Green and Efficient Conversion of CO2 to Methanol by Biomimetic

Mar 27, 2009 - methanol, by encapsulating three dehydrogenases within titania particles ... (formate dehydrogenase, formaldehyde dehydrogenase, alcoho...
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Ind. Eng. Chem. Res. 2009, 48, 4210–4215

Green and Efficient Conversion of CO2 to Methanol by Biomimetic Coimmobilization of Three Dehydrogenases in Protamine-Templated Titania Qianyun Sun, Yanjun Jiang, Zhongyi Jiang,* Lei Zhang, Xiaohui Sun, and Jian Li Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin UniVersity, Tianjin 300072, People’s Republic of China

A green and efficient mutienzyme system was established, which efficiently converted carbon dioxide into methanol, by encapsulating three dehydrogenases within titania particles through a facile and mild biomimetic mineralization process. The enzyme-containing titania particles were characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The results indicated that the enzyme-containing titania particles were amorphous and consisted of interconnected nanospheres with sizes in the range of 400-600 nm. The three encapsulated dehydrogenases (formate dehydrogenase, formaldehyde dehydrogenase, alcohol dehydrogenase) sequentially converted carbon dioxide into HCOOH, CHOH, and CH3OH using NADH as a terminal electron donor for each dehydrogenasecatalyzed reduction. Compared to the open-style system which directly performed the bioconversion using free enzymes in aqueous solution, higher reaction yield in a wider pH and temperature range was obtained by the closed-style coimmobilization multienzyme system. Introduction Due to the growing consensus that anthropogenic emissions of CO2 are causing global climate change and planetary temperature increase, it is urgent to find green and efficient solutions to CO2 utilization. 1,2 In nature, the photosynthesis system has set a perfect example for large-scale CO2 fixation. Inspired by this, tremendous efforts have been devoted to bioconversion and enzymatic conversion of CO2.3-5 In 1999, Obert and Dave discovered the consecutive enzymatic reduction approach of CO2 with three dehydrogenases: formate dehydrogenase (FateDH), formaldehyde dehydrogenase (FaldDH), and alcohol dehydrogenase (ADH) as catalysts, and reduced nicotinamide adenine dinucleotide (NADH) as a terminal electron donor.6 In recent years, our group has carried out a series of investigations on such enzymatic conversion, with the main focus on screening the suitable immobilization methods, designing and preparing the appropriate immobilization carriers, analyzing the relevant reaction kinetics and mass transfer characteristics,anddeterminingtheoptimumreactionconditions.7-9 Since the establishment of green chemistry principles, there has been renewed interest in using these principles to synthesize nanoparticles for enzyme encapsulation.10,11 Nevertheless, quite few studies have realized the green immobilization of enzymes. The most commonly used sol-gel method for enzyme encapsulation often requires strong acid or alkaline catalysts which not only compromise the activity of enzymes but also aggravate environmental deterioration.12 Recently, by mimicking the biomineralization process in nature, the activity of the R5 peptide has been used to induce silica formation and immobilize enzymes in nanostructured silica; this immobilization process was proceeded at near to neutral pH and ambient temperatures and pressures, and no acidic or basic catalyst was involved. It seems that the biomineralization process should constitute a promising alternative for enzyme immobilization.13-16 Compared to silica, titania-based materials showed excellent pHstability,superiormechanicalstrength,andbiocompatibility.17,18 In our previous reports, it was found that protamine could induce * To whom correspondence should be addressed. Tel.: 86-22-2789 2143. Fax: 86-22-2789 2143. E-mail: [email protected].

the formation of titania nanoparticles from water-soluble precursors in a facile way.8 In this study, three dehydrogenases were encapsulated in titania particles through a facile biomimetic titanification. The immobilization process was induced by protamine, and the inorganic phase was formed under mild and ecofriendly conditions. Three dehydrogenases constructed an enzymatic “assembly line” to convert CO2 into methanol. The sequential conversion of CO2 by three enzymes coimmobilized in the biotitania nanoparticles were systematically investigated, and a high yield of methanol was obtained. Moreover, parameters concerning the comprehensive performance of the catalysts, such as activity retention, pH, and temperature stability were briefly assessed. Experimental Section Materials. Protamine sulfate from salmon (P4380), titanium (IV) bis(ammonium lactato) dihydroxide (Ti-BALDH, 388165) (50 wt % in water), formate dehydrogenase (FateDH, 1.4 U/mg, solid), formaldehyde dehydrogenase (FaldDH, 4.4 U/mg, solid), alcohol dehydrogenase (ADH, 303 U/mg, solid), and reduced nicotinamide adenine dinucleotide (98%) were purchased from Sigma (USA). All other chemicals were of analytical grade. Biomimetic Preparation of Titania Containing Encapsulated Dehydrogenases. A stock solution of protamine (20 mg/mL) was prepared in 0.05 M Tris-HCl buffer solution. Ti-BALDH solution was diluted to a final concentration of 0.25 M. The biomimetic preparation of titania containing encapsulated dehydrogenases was conducted by the following procedures: the mixture consisting of 0.5 mL of dehydrogenases stock solution (containing FateDH 4.5 mg, FaldDH 4.5 mg, and ADH 1.0 mg), 0.5 mL of protamine solution, and 1 mL Ti-BALDH solution was prepared and agitated for 5 min at room temperature, the resultant enzyme-containing titania particles were recovered by centrifugation for 5 min (3000 r/min) and then washed three times with deionized water. Encapsulation Efficiency. Supernatant was collected after centrifugation, and the encapsulation efficiency was determined by the following equation:

