using IMAC particles. While the dual-fusion RCAH was immobilized on the IMAC particles through the metal ion affinity interaction of the (His)6 tag, the R5 tag, located on the other end of the immobilized enzyme, was available to induce silica precipitation [20,22,23]. To comprehensively examine the effects of the synthetic R5 peptide sequence, TMOS was acid hydrolyzed with 1 mM HCl to a silicate solution, which was then mixed with the purified enzyme, to induce silicification. As shown in Fig. 4A, the R5 peptide tag rapidly induced silicate aggregation; particles were formed, and enzyme immobilization was complete 30 sec after silicification, indicating that the R5 peptide sequence in the purified enzyme had the ability to induce rapid silicate aggregation. The aggregated particles were analyzed by SEM, and the average diameter of the aggregated particles was approximately 300 nm (Fig. 4B), and it was observed that their surface area was favorable for enzymatic reaction. Elemental analysis revealed that the major component of the particles was silicone, indicating that the dual-fusion RCAH could be simultaneously purified, silicified, and immobilized. 3.3. Stability of biosilicified and immobilized RCAH To ensure long-term usability of the biological catalyst after silicification, the stability of the fusion enzyme was further investigated. The optimal conditions for the non-

Fig. 5. Analysis of the optimal reaction pH of dual-fusion RCAH.

silicified enzyme were first analyzed. As shown in Fig. 5, the optimal pH of the dual-fusion RCAH was 7.0 ~ 7.5, indicating that the enzyme was most stable when the pH was close to neutral. Therefore, pH 7.5 was used as the comparison control value, for further investigations of the stability of the silicified enzyme at different temperatures and pH values, which are also known to affect enzyme stability, and indirectly influence the outcome of the enzymatic reaction. The fusion enzyme had 100% relative activity at 25oC, but exhibited a relative decrease in activity, as the temperature increased. Of note, the enzyme exhibited more than 80% residual activity below 45oC (Fig. 6), suggesting that the temperature stability range was 25 ~ 45oC. When the reaction temperature exceeded 75oC, the non-silicified enzyme exhibited no residual activity, whereas the silicified enzyme exhibited 40% residual activity. Similarly, at 55oC, the residual activity of the non-silicified enzyme was approximately 60%, whereas that of the silicified enzyme

572

Fig. 6. Analysis of the stability of dual-fusion RCAH at different temperatures.

Fig. 7. Analysis of the stability of dual-fusion RCAH at different pH values.

was approximately 80%. This finding indicates that silica encapsulation was efficient in preserving the active enzyme structure, thereby causing a reduction in temperature-induced denaturation. Fig. 7 demonstrates that the fusion enzyme had 100% relative activity at pH 7.0 ~ 7.5 at 25oC. A comparison of various pH values indicated that a neutral environment was most favorable for the stability of the dual-fusion RCAH. The enzyme was quickly inactivated, when the pH became too acidic or too basic. At pH 6 and 8, the non-silicified enzyme retained approximately 80% activity, whereas the silicified enzyme retained more than 90% activity. At pH 5 and 9, the non-silicified enzyme retained approximately 50 ~ 60% residual activity, whereas the silicified enzyme retained more than 80% activity. Finally, at pH 4 and 10, the non-silicified enzyme had only 20% residual activity, whereas the immobilized enzyme had more than 40%

Biotechnology and Bioprocess Engineering 18: 567-574 (2013)

Fig. 8. Analysis of the reusability of silicified dual-fusion RCAH.

