Utilization of Greenhouse Gases - American Chemical Society

Chapter 14 Enzymatic Conversion of Carbon Dioxide to Methanol by Dehydrogenases Encapsulated Downloaded by NORTH CAROLINA STATE UNIV on September 13, 2012 | http://pubs.acs.org Publication Date: June 26, 2003 | doi: 10.1021/bk-2003-0852.ch014

in Sol-Gel Matrix Zhongyi Jiang, Hong Wu, Songwei Xu, and Shufang Huang State Key Laboratory of C Chemistry and Chemical Technology and School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China 1

The effective generation of methanol directly from gaseous carbon dioxide has received considerable attention in recent years since it can recycle the greenhouse gas and produce a clean fuel. Herein, we report an enzymatic approach for carbon dioxide fixation using formate dehydrogenase (F DH), formaldehyde dehydrogenase (F DH) and alcohol dehydrogenase (ADH) co-encapsulated in a silica gel as the catalysts. The gels were prepared by a modified sol-gel process that uses tetramethoxysilane (TMOS) as the precursor and nicotinamide adenine dinucleotide (NADH) as an electron donor. The enzymatic conversion of CO to methanol was carried out at low temperatures and low pressures. The effects of the reaction temperature, pH value, the amount of enzyme and the amount of N A D H on the yields of methanol have been investigated. The highest yield of methanol was 92.1%. The activity of immobilized enzymes was a little lower than that of the free enzymes due to the minor conformation change of the enzyme and the existence of additional diffusion hindrance. ate

ald

2

212

© 2003 American Chemical Society

In Utilization of Greenhouse Gases; Liu, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

213

Downloaded by NORTH CAROLINA STATE UNIV on September 13, 2012 | http://pubs.acs.org Publication Date: June 26, 2003 | doi: 10.1021/bk-2003-0852.ch014

Introduction Effective generation of methanol directly from carbon dioxide has received considerable attention in recent years since it can recycle the greenhouse gas and produce a clean fuel. Hydrogénation of carbon dioxide through heterogeneous catalysis, electrocatalysis and photocatalysis has been the predominant methods investigated (1-4). Herein, we report a novel and promising approach to convert carbon dioxide into methanol through consecutive reduction reactions catalyzed by three different dehydrogenases, which were encapsulated in silica gel matrices (5,6). The whole process consists of three steps (7-9): reduction of C 0 to formate catalyzed by formate dehydrogenase (F DH), reduction of formate to formaldehyde by formaldehyde dehydrogenase (Fai DH), and reduction of formaldehyde to methanol by alcohol dehydrogenase (ADH). Reduced nicotinamide adenine dinucleotide (NADH) acts as a terminal electron donor for each of the dehydrogenase-catalyzed reductions: 2

ate

d

FqfDH

3

F

o l d

DH

ADH

The enzymatic conversion of C 0 to methanol was carried out at low temperatures and low pressures. The suitable procedures for the efficient sol-gel co-encapsulation of the three dehydrogenases have been studied. The effects of the reaction temperature, pH, the amount of enzyme and the amount of N A D H on the yield of methanol have been investigated. The highest yield of methanol was 92.1%. The activity of the immobilized enzymes was a little lower than that of the free enzymes. This is due to the minor conformation change of the enzymes and the existence of additional diffusion hindrance. 2

Experimental Preparation of Immobilized Enzymes The sol-gel encapsulated enzymes were prepared as follows: Tetramethoxysilane (TMOS) was used as the precursor for making the silica sol-gel. The initial sol was prepared by mixing 1.94 g of TMOS and 1.10 g of 3%(v/v) HC1 solution. The mixture was then vigorously mixed for 10 min to form a sol. The 1 gels were prepared by adding 1.0 mL of the stock enzyme solution, which was comprised of 7.0 mg of F^eDH, 2.0 mg of F D H and 2.0 mg of A D H , to 1.0 mL of the sol in a polystyrene cuvette. Typical gelation times were on the order of 50-60 s. The cuvette was then covered with parafilm and the gel was allowed to age at 4 °C for 24-48 h. The 2 gels were prepared by adding 1.0 mL of the stock enzyme solution, which was comprised of 14.0 mg of F D H , 4.0 mg of F D H and 4.0 mg of #

ald

#

ate

ald


214 A D H , to 1.0 mL of the sol in a polystyrene cuvette. Typical gelation times were on the order of 5-10 s. The cuvette was then covered with parafilm and the gel was allowed to age at 4 C for 24-48 h. e