10.1021/ie801931j CCC: $40.75  2009 American Chemical Society Published on Web 03/27/2009

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encapsulation efficiency(%) )

1 - [enzyme]supernatant × 100 [enzyme]dissolve

where [enzyme]supernatant and [enzyme]dissolve were the activity of enzymes in the supernatant and in the original protamine charged enzyme solution, respectively. The enzyme activity was determined by enzymatic conversion reaction. Enzymatic Conversion of CO2 to Methanol. Conversion of CO2 to methanol was conducted in solution phase with free or immobilized dehydrogenases (Scheme 1). A 1.5 mL portion of the solution was bubbled with CO2 for 1 h before the addition of 0.5 mL NADH to initiate the reaction (the actual amount of encapsulated FateDH, FaldDH, and ADH was 2.7, 2.7, and 0.6 mg, respectively, with a final NADH concentration of 0.025-0.1 M). The reaction lasted 8 h for sufficient production of methanol. The methanol concentration was determined via gas chromatography (GC) equipped with a flame ionization detector (FID; Hewlett-Packard, model HP-6890). All results were repeated three times. Recycling Stability and Storage Stability. The encapsulated dehydrogenases were collected after each reaction batch, thoroughly rinsed with Tris-HCl buffer, and utilized in the next reaction cycle. The recycling stability of encapsulated dehydrogenases was evaluated by measuring the enzyme activity in each successive reaction cycle. recycling efficiency(%) ) enzyme activity in the nth cycle × 100 enzyme activity in the 1st cycle Free and encapsulated dehydrogenases were stored at 4 °C for a certain period of time. The storage stability was compared by storage efficiency defined as the ratio of free or encapsulated enzyme activity after storage to their initial activity. storage efficiency(%) )

enzyme activity after storage × 100 initial enzyme activity

Characterization. Scanning electron microscopy (SEM) was performed on a Philips XL30 ESEM instrument at 10-20 kV in vacuum. Elemental analysis was determined by energy dispersive spectroscopy (EDS) analysis attached to the SEM. Fourier transform infrared (FTIR) spectra of the nanoparticles were obtained on a Nicolet-560 spectrometer using the Transmission ESP program. Thirty-two scans were accumulated with a resolution of 4 cm-1 for each spectrum. The surface properties of titania/protamine nanoparticle composite were characterized by X-ray photoelectron spectroscopy (XPS) in a Perkin-Elmer PHI 1600 ESCA system with a monochromatic Mg KR source and a charge neutralizer. Results and Discussion 1. Biomimetic Coimmobilization of the Multienzymes in Protamine-Induced Titania Particles. During the precipitation process of the titania particles, a coimmobilization system was simultaneously formed where the hybrid titania-protamine played the role as the structural scaffold while the three dehydrogenases played the role as the biomolecular machine. Since the enzymatic reaction was performed in a heterogeneous system, the dimension and pore size of the titania particles were both quite important. Scanning electron microscopy (SEM) revealed that the titania consisted of fused particles with size of about 500 nm (Figure 1a). As shown in Figure 2, the pore sizes of the titania particles (about 3.8 nm) were big enough to allow the substrates and products to diffuse in and out but small

Scheme 1. Experimental Setup to Convert CO2 to Methanola

a 1-compressed carbon dioxide cylinder; 2-controlling buffer; 3-reactor; 4-valve.