residual activity. These results confirmed that silicification increased the stability of the enzyme by approximately 20% after immobilization. Silicification of CA not only increased its temperature stability, but also increased its stability to acidic and basic environments. Similar to the results of temperature analysis, immobilization of CA increased its pH stability range. After assessing the stability of the fusion enzyme, the reusability of the silicified enzyme was assessed, by placing the silicified and immobilized enzyme in pure CO2 in the gas phase, so that it was continuously active for approximately 1 h. Samples were then collected for enzyme activity tests, and the experiment was repeated 10 times. The enzyme retained more than 85% residual activity (Fig. 8). Therefore, immobilization of the enzyme by silicification encapsulates it, preventing it from undergoing structural changes due to external influences, thus increasing its stability. This increase in enzyme stability indirectly increased its reusability rate. Therefore, encapsulation of the silicified enzyme through embedding is superior to the low enzymestabilizing forces observed in traditional immobilization methods, thereby confirming that silicified enzymes have a great potential for industrial applications. 3.4. Conversion of industrial CO2 waste To effectively capture CO2 from flue gases, CO2 must undergo a number of transformations, such as dissolution in an aqueous phase, hydration by water, ionization, and carbonate formation. Among these steps, CO2 hydration is the slowest; however, this step can be enhanced by CA. To determine whether the fusion enzyme could be used to convert waste CO2 gas, we investigated its effect on industrial emissions from China Steel Corporation. The conversion rate of the non-silicified enzyme was approximately 31%. The silicified fusion enzyme reduced the CO2 concentration

Biosequestration of Carbon Dioxide using a Silicified Carbonic Anhydrase Catalyst

573

References

Fig. 9. Conversion of industrial waste CO2 gas by dual-fusion RCAH.

from 800 ppm to approximately 42 ppm, and the gas capture rate was approximately 94.7% (Fig. 9). The results indicate that the immobilized enzyme efficiently reduced the CO2 concentrations, suggesting that CA could be utilized for the conversion of industrial waste CO2 gas. Silicification of the enzyme increased its structural stability, and reduced contact between the enzyme and other impurities in the emissions, such as sulfides, thereby protecting the enzymes during conversion reactions.

4. Conclusion This study successfully combined molecular biology and engineering technologies, to develop a novel and highly efficient CO2 biosequestration system, using the newly constructed plasmid, pRCAH-30. In contrast to previously published constructs, the fusion enzyme prepared in this study contains the R5 peptide sequence, which can reliably and rapidly mediate the immobilization of silicified enzymes, thereby simultaneously increasing enzyme stability over wider pH and temperature ranges, and promoting excellent enzyme reusability. Thus, the construct developed in this study can be effectively utilized for sequestration of industrial waste CO2 gas. This system will be studied further, to explore the relationship between CO2 biosequestration apparatuses and algae bioreactors, thereby contributing to an effective solution to tackle the greenhouse effect.

Acknowledgements The authors gratefully acknowledge the financial support from Taiwan’s National Science Council (Grant Nos. 982218-E-131-003 and 99-2221-E-131-019).

