Selecting optimal conditions for Alcalase CLEA-OM for synthesis of dipeptides in organic media

Article abstract of DOI:10.1016/j.molcatb.2011.11.008

In protease-catalyzed peptide synthesis, the availability of water is essential, as a compromise must be made between on the one hand the overall enzymatic activity and, on the other hand, the rate of product synthesis. Water is essential for enzyme activity, but at the same time causes hydrolytic side reactions. We studied the coupling of the carbamoylmethyl ester of N-protected phenylalanine and phenylalanine amide in tetrahydrofuran catalyzed by Alcalase CLEA-OM at a range of water activity (a_w) values, including the coupling in the presence of molecular sieves (i.e. at very low a_w values). The hydrolytic side reaction (in the present system only the hydrolysis of substrate occurs) was found to dominate above an a_w value of about 0.2. To prevent hydrolysis, the presence of molecular sieves was found to be necessary.

Full text of DOI:10.1016/j.molcatb.2011.11.008

Journal of Molecular Catalysis B: Enzymatic 75 (2012) 43–49
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Selecting optimal conditions for Alcalase CLEA-OM for synthesis of dipeptides in
organic media
P. Vossenberga,∗, H.H. Beeftinka, T. Nuijensb, M.A. Cohen Stuartc, J. Trampera
a Bioprocess Engineering, Wageningen University, P.O. Box 8129, 6700 EV, Wageningen, The Netherlands
b DSM Innovative Synthesis B.V., P.O. Box 18, 6160 MD, Geleen, The Netherlands
c Laboratory of Physical Chemistry and Colloid Science, Wageningen University, P.O. Box 8038, 6700 EK, Wageningen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2011
Received in revised form 7 November 2011
Accepted 8 November 2011
Available online 17 November 2011
In protease-catalyzed peptide synthesis, the availability of water is essential, as a compromise must be
made between on the one hand the overall enzymatic activity and, on the other hand, the rate of product
synthesis. Water is essential for enzyme activity, but at the same time causes hydrolytic side reactions.
We studied the coupling of the carbamoylmethyl ester of N-protected phenylalanine and phenylalanine
amide in tetrahydrofuran catalyzed by Alcalase CLEA-OM at a range of water activity (aw) values, including
the coupling in the presence of molecular sieves (i.e. at very low aw values). The hydrolytic side reaction
(in the present system only the hydrolysis of substrate occurs) was found to dominate above an aw value
of about 0.2. To prevent hydrolysis, the presence of molecular sieves was found to be necessary.
© 2011 Elsevier B.V. All rights reserved.
Keywords:
Protease
Dipeptide synthesis
Water activity
Molecular sieves
Solvent
1. Introduction
activity [9]. Nevertheless, in the case of peptide synthesis (or
any other reaction in which water is a substrate in hydrolytic
Natural and non-natural peptides are important for pharmaceu-
tical and food applications [1]. These peptides may be synthesized
enzymatically. A possible peptide synthesis route, which is inves-
tigated in the frame of the Dutch national research program
“Integration of Biosynthesis and Organic Synthesis (IBOS),” starts
with activating the C-terminus of an N-protected amino acid using
a lipase. Subsequently, this activated amino acid is coupled with
an amino acid amide to give a dipeptide using a protease (Fig. 1,
reaction 1) [2–5]. If ‘free’ water is present in the reaction medium,
hydrolytic side reactions may proceed in addition to the coupling
reaction (Fig. 1, reactions 2 and 3). To prevent the spontaneous
hydrolysis of the activated amino acid (Fig. 1, reaction 2) as well
as the enzyme-catalyzed hydrolysis of the activated amino acid,
of the formed dipeptide (Fig. 1, reaction 3), and of the protease
itself (autolysis), the availability of water in the system should be
minimized. To achieve this, the coupling reaction can be carried
out in a neat organic solvent. One should expect, however, that
the organic solvent should not be completely dry as enzymes need
some water to maintain their catalytically active conformation
[6–8]. In a certain range, the more water, the greater the enzymatic
reactions, e.g. transesterification) a compromise, with respect to
the amount of water in the system, must be made between the
overall enzymatic activity and the rate of specifically the product
synthesis [10].