enough to substantially prevent the entrapped dehydrogenases from migrating out of the titania particles.8 The morphology of enzyme-containing titania particles was similar to that of previous silica particles.14 Energy dispersive spectroscopy (EDS) analysis accompanying SEM characterization indicated that the titania particles were enriched in carbon and sulfur, as well as oxygen and titanium (Figure 1b). The presence of carbon in the EDS spectrum was consistent with a precipitate consisting of an organic/inorganic composite of protein within a titania matrix. The sulfate introduced with protamineanddehydrogenasesprovidedasourceofsulfurswhereas the titanium and oxygen peaks were clearly associated with the inorganic precursor. Figure 3 presented the FTIR spectrum of titania particles containing enzymes synthesized in the presence of protamine. The typical peaks of titania (Ti-O-Ti) stretching frequencies from 450 to 700 cm-1, could be clearly observed. Additionally, there was a broad absorbance near 3420 cm-1 as well as distinct peaks around 1650 cm-1, which could be attributed to absorbance of the amide and suggested that protamine and the enzymes were indeed encapsulated within the nanoparticles.8,19 As shown in Figure 4a, there are C, O, Ti, and N elements in the XPS survey spectrum. Three synthetic components (C1, C2, and C3) were corresponding to the following bonds: aliphatic CHs*CH (BE ) 285.0 eV), oxidrilic *CsOH, and amidic Ns*CHsCO (BE ) 286.4 eV) and NsCHs*CO (BE ) 288.3 eV) in the amidic group, where the respective carbon species were marked by asterisks. It was found that the O 1s XPS spectrum was composed of three peaks at 529.8, 533.2, and 532 eV, which can be assigned to TisOsTi, OdC, and CsO bonds, respectively. The analysis of the binding energies of the N 1s (BE ) 400.7 eV) and O 1s (BE ) 532.9 eV) peaks confirmed that these elements were also attributable to protamine and dehydrogenases. Excluding the aliphatic carbon C1, which could be originated from the surface contamination, all other chemical species (C2, C3, N, and O) could be used as fingerprints of the protein (protamine or enzymes). Compared to the composites without enzymes, the O/C and N/C ratios for composites containing enzymes were both much lower owing to the excess of C element in the dehydrogenases, which confirmed the encapsulation of the enzymes (Figure 3a). Furthermore, it was observed that the binding energies of Ti 2p3/2 and 2p1/2 were centered at 458.8 and 464.6 eV, respectively, which confirmed that the titanium occurs in the form of titania.20,21 To judge whether the enzymes were adsorbed on surfaces or trapped within the matrix of the titania, particles without enzymes were prepared and then incubated in dehydrogenases solution for a certain period. After washed three times, no enzyme was retained onto the particles, which confirmed that

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Figure 1. SEM micrograph of enzyme-containing titania particles (a) and EDS spectrum of the titania particles (b).

Figure 2. Pore size distribution curves by the BJH (Barrett-Joiner-Halenda) method. (inset) Nitrogen adsorption-desorption isotherms of titania after calcination: protamine-induced titania particles.

Figure 3. FTIR spectrum of the multienzyme-containing titania particles.

enzyme physically encapsulated within the titania matrix during the titania formation. 2. Conversion of CO2 to Methanol Catalyzed by Coimmobilized Multienzymes. The development of versatile and efficient coimmobilization system is of great interest in enzyme catalysis and controlled drug delivery.22,23 The strategy developed in this study may provide a facile and green approach for the encapsulation of multienzymes. Figure 5 illustrated the enzymatic reduction of CO2 as a function of concentration of NADH. Initially, methanol was not detected when NADH was not added, but its amount increased progressively at elevated NADH concentration, indicating that NADH serves as a limiting reagent in the overall reduction. Due to this fact, the yield of the methanol production was calculated based on the above-