Characterization The size of gel particles was determined using a Materizer 2000 instrument (Malvern Instrumente Ltd., U K ) . A l l adsorption measurements of the dehydrogenases encapsulated in the aqueous TMOS-based silica gels were made with an UV-3010 Spectrometer (Hitachi, Japan). The pore size and surface areas were obtained using a CHEMBET-3000. Enzymatic Reactions The aged gels were put into a dialysis membrane using 250 mL of 0.1 M phosphate buffer at pH of 7 as the dialysis solution, and then placed in a refrigerator at 4 C with frequent change of the 0.1 M phosphate buffer. The dialysis lasted 24-48 h in order to completely remove the methanol or ethanol generated in the sol-gel process. Through dialysis 0.1 mL of N A D H solution diffused into the gel (the final concentration of N A D H varied from 0.025 to 0.1 M). The sample containing the gel and the N A D H solution was left undisturbed for 48 h. C 0 was then bubbled for 8 h through this mixture in order to produce methanol. The concentration of methanol produced was determined by gas chromatography (HP 6890). A calibration curve was established for aqueous methanol solutions at known concentrations of methanol. e

2

Results and Discussions Characterization of 1* Gel and 2 * Gel Table 1 gives the values of the relative absorbance (k) of the spectra and the corresponding spectral shift ( άλ ) for the 1 gel and the 2 gel. Both k and άλ were determined based on the characteristic absorption peak of the corresponding dehydrogenases in buffer solution, relative to those in the gels. Dehydrogenases encapsulated in TMOS-based gels exhibited no or little spectral shift, indicating that the enzyme conformation was preserved to a considerable extent after encapsulation (70). The change in spectra intensity, nevertheless, suggested an effect on the enzyme encapsulation by the sol-gel process (11,12). The surface areas of the 1 gel and the 2 gel were 260 m /g and 200 m /g, respectively, whereas the surface areas of the plain TMOS gel that contained no dehydrogenases was 280 m /g. The small decrease in surface area was probably #

#

#

#

2

2


2

215 due to the difference in the microenvironment created by the interactions of the enzymes with the matrices, which involved van der Waals forces and hydrogen bonding. The average pore size of the 1 gel and the 2 gel were 3.4 nm and 2.6 nm, respectively, whereas the average pore size of the plain TMOS gel was 2.4 nm. This shows that dehydrogenases acted as the template molecules to modify the sol-gel process (13,14). #

#


Table 1. Comparison of the spectral shift ( Δλ , nm) and the relative change of adsorbance intensity (k,%) of dehydrogenases encapsulated in the Gel 2*

k 26.5 40.8

Αλ 0 -0.5

Enzymatic Reactions The yields of methanol were calculated based on the amount of N A D H . As seen from the reaction scheme mentioned above, 3 mol of N A D H are consumed per mol of methanol produced. As such, for a 100% methanol yield, the moles of methanol produced should be 1/3 of the amount of N A D G added. Therefore: Yield of methanol (%)=3*moles of methanol product/initial moles of N A D H X100% From the preliminary experimental results, the feasibility of the enzymatic conversion of carbon dioxide to methanol has been successfully proved. The production of methanol is due to the enzymatic reactions and not due to the possible hydrolysis of the residual methoxides present in the TMOS sol-gels. This was confirmed by a control experiments performed with plain T M O S solgels. The plain TMOS sol-gels, without the encapsulated hydrogenases, did not produce any methanol under the identical reaction conditions. In addition, it was also found that all four species (i.e. F D H , F ^ D H , A D H , N A D H ) must be present to generate methanol. This was established by preparing several sol-gel with systematic exclusion of one or more of the four components. It was observed that the sol-gels prepared without any one of the four components failed to produce methanol. The highest yield of methanol using the 1 gel as the catalyst was 92.1%. Under the same reaction conditions, the yield of methanol using dehydrogenases in the free form as the catalyst was 98.1%. The decrease in the activity of the immobilized enzymes was mainly due to the minor conformation change of the enzymes and the extra diffusion hindrance for the substrate and product. ate

#


216 Effects of the Enzyme Amount Encapsulated in the Gels on the Methanol Yields #

#

Table 2 gives the methanol yield comparison for the 1 gel and 2 gel. The reason of the yield of methanol for the 2* gel is lower than that for the 1 gel is explained as follows: for the 2 gel, the gelation time was 5-10 s; for the 1 gel, the gelation time was 50-60 s. The faster gelation induces a more uneven distribution of the hydrogenases in the gel and also an un-favored mass transfer due to the decrease in pore size and surface area. #


#

#

#

Table 2. Effects of enzyme amount encapsulated in the 1* gel and the 2 gel on the enzymatic conversions of C 0 2

