Enzymatic conversion of carbon dioxide to methanol: Enhanced methanol production in silica sol-gel matrices [5]

Full text of DOI:10.1021/ja991899r

12192
J. Am. Chem. Soc. 1999, 121, 12192-12193
Enzymatic Conversion of Carbon Dioxide to
Methanol: Enhanced Methanol Production in Silica
Sol-Gel Matrices
shown to be effective matrices for stability and reactivity of
different proteins, enzymes, and other biosystems with retention
of biological reactivity upon encapsulation. Indeed, when the
enzymes are encapsulated in the porous silica sol-gel matrix, it
is found that the yield of methanol production is substantially
increased as compared to that in solution media.
7
Robyn Obert and Bakul C. Dave*
The reaction was studied in the solution phase by using an
enzyme stock solution that was comprised of 10 mg/mL of each
enzyme dissolved in 0.1 M phosphate buffer at pH 7. The reaction
mixtures used in this study were prepared by adding 1.0 mL of
the enzyme stock solution to 1.0 mL of NADH solution in a
polystyrene cuvette such that the final concentration of NADH
varied from 0.025 to 0.1 M. The cuvette was covered with
Parafilm, and gaseous CO was then bubbled through this solution
2
for 3 h using a small nozzle with an approximate outlet diameter
of 0.5 mm through a hole made in the Parafilm. The extended
time for bubbling of CO ensured that reaction was allowed to
2
Department of Chemistry and Biochemistry
Southern Illinois UniVersity at Carbondale
Carbondale, Illinois 62901-4409
ReceiVed June 8, 1999
2
Strategies for effective conversion of atmospheric CO to
methanol offer promising new technologies not only for recycling
of the greenhouse gas but also for an efficient production of fuel
1
alternatives. Partial hydrogenation of carbon dioxide has been
2
accomplished by means of heterogeneous catalysis, electroca-
3
4
talysis, and photocatalysis. Oxide-based catalysts are predomi-
nantly used for industrial fixation of carbon dioxide.2 A unique
approach in this direction involves the use of enzymes as catalysts
for conversion of carbon dioxide to methanol.5 The use of
enzymes is particularly appealing since it provides a facile low-
temperature route for generation of methanol directly from
gaseous carbon dioxide.
go to completion and equilibrium was established. The Parafilm
cover was used to prevent extensive loss of methanol produced
due to evaporation.
-4
Quantitative measurement of methanol was carried out by using
gas chromatography (GC). A calibration curve was established
for aqueous methanolic solutions with known concentrations of
methanol ranging from 0.001 to 0.05 M. To evaluate the
concentration of methanol produced as a result of the enzyme-
catalyzed reaction, 1.0 µL of the final reaction solution was used
for GC measurements. The concentration of methanol was
calculated by using peak areas for the characteristic methanol band
in the chromatogram.
Herein, we report an enzymatically coupled sequential reduction
of carbon dioxide to methanol by using a series of reactions
catalyzed by three different dehydrogenases. Overall, the process
2
involves an initial reduction of CO to formate catalyzed by
formate dehydrogenase (FateDH), followed by reduction of formate
to formaldehyde by formaldehyde dehydrogenase (FaldDH), and
finally formaldehyde is reduced to methanol by alcohol dehy-
drogenase (ADH). In this process, reduced nicotinamide adenine
dinucleotide (NADH) acts as a terminal electron donor for each
dehydrogenase-catalyzed reduction. The overall reaction process
is shown in Scheme 1.
The sol-gel encapsulated samples were prepared by using the
biocompatible synthesis method previously reported in the
7a
literature. Tetramethoxysilane (TMOS) was used as precursor
for making the silica sol-gel. The initial sol was prepared by
mixing 3.82 g of TMOS, 0.85 g of water, and 0.055 g of 0.04 M
HCl. The mixture was then sonicated for 20 min to form sol.
The gels were prepared by adding 1.0 mL of the enzyme stock
solution to 1.0 mL of the sol in a polystyrene cuvette. Typical
gelation times are on the order of 10-30 s. The cuvette was then
covered with Parafilm and gel was allowed to age at 4 °C for 24
h. After the initial aging process, the gels shrink and can be
removed from the cuvette. The aged gel was then transferred to
a beaker containing 250 mL of 0.1 M phosphate buffer at pH 7
and placed in refrigerator at 4 °C for 24 h. The gel was then
transferred to another 250 mL beaker containing fresh 0.1 M
phosphate buffer at pH 7 and was placed in the refrigerator for
another 24 h. This step was repeated once more, for a total of 72
h of soaking in the buffer bath, to ensure complete removal of
2
Our strategy for CO reduction takes advantage of the fact that
dehydrogenases can effectively catalyze the reverse reactions (i.e.
reduction) in the presence of suitable electron donors.5 The ability
of the dehydrogenases to catalyze the reverse reactions in the
presence of an excess of NADH is well-established. Additionally,
since the process involves a sequential reaction of in situ generated
substrates with three different enzymes, it was expected that
confinement of the system in a porous matrix would result in an
enhanced probability of primary reaction events due to an overall
increase in local concentration of reactants within the nanopores
of the sol-gel processed glasses. The silica sol-gels have been
,6
*
To whom correspondence should be addressed.