mentioned reaction, 3 mol of NADH were consumed per mol of methanol produced. As such, for 100% yield, the moles of methanol produced should be 1/3 of the NADH added. 6,7,24 Under optimal conditions (35 °C and pH 7.0), the enzyme activity of free and encapsulated multienzymes in titania particles was investigated. As shown in Figure 6, the bioconversion process was monitored by measuring the methanol with the lapse of time. The reaction rate and the final conversion using encapsulated dehydrogenases were higher than those using free dehydrogenases. The equilibrium conversion using free dehydrogenases was obtained in 4 h, whereas that in the case of encapsulated dehydrogenases was 8 h. Additionally, as shown in Figure 7, the yield of methanol was very low in the aqueous solution phase (5-10%). Under identical conditions, the yields of methanol ranged from 35% to 60% in the coimmobilization system. The enhanced yield could be owing to the confinement effect of the titania matrix, which immobilized the multienzymes within the nanoparticles and might alter the kinetics of the reaction.6 Recently, a conceptual integrated architecture “pseudocell factory” was proposed, which could be defined as a multifunctional system that mimicked living cell as well as using biomolecules in an artificial environment or device.25 As shown in Scheme 2, the enzyme containing titania particles could be regarded as a pseudocell factory, which is comprised of three essential components: (1) the titania particle as the structural scaffold; (2) the three dehydrogenases as biochemical machinery; (3) the transport of substrates or products. For the reaction system in this work, the transformation from CO2 to methanol involved the formation of two intermediates. For a separately encapsulated or free multienzymes system, the intermediates had to be generated by one enzyme and then diffuse a relatively long distance to interact with another enzyme until the formation of methanol, and it could be considered that the concentration of the substrates and intermediates were low and homogeneous.6,7,24 In contrast, by encapsulating multienzymes within the particle, an enzymatic assembly line was constructed, which could alter the incoming substrates or “manufacture” the product by the immobilized multiple enzymes because their active sites were accessible only from the interior of the system.25 The particles elevated the local concentration of the intermediate products and integrated three enzymes within a defined small area which substantially reduced the distance for the intermediate chemicals needed to travel between the active sites of the enzymes as compared to free enzyme system and, thus, increased the overall rate of methanol production.6,7,24 A comparison of production yield based on different amount of NADH added indicated that the increased cofactor concentration leads to the increased reaction rate, but the overall reaction

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Figure 5. Plot of methanol produced as a function of terminal electron donor (NADH) present in aqueous solution and titania particles.

Figure 6. Plot of methanol produced as a function of reaction time in aqueous solution and titania particles.

Figure 7. Plot of methanol yield as a function of terminal electron donor (NADH) present in aqueous solution and titania particles.

Figure 4. XPS spectrum (a) and high resolution spectra C 1s (b), N 1s (c), O 1s (d), and Ti 2p (e) of the multienzyme-containing titania particles.

equilibrium may become limiting and thus the utilization of the cofactor decreased (Figures 5). It should be noted that the overall yield of the reaction was decreased at higher concentration of NADH presumably due to an increased tendency of the reverse reaction.6

Additionally, the biomimetic mineralization process, which occurred at neutral pH and mild temperature, may preserve the activity of enzymes more effectively. The comparison was made between the multienzymes immobilized by traditional sol-gel and biomimetic mineralization process, which revealed that under identical conditions, amount of methanol catalyzed by each mole of enzymes was higher in the biomimetic titania particles than that in traditional sol-gel silica.6 The higher yield of methanol raised the question of whether these reactions were influenced by the titania matrix, which was examined by the following control experiments. The particles without enzyme did not generate any methanol under identical conditions. Furthermore, several kinds of titania particles with systematic exclusion of one or more of the four species (i.e., FateDH, FaldDH, ADH, and NADH) were prepared in order to confirm whether all four species must be present for methanol production. It was observed that the reaction system without any of the four components failed to produce methanol. From

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Figure 8. Effect of temperature on the methanol yield catalyzed by coimmobilized dehydrogenases.

Figure 9. Effect of pH on the methanol yield catalyzed by coimmobilized dehydrogenases.

Scheme 2. Coimmobilized Multienzyme System

Figure 10. Recycling stability of coimmobilized dehydrogenases.

the results of these control experiments, it could be concluded that the production of methanol came from the enzymic reaction, but not from the catalysis of titania particles. Next, the methanol yield at a series of temperatures catalyzed by free and immobilized enzyme was investigated. Meanwhile, the effect of temperature on the activity of immobilized dehydrogenases for the reduction was studied at pH 7.0 in 0.05 M tris-HCl buffer. It is reported that the optimum temperature of enzymatic reaction catalyzed by the three dehydrogenases was37(FateDH),37(FaldDH),and25°C(ADH),respectively.26-28 As shown in Figure 8, both the immobilized dehydrogenases and the free dehydrogenases had a high activity in the range of 27-37 °C, which was a combined optimization of the three kinds of enzymes. In addition, the reduction catalyzed by immobilized enzymes exhibited a much higher yield at wider range of temperatures than the free enzymes which could also be explained by the confinement effect of the multienzyme system discussed above.29,30 The effect of pH on the activity of dehydrogenases for the reduction of CO2 to methanol was studied at 37 °C in 0.05 M tris-HCl buffer. The optimum pH values of the enzymatic reaction catalyzed by the three dehydrogenases was observed atpH7.0(FateDH),7.0(FaldDH),and7.0(ADH),respectively.26-28 Figure 9 demonstrates the maximum yields of methanol catalyzed by both immobilized and free dehydrogenases were at pH 7.0. In general, changes in pH affected the charges carried by different amino acid residues, leading to enzyme unfolding,

