J . Org. Chem. 1996, 61, 2125-2128
2125
Cr oss-Lin k ed LDH Cr ysta ls for La cta te Syn th esis Cou p led to
Electr oen zym a tic Regen er a tion of NADH
,
§
‡
Susan B. Sobolov,* Mihaela Draganoiu Leonida, Anita Bartoszko-Malik, Kamen I. Voivodov,
Frank McKinney, J ason Kim, and Albert J . Fry*,
†
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459
Received J uly 31, 1995 (Revised Manuscript Received November 27, 1995X)
Lactate dehydrogenase (LDH) was crystallized from concentrated ammonium sulfate solution and
cross-linked with glutaraldehyde to afford long-lived enzymatically active cross-linked crystals (LDH-
CLC). The crystals were employed in an electrolytic cell for lactate production from pyruvate, in
which the cathode consisted of a carbon electrode containing a coating of lipoamide dehydrogenase
(LiDH) immobilized with methyl viologen under a Nafion membrane. This cell was more effective
than a similar cell containing LDH in soluble form. An even greater improvement in performance
was achieved by chemically binding a viologen derivative to the LiDH and using an electrode based
on this modified enzyme in a cell containing LDH-CLC. The activity of the LDH-CLC is much less
sensitive to pH than that of the soluble enzyme.
1
The high cost and instability of the cofactors required
in enzymatic syntheses justifies efforts to regenerate
them. Since 1980, a number of experiments have been
reported in which NADH has been regenerated using
Many studies have been reported of ways to immobilize
enzymes under a variety of conditions to extend their
lifetimes for practical use. There are a number of general
approaches to this problem, but each has disadvantages
2
,3
10
electrochemical methods.
duction of NAD does not produce NADH; instead, a
catalytically inactive dimer is formed. A common solu-
tion to this problem is to utilize two enzymes in the
electrosynthesis; one enzyme transfers a reducing equiva-
Direct electrochemical re-
of one sort or another. In general, enzymes are much
+
more stable in the crystalline state than in solution. For
this reason, enzyme crystallization or precipitation is
useful not only as a method of purification but also for
long-term storage. This makes the crystalline form of
the enzyme very interesting from a practical point of
view. However, even under the most favorable condi-
tions, enzyme crystals remain mechanically fragile and
this affects their operational stability. The solution to
2
+
lent from an electrogenerated redox mediator to NAD ,
while a second enzyme carries out the actual synthesis
using the NADH produced in the first step.4
,5
The
synthetic sequence is illustrated in Figure 1, where the
first enzyme is lipoamide dehydrogenase (LiDH), the
second enzyme is lactate dehydrogenase (LDH), and the
redox mediator is methyl viologen (MV ). A problem
encountered in all such electroenzymatic reactions is the
instability of the enzymes in solution. We and other
research groups have shown that the first (NADH-
the fragility problem has been found to be cross-linking
of enzyme crystals in an insoluble matrix.1
1-14
This is
2
+ 2
usually carried out by cross-linking amino groups on the
enzyme with glutaraldehyde, a practice used by crystal-
lographers as early as the 1960s.14 Cross-linking stabi-
lizes the crystalline structure of the enzyme by forming
immobilized enzyme particles which retain the enzymatic
activity of crystals. Recent studies have shown cross-
linked enzyme crystals (CLC) to be highly pure, very
active, stable, and insoluble biocatalysts.1
regenerating) enzyme can be stabilized by immobilizing
it on the electrode surface.1
,6-8
However, the instability
of the second enzyme remains a problem. For this
reason, it is generally necessary to add fresh enzyme to
1-13
In our previously reported experiments,6,7 the electro-
enzymatic regeneration of NADH was coupled with
electroenzymatic syntheses of several organic acids.
Lactic, malic, and glutamic acids were prepared success-
fully utilizing the appropriate substrates and redox
enzymes. Because of the instability of the soluble
enzymes studied, it was necessary to add fresh enzyme
to the cell every other day in long-term electrosynthetic
reactions. As a solution to this problem, we describe here
the electrolysis solution periodically during the course
It is this problem which
we wish to address in the present paper.
of an extended electrolysis.1
-3,9
†
Address correspondence to Department of Chemistry, Wesleyan
University, Middletown, CT 06459. Phone (860) 685-2622; FAX (860)
6
85-2211; e-mail afry@wesleyan.edu.
Current address: Pfizer Corp., Eastern Point Dr., Groton, CT
§
0
6340. Phone (860) 441-1516; FAX (860) 441-6543.
Current address: Department of Food Science and Technology,
†
University of California, Davis, CA.
X
Abstract published in Advance ACS Abstracts, February 15, 1996.
(
1) Current prices (Sigma) of NADH are $13 000-$25 000 or more
per mole, depending on purity.
2) Fry, A. J .; Sobolov, S. B.; Leonida, M. D.; Voivodov, K. I.
Tetrahedron Lett. 1994, 35, 5607.
(9) Grimes, M. T.; Drueckhammer, D. G. J . Org. Chem. 1993, 58,
6148.
(10) (a) Zaborsky, O. R. Immobilized Enzymes, CRC Press: Cleve-
land, 1973. (b) Wong, C.-H.; Whitesides, G. M., Elsevier: Tarrytown,
NY, 1994.
(11) St. Clair, N. L.; Navia, M. A. J . Am. Chem. Soc. 1992, 114, 7314.
(12) Navia, M. A.; St. Clair, N. L.; Griffith, J . P. in Stability and
Stabilization of Enzymes (Proceedings of Int. Symposium); van den
Tweel, W. J . J .; Harder, A., Buitelaar, R. M., Eds.; Elsevier Science
Publishers B.V.: Maastricht, The Netherlands, 1993; p 63.
(13) Sobolov, S. B.; Bartoszko-Malik, A.; Oeschger, T. R.; Montel-
bano, M. M. Tetrahedron Lett. 1994, 35, 7751.
(14) (a) Quiocho, F. A.; Richards, F. M. Proc. Natl. Acad. Sci. U.S.A.
1964, 52, 833. (b) Quiocho, F. A.; Richards, F. M. Biochemistry 1966,
5, 4062. (c) Quiocho, F. A.; Bishop, W. H.; Richards, F. M. Proc. Natl.
Acad. Sci. U.S.A. 1967, 57, 525.
(
(
(
3) Blaedel, W. J .; Engstrom, R. C. Anal. Chem. 1980, 52, 1691.
4) Roerig, P.; Wolff, C. M.; Schwing, J . P. Anal. Chim. Acta 1983,
1
53, 181.
5) DiCosimo, R.; Wong, C.-H.; Daniels, L.; Whitesides, G. M. J . Org.
Chem. 1981, 46, 4622.
6) Fry, A. J .; ; Sobolov, S. B.; Leonida, M. D.; Voivodov, K. I. Denki
Kagaku 1994, 82, 1260.
7) Voivodov, K. I.; Sobolov, S. B.; Leonida, M. D.; Fry, A. J . Bioorg.
Med. Chem. Lett. 1995, 5, 681.
8) Chang, H.; Matsue, T.; Uchida, I. In Electroorganic Synthesis-
(
(
(
(
Festschrift for Manuel M. Baizer; Little, R. D., Weinberg, N. L., Eds.;
Dekker: New York, 1991, p 281.
0
022-3263/96/1961-2125$12.00/0 © 1996 American Chemical Society