Thermodynamic water activity (aw) is a measure of the avail-
ability of water in a system and reflects the extent of the
interactions of water with other system components. It thus
depends on the hydrophilic or hydrophobic nature of the solvent.
To hydrophilic solvents a lot more water has to be added than
to hydrophobic solvents to achieve the same aw. The aw value
determines the hydration level of the enzyme and thereby also its
activity [11,12].
The present study focuses on the coupling of the carbamoyl-
methyl ester of phenylalanine (of which the amino group was
benzyloxycarbonyl-protected, Z-Phe-OCam), and phenylalanine
amide (Phe-NH2). A carbamoylmethyl ester was used instead of
the more regularly used methyl or ethyl esters due to its posi-
tive effect on the coupling rate [13]. The coupling was catalyzed
by Alcalase CLEA-OM in tetrahydrofuran (THF) at different aw val-
ues. Protease-catalyzed peptide synthesis in monophasic organic
solvents has been studied previously [4,14–17] including studies
investigating the effect of water concentration on peptide synthesis
catalyzed by different proteases: chymotrypsin [6,18–24], subtil-
isin Carlsberg [13,20,25,26], subtilisin BPN’ [18], Alcalase [27–29],
and papain [30]. Nevertheless, the present contribution is to our
∗
Corresponding author. Tel.: +31 317 482954.
E-mail addresses: petra.vossenberg@wur.nl, petravossenberg@gmx.de
(P. Vossenberg).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.11.008

44
P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 43–49
Fig. 1. Coupling of an N-protected, C-terminally activated amino acid with an amino acid amide to give a dipeptide (1), hydrolysis of an N-protected, C-terminally activated
amino acid (2), and hydrolysis of the formed dipeptide (3). X1 and X2 can be any amino acid side chain, but are in this study both a benzyl side chain. R is an activating group.
In this study R is carbamoylmethyl.
knowledge the first to investigate the coupling of Z-Phe-OCam and
Phe-NH2 in THF using Alcalase CLEA-OM at a range of aw values,
including the coupling in the presence of molecular sieves (i.e. at
very low aw values).
2.3. Setting water activity
The aw was set by adding an appropriate amount of water to the
THF, to which the enzyme and substrates were added. The amount
of water that needed to be added to achieve a certain aw was cal-
culated using the activity coefficient (ꢀw) and the mole fraction of
The aim of our study was to investigate up to which aw value the
rate of peptide synthesis could be increased without significantly
increasing the rate of hydrolysis, i.e. without significantly decreas-
ing the synthesis/hydrolysis (S/H) ratio. The aw was varied from ∼0
to 0.95 (in THF these aw values correspond to 0–11%, v/v, water).
In addition, the pH of the buffer used to wash Alcalase CLEA-OM
was varied from 7 to 11 to examine whether this had an effect on
subsequent coupling in near dry conditions. Furthermore, the pH
of the buffer that was added directly to the reaction medium, to
achieve an aw of 0.6, was varied to observe whether this had an
effect on the synthetic and hydrolytic reactions that take place at
this aw. The effect of the concentration of molecular sieves present
in the reaction medium was studied in order to study the dipeptide
synthesis at aw values near 0.