Gel

pH

1# 2# 1# 2#

7.0 7.0 7.0 7.0

Temperatur e/V 37 37 37 37

NADH amount Mmol 100 100 150 150

Pressure /Mpa 0.3 0.3 0.3 0.3

Yield /% 92.1 52.3 42.2 35.1

Effects of Reaction Temperatures on the Methanol Yields The three dehydrogenases have different optimum reaction temperatures. The optimum temperatures were 37 C for FaeDH and FaiJDH, whereas the optimum temperatures for A D H was 25 °C. The experiments were carried out at 25 C and 37 C in order to find the optimum temperature for the three enzyme coimmobilized in the same gel. The experimental results are shown in Table 3. They showed that the better reaction temperature is 37 °C. e

e

e

Table 3. Effects of temperature on the enzymatic conversions of C 0 Temperature /V

PH

NADH amount Mmol

Pressure /Mpa

Yield /%

25 37

7.0 7.0 7.0 7.0

0.3 0.3 0.5 0.3

30 92.1

25 37

100 100 150 150

27 42.2


2

217 Effects of p H Values on the Methanol Yields


Table 4 shows the effect of pH on the methanol yield. The highest methanol yield was obtained at pH=7.0. According to the tentative analysis of the reaction mechanism for dehydrogenase, the reduction uses N A D H as the electron donor and needs the existence of a proton. The lower pH was more favorable for protonation and thus more favorable for the increase in methanol yield.

Table 4. Effects of p H on the enzymatic conversions of C 0 Temperature /V 37 37 37

PH 7.0 7.5 8.0

NADH amount Mmol 100 100 100

Pressure /Mpa 0.3 0.3 0.3

2

Yield m 92.1 66.9 49.5

Effect of the Amount of N A D H on the Methanol Yield The effects of the amount of N A D H on the methanol yields are shown in Table 5. It is important to note that the overall yield of the enzymatic reactions was lower at higher concentrations of N A D H . This is presumably due to an increased tendency of this system to undergo the reverse reaction (i.e., conversion of methanol to carbon dioxide)(/5,/d)-

Table 5. Effects of the amount of N A D H on the enzymatic conversions NADH amount Mmol 50 100 150

Temperature /V 25 37 37

pH 8.0 8.0 8.0

Pressure IMPa 0.3 0.5 0.5

Yield /% 68.8 49.5 32.0

Conclusions The feasibility of enzymatic conversion of carbon dioxide to methanol is tentatively explored. The consecutive reduction of carbon dioxide by three different dehydrogenases encapsulated in a TMOS sol-gel matrix results in a


218 high yield of methanol. This will open up a new avenue not only for on-site production of methanol from cheap raw materials but also for the efficient fixation of the greenhouse gas carbon dioxide.


Acknowledgement The financial supports from the National Natural Science Foundation of China (under the contract 20176039) and from the 985 Project of State Key Laboratory of CI Chemistry and Technology of Tianjin University are greatly appreciated.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

References Wieser, M.; Yoshida, T.; Nagasawa, T. J. Mol Cata. B: Enzymatic. 2001, 11, 179-184. Kuwabata, S.; Tsuda, R., Yoneyama, H . J. Am Chem. Soc. 1994, 116, 5437-5443. Heleg, V.; Willner, I.; Chem. Commun. 1994,18, 2113-2114. Aresta,M.;Quaranta, E.; Liberio, R. Trtrahedron 1998, 54, 8841-8846. Obert, R.; Dave, B.C. J. Am. Chem. Soc. 1999,121,12192-12193. Kumar, Α.; Malhotra, R.; Malhotra, B.D. Anayltica Chimica Acta 2002, 414, 43-50. Gill, I.; Ballesteros, A. Trends in Biotechnology ,2000,75, 282-296. Kutzenko, A . S.; Lamzin, V . S.; Popov, V . O. FEBS Letters 1998, 423, 105-109. Palmore, G.T. R.; Bertschy, H.; Bergens, S. H . J. Electroanalytical Chem. 1998, 443, 155-161. Pocker, Y.; Page, J.D.;Li,H . Chemical-Biological Interactions 2001, 130132, 371-381. Dunn, B.; Miller, J.M.;Dave, B.C. Acat mater. 1998, 46, 737-741. Bhatia,R.B.;Brinker,C.J. Chem.Mater. 2000,12,2434-2441. Liu, D.M.;Chen, I-W. Acta mater. 1999, 47,4535-4544. Wei, Y . ; Xu, J-G.; Feng, Q-W. Mater. Lett. 2000, 44,6-11. L i , C-I.; Lin, Y - H ; Shih, C-L. Biosensors and Bioelectronics 2002,17,323330. Pocker, Y . Chemical-Biological Interactions 2001,130-132, 383-393.


Utilization of Greenhouse Gases - American Chemical Society

Recommend Documents