1) (a) Catalytic ActiVation of Carbon Dioxide; Ayers, W. M., Ed.; ACS
Symp. Ser. No. 363; American Chemical Society: Washington, DC, 1988.
b) Methanol Production and Use; Cheng, W.-H., Kung, H. H., Eds.; Marcel
Dekker: New York, 1994.
2) (a) Fan, L.; Fujimoto, K. Chem. Lett. 1994, 105. (b) Eliasson, B.;
Kogelschatz, U.; Xue, B.; Zhou, L.-M. Ind. Eng. Chem. Res. 1998, 37, 3350.
c) Fan, L.; Fujimoto, K. Energy ConVers. Mgmt. 1995, 36, 633. (d) Fan, L.;
Fujimoto, K. Appl. Catal. A: Gen. 1993, 106, 1. (e) Fujitani, T.; Saito, M.;
Kanai, Y.; Watanabe, T.; Nakamura, J.; Uchijima, T. Appl. Catal. A: Gen.
(
methanol generated due to hydrolysis of TMOS during the sol-
gel process.8
(
After the initial equilibration, the gel was transferred to a
standard polystyrene cuvette followed by addition of 1.0 mL of
NADH solution (the final concentration of NADH varied from
(
(
0
.025 to 0.1 M). To allow the NADH to diffuse into the gel, the
sample containing the gel and the NADH solution was left
undisturbed for 48 h. To this mixture, CO was then bubbled for
h for production of methanol. The concentration of methanol
1
995, 106, 199.
2
(
3) (a) Azuma, H.; Hashimoto, K.; Hiramoto, M.; Sakata, T. J. Electrochem.
3
Soc. 1990, 137, 1772. (b) Noda, H.; Ikeda, S.; Oda, Y.; Inai, K.; Maeda, M.;
Itoh, K. Bull. Chem. Soc. Jpn. 1990, 63, 2459. (c) Beley, M.; Collins, J. P.;
Ruppert, R.; Sauvage, J. P. J. Am. Chem. Soc. 1986, 108, 7461. (d) Lieber,
C. M.; Lewis, N. S. J. Am. Chem. Soc. 1984, 106, 5033. (e) Bolinger, C. M.;
Story, N.; Meyer, T. J. Inorg. Chem. 1988, 27, 4582.
produced was determined using GC by taking a 1.0 µL aliquot
of the solution.
The results for methanol production in solution and the sol-
gel system are shown in Figure 1. The amount of each enzyme
(
4) (a) Heleg, V.; Willner, I. J. Chem. Soc., Chem. Commun. 1994, 2113.
b) Kuwabata, S.; Nishida, K.; Tsuda, R.; Inoue, H.; Yoneyama, H. J.
(
Electrochem. Soc. 1994, 141, 1498. (c) Heleg-Shabtal, V.; Zahavy, E.; Willner,
I. Energy ConVers. Mgmt. 1995, 36, 609.
(7) (a) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, S. A.; Dunn,
B. S.; Valentine, J. S.; Zink, J. I. Science 1992, 255, 1113. (b) Dave, B. C.;
Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120A. (c) Avnir,
D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605. (d) Wang,
R.; Narang, U.; Prasad, P. N.; Bright, F. V. Anal. Chem. 1993, 65, 2671.
(
5) Kuwabata, S.; Tsuda, R.; Yoneyama, H. J. Am. Chem. Soc. 1994, 116,
5
437.
(
6) (a) Ruschig, U.; M u¨ ller, U.; Willnow, P.; H o¨ pner, T. Eur. J. Biochem.
1
976, 70, 325. (b) Parkinson, B. A.; Weaver, P. F. Nature 1984, 309, 148. (c)
(8) GC of the external solution prior to addition of NADH and CO
2
was
Mandler, D.; Willner, I. J. Chem. Soc., Perkin Trans. 1988, 997.
used to ascertain that there was no residual methanol present in the system.
1
0.1021/ja991899r CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/09/1999

Communications to the Editor
J. Am. Chem. Soc., Vol. 121, No. 51, 1999 12193
Scheme 1
that the enhanced production of methanol is an intrinsic feature
of the nanoconfined reaction system brought about by subtle
influence of the sol-gel matrix.
The results obtained in this study indicate that confinement of
the multienzyme system in the nanopores of silica sol-gels alters
the reaction thermodynamics and final equilibrium of the reaction.