water (xw
)
aw = ꢀwxw
(1)
ꢀw can be calculated from [31–33]:
ln(ꢀw) = − ln(xw + ꢁws(1 − xw)) +
ꢀ
ꢁ
ꢁws
ꢁsw
(1 − xw
)
−
(2)
(3)
(4)
xw + ꢁws(1 − xw
)
ꢁswxw + (1 − xw
)
where
ꢀ
ꢁ
Vs
−(ꢂws − ꢂww
)
ꢁws
=
exp
Vw
RT
and
ꢀ
ꢁ
2. Materials and methods
Vw
Vs
−(ꢂsw − ꢂss
)
ꢁsw
=
exp
RT
2.1. Enzymes
where Vs and Vw are the molar volumes of the organic solvent, in
this case THF and water, respectively. Vs is 81.55 ml mol−1 at 25 ◦C
and Vw 18.07 ml mol−1 [31]. For a THF-water mixture the value of
the ꢂws − ꢂww Wilson coefficient is 7603 J mol−1 and the value of
the ꢂsw − ꢂss Wilson coefficient is 11,212 J mol−1 [31]. R is the gas
constant (8.314 J mol−1 K−1) and T the absolute temperature (K).
The above calculation is based on a binary system, in this case
water and THF. For the enzymatic peptide synthesis the system
contained additional compounds such as substrates, an enzyme
formulation, and, in time, products. As the concentration of sub-
strates (53 mM Phe-NH2 and 37 mM Z-Phe-OCam) and the amount
of enzyme added to the reaction mixture (6 mg ml−1) is rather low,
we assume the aw of our system to be similar to the calculated aw
based on a binary system.
Alcalase® (protease from Bacillus licheniformis) CLEA-OM® (for-
mulation optimized for use in organic media) was obtained from
CLEA Technologies (Delft, The Netherlands). Alcalase CLEA-OM con-
tains the enzyme Alcalase from Novozyme Corp.
2.2. Chemicals
All chemicals used were reagent or analytical grades. tert-
˚
Butanol (t-BuOH) and THF were dried over 3 A molecular sieves,
8–12 mesh beads (Sigma–Aldrich), for at least 1 day prior to use.
t-BuOH was pre-heated to a liquid (40 ◦C) prior to use. Z-Phe-
OCam and Z-Phe-Phe-NH2 were a kind gift from DSM (Geleen,
The Netherlands). Phe-NH2, N-protected phenylalanine (Z-Phe-
OH), and N-protected phenylalanyl-phenylalanine (Z-Phe-Phe-OH)
were purchased from Bachem (Weil am Rhein, Germany).
The water content of the THF solutions was measured by
Karl–Fischer titration (Mettler Toledo DL38; Tiel, The Netherlands)
after the incubation period, in which dipeptide synthesis could take

P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 43–49
45
place. Based on this measured water content, the aw of the reaction
mixtures was recalculated using the equations above.
were incubated at 25 ◦C without enzyme at aw values ranging from
0.4 to 0.95. Samples, for HPLC analysis, were taken after 24 h, and
after 6 or 8 days of incubation time.
To investigate whether hydrolysis of the dipeptide, Z-Phe-Phe-
NH2, takes place in our system, Z-Phe-Phe-OH was incubated at
25 ◦C with 1.7 mg of Alcalase CLEA-OM, that was washed succes-
sively with 1 ml anhydrous t-BuOH and THF prior to use, at a range
of aw values (aw = 0–0.95). The possible hydrolysis of Z-Phe-Phe-OH
was assumed to be similar to Z-Phe-Phe-NH2. Samples, for HPLC
analysis, were taken after 24 h and 7 days of incubation time.
We chose to set the aw by adding an appropriate amount of
water to the THF instead of equilibrating with saturated salt solu-
tions or salt hydrate pairs [34]. When THF was equilibrated with
a saturated salt solution for 24 h, a lot of THF evaporated and was
found in the salt solution. When solvents dissolute in the salt solu-
tion it can alter the aw of the salt solution [35]. In addition, we
did not want to add the salt hydrate pairs directly to the sys-
tem containing the enzyme because the salt hydrates may not be
compatible with the enzyme [35] and the salt hydrates may have
acid–base effects on the enzyme [36].
