D. Jung, M. Hartmann / Catalysis Today 157 (2010) 378–383
381
Table 1
Steady-state oxindole concentration at the reactor outlet and the calculated space-time-yield (STY) for different catalysts.
cconst., oxindole (mmol L−1)
STY (mmoloxindole h
−1
−1
gcatalyst )
sample
Activity (U)
CPO-SBA-15
150
150
23
0.16
0.27
0.55
0.78
0.007
0.010
0.346
0.243
CPO-GA-ATS-SBA-15
CPO-CLEA-MCF
CPO-CLEA-MCF
46
dole yield is significantly reduced from 23% to about 11.5% over
the mixture of 150 U CPO-GA-ATS-SBA-15 and 7.5 U GOx-GA-ATS-
SBA-15. After more than 48 h a final yield of 8.3% is obtained. The
roughly constant 2-oxindole yield is reached earlier as in case
of the adsorbed enzymes. Thus, we have to conclude that even
functionalized SBA-15 materials contain a certain amount of only
physisorbed enzymes, which were not removed by the washing
procedure employed during the catalyst preparation. This weak
interaction between the mesoporous host and the enzyme results
in rapid leaching of the enzyme during the first few hours of time-
on-stream, which is indicated by a strong decrease of the oxindole
yield.
Here, we discuss only leaching of the enzyme as the major cause
of the observed decrease in 2-oxindole concentration in the prod-
uct stream. In our opinion, enzyme deactivation plays only a minor
role. In the later case, deactivation will continue with time-on-
stream and a constant product flux is not achievable. This is indeed
observed for external hydrogen peroxide addition as shown in Fig. 3
Nevertheless, an increase in activity of immobilized CPO used by a
factor of two (from 23 to 46 U) and a three-fold increase in activity of
immobilized GOx (from 7 to 21 U) results in an enhanced final oxin-
dole yield by a factor of 1.4 (from 14% to 20%). It appears that in the
first 10 h the oxindole yield is higher for the smaller amount of cat-
alyst (23 U CPO-CLEA-MCF + 7 U GOx-CLEA-MCF). Both enhanced
enzyme leaching as well as faster deactivation due to a higher
local H O concentration for 46 U CPO-CLEA-MCF + 21 U GOx-CLEA-
MCF may contribute to the observed behavior. Moreover, the use
of a larger amount of catalyst results in a noticeable increase in
the length of the packed bed of the heterogeneous biocatalysts in
the reactor. Furthermore, a gradient in the indole–2-oxindole ratio
from the top to the bottom of the fixed-bed reactor is the conse-
quence of pumping the indole solution over the packed bed. The
concentration of indole is reduced from the top to the bottom of the
reactor. However, internal and external mass transfer limitations
also have to be considered.
2
2
Table 1 compares the different catalysts with respect to the
resulting final oxindole concentration at the reactor outlet and
the corresponding space-time-yield (STY). The space-time-yields of
physisorbed CPO and covalently anchored CPO on SBA-15 amount
(
right). Leaching of CPO and its deactivation with time leads to
a steady decrease of 2-oxindole yield down to zero within 75 h.
Furthermore, in our previous publication, we have demonstrated
employing SANS experiments that leaching of the enzymes from
the mesoporous host is the main cause for the reduction of the
oxindole yield [11].
−
1
−1
−1 −1
to 0.007 mmol h
g
and to 0.010 mmol h g , respectively. A
significant increase of the space-time-yield is realized by using the
cross-linked CPO in the mesoporous cages of MCFs. Here, 0.346
−
1
−1
However, it appears that based on units, that immobilized CPO
has to be filled into the reactor in excess compared to immobi-
lized GOx. According to Fig. 1, one catalytic cycle of GOx generates
one molecule of H O , while one molecule of indole is oxidized
and 0.243 mmol h
g are obtained for 23 U CPO-CLEA-MCF and
46 U CPO-CLEA-MCF, respectively. These results clearly confirm
that the CLEA-MCF biocatalysts show higher product yields and
better space-time-yields compared to physisorbed CPO on SBA-15.
That is because the MCF support (i) allows a higher enzyme loading
resulting in higher activity and (ii) offers an increased availabil-
ity of the enzyme for the substrate due to its three-dimensional
pore structure. Furthermore, as shown in Table 1, an increase in the
amount of catalyst results in higher product yields but in reduced
space-time-yields.
2
2
per catalytic cycle of CPO. The key to an optimum process is the
maximum utilization of H O2 generated for the catalytic oxidation
2
of CPO. Because the accessibility of the immobilized CPO for H O2
2
is reduced due to the immobilization onto SBA-15 exhibiting an
ordered structure consisting of pore channels, immobilized CPO
is added in excess. Using mesocellular foams as enzyme support,
the excess of immobilized CPO may be reduced in the reactor, pre-
sumably because the pore structure of MCF materials has various
pore entrances at the outer surface of the catalyst particle and the
diffusion of the reactants to the active sites is facilitated.
In contrast to the performance of enzymes physisorbed onto
SBA-15, the initial oxindole yield of the biocatalyst prepared by
physisorption on MCF amounts to only 17% and drops to almost
zero after 12 h. Due to the large-pore cages and entrances of the
MCF material, the enzymes cannot be partially stabilized as in the
case of SBA-15. The diameter of the SBA-15 pore channels has been
adjusted to a diameter closed to the enzyme size, which fits the
enzymes ideally. Therefore, the indole conversion using CPO-SBA-
In principle, the turnover number (TON) in enzyme catalysis
denotes the maximum value of the rate per catalytic site at sat-
uration of the enzyme by the reacting substrate as defined by the
Michaelis–Menten kinetics. Subsequently, the rate referred to the
number of catalytic sites is known as turnover rate or turnover
frequency (TOF) which is defined as the number of repetitions of
the catalytic cycle per time. The difficulty in determining the TOF
involves not only the measurement of the reaction rate, but also
the counting of the active sites, which is a sophisticated problem.
The turnover frequency TOFtotal (Table 2) is determined using
the total amount of enzymes immobilized, which was accurately
quantified by chemical analysis. We have to point out that the
TOF calculated here is related to the amount of CPO used in the
fixed-bed reactor at the start of the catalytic run and not to the
amount of CPO that remained on the support after leaching, when
a constant 2-oxindole flux is achieved. The effective activity, that
is the activity per amount of enzyme immobilized, was calculated
to determine the amount of enzymes which are active and avail-
able. The quotient of the effective activity of the immobilized CPO
and the native CPO estimates the amount of accessible and active
enzymes. Nevertheless, since the activity is determined employing
the MCD activity assay, it has to be assumed that the number of
immobilized CPO enzymes which are accessible and active do not
1
5 results in a final constant oxindole yield of 3.5 %, while the yield
decreases to zero over CPO-MCF.
The oxindole yield over catalysts containing cross-linked
enzymes in the mesopores of MCFs is shown in Fig. 3 (right). The
initial activity of both runs amounts to about 35% and drops to
constant yield within the first 24 h. For a catalyst mixture of 23 U
CPO-CLEA-MCF and 7 U GOx-CLEA-MCF, a final yield of 2-oxindole
of 14% is reached, while for the mixture of 46 U CPO-CLEA-MCF and
2
1 U GOx-CLEA-MCF a yield of 20% is achieved. As demonstrated
before, an enhancement in enzyme activity due to an increase of the
amount of biocatalyst used results in a rise of the 2-oxindole yield.