The yield of the reaction is very low in the solution phase (10-
20%). However, for the same concentration of NADH, it is seen
that in the sol-gel, the production of methanol is significantly
enhanced as compared to solution with yields ranging from 40
to 90% indicating that the overall equilibrium is shifted more
toward the products. It is important to note that the overall yield
of the reaction is lowered at higher concentration of NADH
presumably due to an increased tendency of the system to undergo
the reverse reaction (i.e. conversion of methanol to carbon
dioxide).
Although at present a detailed understanding of the reaction
kinetics of the system remains to be fully evaluated, the
enhancement of methanol production in sol-gel can be tentatively
attributed to confinement and matrix effects.10 The immobilized
system in the nanopores of a sol-gel matrix is characterized by
limited pore volume. As such an increased local concentration
of enzymes and reactants is likely to prevail in the porous structure
of silica sol-gel.10 As a result, the overall efficiency of enzyme
reactions is enhanced such that the final equilibrium is shifted
more toward the product. The overall reaction involves conversion
of a gas to a liquid, and such a shift in equilibrium toward products
is most likely consistent with reduction in available volume as a
result of confinement.
Figure 1. Plot of methanol produced as a function of terminal electron
donor (NADH) present in solution and sol-gel matrix.
Table 1. Relative Comparison of Methanol Production in Solution
and Sol-Gel
solution
sol-gel
NAD
MeOH MeOH/
MeOH MeOH/
(µmol)
a
NADH % yielda
(µmol) (µmol) NADH % yield
5
0
1.3 ( 0.7 0.02
7.0 ( 0.9 0.07
7.8 15.2 ( 0.4 0.30
21.0 26.6 ( 0.6 0.26
20.4 28.5 ( 0.7 0.19
16.8 29.2 ( 0.6 0.15
91.2
79.8
57.0
43.8
1
1
00
50 10.2 ( 0.6 0.07
00 11.2 ( 0.9 0.05
2
a
%
yield ) [moles of MeOH/{0.33(moles of NADH)}] × 100.
in solution and sol-gels is 5 mg, and the amount of methanol
generated with respect to variation of NADH enables monitoring
of the overall efficiency of the reaction. The moles of methanol
produced are plotted as a function of the moles of the terminal
electron donor (NADH). Since NADH serves as a limiting reagent
in the overall reaction, it provides a relative measure of the
efficiency of the reaction and the yield of the methanol production.
As can be seen from Scheme 1, 3 mol of NADH are consumed
per mol of methanol produced. As such, for 100% yield, the moles
of methanol produced should be 1/3 of the NADH added. Table
In summary, the primary feasibility of enzyme catalysis for
1
1
efficient conversion of carbon dioxide to methanol is reported.
A sequential reduction of carbon dioxide by three different
dehydrogenases encapsulated in sol-gel matrix results in en-
hanced yields for generation of methanol. The efficient production
of methanol provides a facile pathway not only for on-site
generation of methanol from readily available resources but also
for potential applications related to energy technology and
environmental fixation of carbon dioxide.
1
compares the relative yield of methanol per mole of NADH
Acknowledgment. Financial support for this work from the Materials
Technology Center and the Office of Research Development and
Administration at SIU, Carbondale is gratefully acknowledged. R.O.
thanks the Department of Chemistry at SIU, Carbondale for an under-
graduate summer research fellowship.
added. The overall yield of the reaction in solution is very low.
However, in sol-gels, the production of methanol is substantially
enhanced.
The enhanced production of methanol is due to enzymatic
reactions and not due to probable hydrolysis of residual meth-
oxides present in the TMOS sol-gels. This is confirmed by
control experiments performed with plain TMOS sol-gels. The
sol-gel processed glasses without the encapsulated enzymes do
not generate any methanol under identical conditions of treatment
with carbon dioxide. Furthermore, it is found in order to generate
methanol all four species (i.e. FateDH, FaldDH, ADH, and NADH)
must be present. This was established by preparing several sol-
gels with systematic exclusion of one or more of the four
components. It is observed that sol-gels prepared without any9
of the four components fail to show any production of methanol.
Thus, from the results on control experiments, it can be concluded
JA991899R
(
9) TMOS sol-gels containing the following dopants were studied: (a)
NADH; (b) ADH + NADH; (c) FateDH + NADH; (d) FateDH + ADH; (e)
ateDH + FaldDH + ADH; (f) FaldDH + ADH + NADH; (g) FateDH + ADH
+ NADH; and (h) FateDH + FaldDH + NADH.
10) (a) Dave, B. C.; Dunn, B.; Zink, J. I. In Access in Nanoporous
F
(
Materials; Pinnavia, T., Ed.; Plenum Press: New York, 1995; pp 80-93. (b)
Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink J. I. In Nanotechnology; Chow,
G.-M., Gonsalves, K. E., Eds.; American Chemical Society: Washington, DC,
1
996; pp 351-365.
(
11) The sol-gel processed glasses retain full activity for months if they
are stored at 4 °C in pH 7 phosphate buffer. However, drying leads to rapid
loss of activity and the gels become inactive after a week.