1. Dreybrodt, W., J. Lauckner, Z. H. Liu, U. Svensson, and D. Buhman (1996) The kinetics of the reaction CO2 + H2O → H + + HCO3−, as one of the rate limiting steps for the dissolution of calcite in the system H2O-CO2-CaCO3. Geochimica et Cosmochimica Acta. 60: 3375-3381. 2. Bond, G. M., J. Stringer, D. K. Brandvold, F. A. Simsek, M. G. Medina, and G. Egeland (2001) Development of integrated system for biomimetic CO2 sequestration using the enzyme carbonic anhydrase. Energy Fuels 15: 309-316. 3. Liu, N., G. M. Bond, A. Abel, B. J. McPherson, and J. Stringer (2005) Biomimetic sequestration of CO2 on carbonate form: Role of produced waters and other brines. Fuel Proc. Technol. 86: 1615-1625. 4. Mirjafari, P., K. Asghari, and N. Mahinpey (2007) Investigating the application of enzyme carbonic anhydrase for CO2 sequestration purposes. Ind. Eng. Chem. Res. 46: 921-926. 5. Cheng, L. -Y., Y. -T. Long, H. -B. Kraatz, and H. Tian (2011) Evaluation of an immobilized artificial carbonic anhydrase model for CO2 sequestration. Chem. Sci. 2: 1515-1518. 6. Lindskog, S. (1997) Structure and mechanism of carbonic anhydrase. Pharmacol. Ther. 74: 1-20. 7. Badjic J. D. and N. M. Kostic (1999) Effects of encapsulation in sol-gel silica glass on esterase activity, conformational stability, and unfolding of bovine carbonic anhydrase II. Chem. Mater. 11: 3671-3679. 8. Hosseinkhani, S. and M. N. Gorgani (2003) Partial unfolding of carbonic anhydrase provides a method for its immobilization on hydrophobic adsorbents and protects it against irreversible thermoinactivation. Enz. Microb. Technol. 33: 179-184. 9. Drevon G. F., C. Unbarke, and A. J. Russell (2003) Enzyme-containing michael-adduct-based coatings. Biomacromol. 4: 675682. 10. Cheng L. H., L. Zhang, H. L. Chen, and C. J. Gao (2008) Hollow fiber contained hydrogel-CA membrane contactor for carbon dioxide removal from the enclosed spaces. J. Memb. Sci. 324: 3343. 11. Ozdemir, E. (2009) Biomimetic CO2 sequestration: 1. Immobilization of carbonic anhydrase within polyurethane foam. Energy Fuels 23: 5725-5730. 12. Ellerby, L. M., C. R. Nishida, F. Nishida, S. A. Yamanaka, B. Dunn, J. S. Valentine, and J. I. Zink (1992) Encapsulation of proteins in transparent porous silicate glasses prepared by the sol-gel method. Sci. 255: 1113-1115. 13. Gil, I. and A. Ballesteros (2000) Bioencapsulation within synthetic polymers (Part 1): Sol-gel encapsulated biologicals. Trends Biotechnol. 18: 282-296. 14. Besanger, T. R., Y. Chen, A. K. Deisingh, R. Hodgson, W. Jin, S. Mayer, M. A. Brook, and J. D. Brennan (2003) Screening of inhibitors using enzymes entrapped in sol-gel-derived materials. Anal. Chem. 75: 2382-2391. 15. Cruz-Aguado, J. A., Y. Chen, Z. Zhang, N. H. Elowe, M. A. Brook, and J. D. Brennan (2004) Ultrasensitive ATP detection using firefly luciferase entrapped in sugar-modified sol-gelderived silica. J. Am. Chem. Soc. 126: 6878-6879. 16. Roth, K. M., Y. Zhou, W. Yang, and D. E. Morse (2005) Bifunctional small molecules are biomimetic catalysts for silica synthesis at neutral pH. J. Am. Chem. Soc. 127: 325-330. 17. Kroger, N., R. Deutzmann, and M. Sumper (1999) Polycationic peptides from diatom biosilica that direct silica nanosphere formation. Sci. 286: 1129-1132. 18. Kroger, N., R. Deutzmann, C. Bergsdorf, and M. Sumper (2000) Species-specific polyamines from diatoms control silica mor-

574

phology. Proc. Natl. Acad. Sci. U S A. 97: 14133-14138. 19. Kroger, N., S. Lorenz, E. Brunner, and M. Sumper (2002) Selfassembly of highly phosphorylated silaffins and the function in biosilica morphogenesis. Sci. 298: 584-586. 20. Chien, L. J. and C. K. Lee (2008) Biosilicification of DualFusion Enzyme Immobilized on Magnetic Nanoparticle. Biotechnol. Bioeng. 100: 223-230. 21. Hou, W. C., H. J. Chen, and Y. H. Lin (2000) Dioscorins from different Dioscorea species all exhibit both carbonic anhydrase and trypsin inhibitor activities. Bot. Bull. Acad. Sinica. 41: 191196.

Biotechnology and Bioprocess Engineering 18: 567-574 (2013)

22. Luckarift H. R., J. C. Spain, R. R. Naik, and M. O. Stone (2004) Enzyme immobilization in a biomimetic silica support. Nat. Biotechnol. 22: 211-213. 23. Luckarift, H. R., G. R. Johnson, and J. C. Spain (2006) Silicaimmobilized enzyme reactors; application to cholinesterase-inhibition studies. J. Chromatogr. B Biomed. Appl. 843: 310-316. 24. Khalifah, R. G. and D. N. Silverman (1990) Carbonic anhydrase kinetics and molecular function. pp. 49-70. In: S. J. Dodgson, R. E. Tashian, G. Gros, and N. D. Carter (eds.). The carbonic anhydrase: Cellular physiology and molecular genetics. Plenum press, NY, USA.