Figure 11. Storage stability of coimmobilized dehydrogenases.

which resulted in the loss of native structure and functionality of enzyme. However, the reaction showed high yield at pH 6.0 and 8.0. The reasons for the resistance of encapsulated enzymes against the acidic and alkaline changes in medium were tentatively analyzed as follows: (1) the physical cage confinement; (2) the pI value of titania was about 6; therefore in acidic medium, the positively charged titania shell could repel the H+ ions to some extent, assisting in preventing the H+ from diffusing into and contacting with the enzyme. On the contrary, in alkaline medium, the added protection ability against hydroxyls could be attributed to the negative charge repulsion of the whole system: enzymes and titania.31,32 Figure 10 revealed that more than 50% of its initial activity was retained after eight cycles for dehydrogenases encapsulated in the titania particles. The high recycling stability could be attributed to the complete confinement of titania particles. Given the pore size of the titania (about 3.8 nm), enzyme molecules with the size of 7 nm were unlikely to migrate into the exterior of the titania particles. The loss of yield might be caused by

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the breakage of the particles after multiple operations under continuous stirring.33 The yield of methanol catalyzed by dehydrogenases entrapped in titania particles was measured after storage for a specified time period. As Figure 11 revealed, the entrapped enzyme demonstrated better long-term stability than in solution, likely because of the protection from unfolding within a rigid titania pocket, as well as the antibiotic function of protamine and titania.18,33 Conclusions In this study, a green and efficient enzymatic system was constructed through a biomimetic approach in which three different dehydrogenases worked as an enzymatic assembly line, efficiently converting carbon dioxide into methanol under mild conditions. Compared to the free enzyme system, the immobilized enzymes showed enhanced yields for production of methanol due to the more favorable molecular interactions among enzymes in a nanoscale environment. Furthermore, the coimmobilized multienzymes exhibited superior catalytic activity and stability. Hopefully, such a kind of integrated multienzyme system will provide a novel solution for green and efficient utilization of carbon dioxide. Acknowledgment The authors are thankful for the financial support from the National High-Tech Research and Development Plan (No. 2007AA10Z305), the Cross-Century Talent Raising Program of Ministry of Education of China, the program for Changjiang Scholars and Innovative Research Team in University (PCSIRT), and the Program of Introducing Talents of Discipline to Universities (No. B06006) Literature Cited (1) Aresta, M.; Dibenedetto, A. Utilisation of CO2 as a Chemical Feedstock: Opportunities and Challenges. Dalton Trans. 2007, 2975. (2) Sakakura, T.; Choi, J. C.; Yasuda, H. Transformation of Carbon Dioxide. Chem. ReV. 2007, 107, 2365. (3) Aresta, M.; Dibenedetto, A.; Pastore, C. Biotechnology to Develop Innovative Syntheses Using CO2. EnViron. Chem. Lett. 2005, 3, 113. (4) Aresta, M.; Dibenedetto, A. Development of Environmentally Friendly Syntheses: Use of Enzymes and Biomimetic Systems for the Direct Carboxylation of Organic Substrates. ReV. Mol. Biotechnol. 2002, 90, 113. (5) Aresta, M.; Dibenedetto, A. Mixed Anhydrides: Key Intermediates in Carbamates Forming Processes of Industrial Interest. Chem.sEur. J. 2002, 8, 685. (6) Obert, R.; Dave, B. C. Enzymatic Conversion of Carbon Dioxide to Methanol:Enhanced Methanol Production in Silica Sol-Gel Matrices. J. Am. Chem. Soc. 1999, 121, 12192. (7) Xu, S.; Lu, Y.; Li, J.; Jiang, Z. Efficient Conversion of CO2 to Methanol Catalyzed by Three Dehydrogenases Co-encapsulated in an Alginate-Silica (ALG- SiO2) Hybrid Gel. Ind. Eng. Chem. Res. 2006, 45, 4567. (8) Jiang, Y.; Yang, D.; Zhang, L. Biomimetic Synthesis of Titania NanoparticlesInduced by Protamine. Dalton Trans. 2008, 4165. (9) Zhang, Y.; Wu, H.; Li, J. Protamine-Templated Biomimetic Hybrid Capsules:Efficient and Stable Carrier for Enzyme Encapsulation. Chem. Mater. 2008, 20, 1041. (10) Albrecht, M. A.; Evans, C. W.; Raston, C. L. Green Chemistry and the Health Implications of Nanoparticles. Green Chem. 2006, 8, 417.

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ReceiVed for reView December 15, 2008 ReVised manuscript receiVed March 5, 2009 Accepted March 10, 2009 IE801931J