COMMUNICATION
tioned above.[3] It is therefore doubtful whether these three
concepts can be generally applied.
been proposed for cysteine[8] and a high reactivity between
cysteine and heavy metals had also already been observed
for other enzymes.[10] Moreover, the inactivating effects of
tryptophan and histidine are very strong, since the heteroar-
omatic rings can insert into the rhodium coordination
sphere. Other amino acids have less influence on the catalyt-
ic activity of the mediator, although in this case the a-amino
groups may also interact with the mediator. In a protein,
these groups would not be reactive as part of the peptide
bonds. This becomes apparent on the basis of the high resid-
ual activity with proline, which does not contain a free
amino group.
In the alcohol dehydrogenase from Lactobacillus brevis
(Lb-ADH), which is frequently used in both laboratory
scale and commercial scale, eight of these inactivating
amino acids are present[11] (2Cys, 4His and 2Trp), so that in-
activation is inevitable.
Since enzyme activity was still present at the end of the
reaction when a nonreactive protein was added, we thus
concluded that the termination of the reaction cannot be
brought about solely by the inactivated enzyme but also by
inactivated mediator. As yet, the stability of the mediator
under reaction conditions has received little attention in the
literature. To investigate the interactions between the medi-
ator and the amino acids in more detail, we determined the
influence of amino acids on the mediator activity by cyclic
voltammetry. In the presence of amino acids, an in part dras-
tic reduction in the cathodic peak current was detected,
which is directly correlated with the catalytic activity of the
mediator. Figure 1 shows the ratio of the peak currents in
the presence and absence of amino acids.
These findings make it clear why a stable electroenzymat-
ic reaction system is so difficult to establish. The inactivation
of the mediator by tryptophan cannot be prevented by mod-
ifying the thiol and amino groups.[6] Nucleophilic additives
cannot stabilise the reaction system either since the free co-
ordination site at the mediator, which is necessary for cata-
lytic activity, can always also react with the above-men-
tioned amino acids.[9] The addition of a nonreactive protein
leads to the retention of some of the enzyme activity but
only by completely inactivating the mediator, which likewise
leads to a termination of the reaction.[2] However, restricting
the choice of enzymes to those without these amino acids
present at accessible sites clearly negates a general applica-
bility of the process.
The only possibility of stabilising the enzyme and media-
tor and of establishing an efficient process is to create a spa-
tial separation. A compartmentalisation of the electrochemi-
cal activation of the mediator and cofactor reduction, on the
one hand, and the enzymatic synthesis reaction, on the
other hand, prevents direct contact between the enzyme and
mediator and should thus completely prevent inactivation.
This separation is made more difficult by the fact that the
cofactor must be able to reach both reaction chambers for
electron transfer. Since the mediator and cofactor are very
similar with respect to molecular weight and polarity,[3] the
molecular weight of the mediator has to be increased to ach-
ieve selective membrane retention.
Figure 1. Activity loss of the mediator in the presence of amino acids. Re-
action conditions: 50 mmolLꢀ1 phosphate buffer, pH of 7; 2 mmolLꢀ1
mediator; 5 mmolLꢀ1 amino acid; potential 0 mV to ꢀ1000 mV versus
AgjAgCl; 100 mVsꢀ1
.
The measurements support the hypothesis that the activity
of the mediator is reduced in the presence of proteins.
Above all, the amino acids cysteine, histidine and trypto-
phan drastically reduce the activity of the mediator.[3] Even
if the nucleophilicity of the amino acids can vary as part of
a protein, nevertheless it is to be expected that proteins that
have these amino acids at accessible positions will inactivate
themselves and the mediator within a short period by reac-
tion with the mediator.
The water-soluble polymer bonding of a catalyst for reten-
tion in the reactor chamber has already become well estab-
lished for a large number of processes.[12,13] [Rh
ACTHNUGRTENUNG(bpy)] has al-
This is thus the first time that a systematic study has been
conducted to investigate the aspect of mediator stability in
connection with an enzyme reaction. These results enabled
us to assign the inactivation to certain amino acids and to
correct the previous hypothesis, which generally stated that
the basic amino acids and cysteine are involved in the inacti-
vation.[8] Whereas, as expected, cysteine and histidine led to
high losses of mediator activity, the effect was less pro-
nounced for the other two basic amino acids lysine and argi-
nine. A possible reaction with the mediator had already
ready been polymer-bound but only with a very high loss of
activity.[5,14] Since polymer bonding has to date only focused
on the retention of the mediator in the reactor chamber, in
which the enzyme was also present, the mediators only dis-
played minor stabilities.[15]
In contrast, we have developed a new synthesis route
leading to a hydrolysis-stable block polymer by the polycon-
densation of 2,2’-bipyridine-4,4’-di-aldehyde and a, w-func-
tionalised amino polyethylene glycol (M=6000 gmolꢀ1) with
subsequent imine reduction by sodium borohydride (see
Chem. Eur. J. 2009, 15, 4998 – 5001
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4999
Hildebrand, Falk