2.6. HPLC analysis
2.4. Enzymatic peptide synthesis
The amounts of dipeptide (Z-Phe-Phe-NH2), Z-Phe-OCam, and
Z-Phe-OH were analyzed by HPLC (Thermo Separation Products
P4000 pump and AS3000 autosampler) using a reversed-phase col-
umn (Inertsil ODS-3, C18, 5 ␮m, 150 mm × 4.6 mm) at 40 ◦C. UV
detection was performed at 220 nm using an Ultimate 3000 Diode
Array Detector (Dionex). The gradient program was: 0–25 min lin-
ear gradient ramp from 5% to 98% eluent B, 25–29 min linear
gradient ramp back to 5% eluent B, 29–40 min 5% eluent B (elu-
ent A: 0.5 ml l−1 methane sulfonic acid (MSA) in H2O, eluent B:
0.5 ml l−1MSA in acetonitrile). The flow was 1 ml min−1. Injection
volumes were 20 ␮l. Quantitative analysis was performed using
calibration curves of Z-Phe-Phe-NH2, Z-Phe-OCam, and Z-Phe-OH.
HPLC analysis was not a problem for the system in which CLEA-
OM and molecular sieve beads, or CLEA-OM and molecular sieve
powder were used, as enough reaction liquid remained for the
analysis.
Enzymatic reactions were carried out at 25 ◦C in 1.5 ml glass
vials placed in a blood rotator spinning at 15 rpm. Alcalase CLEA-
OM was washed successively with 1 ml of anhydrous t-BuOH and
1 ml of THF to remove residual water. This washing step involved
adding the solvent to the Alcalase formulation, shaking the sam-
ple, allowing the sample to settle, and removing the solvent using
a pipette. This method was analogous to the procedure used to
produce propanol-rinsed enzyme preparations [37,38].
For some experiments, Alcalase CLEA-OM was washed with 1 ml
of Milli-Q or buffer prior to washing with t-BuOH and THF.
In an attempt to achieve a completely dry Alcalase CLEA-OM
˚
formulation, 75 mg 3 A molecular sieves, 8–12 mesh beads, were
added to 1.7 mg of solid Alcalase CLEA-OM particles. The mixture
was stored at 4 ◦C for 18 h prior to use.
In order to dry Alcalase CLEA-OM in the presence of THF, differ-
˚
ent amounts of 3 A molecular sieve powder (Sigma–Aldrich) were
added to the enzyme preparation that was incubated in THF at 25 ◦C
for 24 h prior to use.
3. Results and discussion
For the enzymatic peptide synthesis, 150 ␮l THF containing
16 ␮mol Phe-NH2 and 150 ␮l THF containing 11 ␮mol Z-Phe-OCam
were added to 1.7 mg of the enzyme. To set the aw of the reaction
mixture, either molecular sieves or an appropriate amount of water
were added. Samples (50 ␮l) were diluted with 700 ␮l dimethyl
sulfoxide (DMSO) before HPLC analysis.
3.1. Substrate and dipeptide (product) hydrolysis
To investigate whether hydrolysis of the substrate, Z-Phe-OCam
(Fig. 1, reaction 2), takes place in our system, Z-Phe-OCam was
incubated with Alcalase CLEA-OM, that was not washed with anhy-
drous t-BuOH and THF prior to use. After 24 h of incubation in
the absence of both molecular sieves and additional water, Z-Phe-
OH was detected. The original Alcalase CLEA-OM formulation thus
contains enough water for hydrolysis of Z-Phe-OCam to take place.
Z-Phe-OCam was also incubated without enzyme at aw values
ranging from 0.4 to 0.95, to test spontaneous hydrolysis. At aw val-
ues ranging from 0.4 to 0.8 no Z-Phe-OH was detected after 6 days
of incubation. At an aw value of 0.95, 5% of the Z-Phe-OCam was
hydrolyzed after 24 h and 35% after 8 days of incubation. So only
above an aw value of minimally 0.8 some measurable spontaneous
hydrolysis of the substrate occurs.
To investigate whether hydrolysis of the dipeptide, Z-Phe-Phe-
NH2 (Fig. 1, reaction 3), takes place in our system, Z-Phe-Phe-OH
was incubated with Alcalase CLEA-OM, that was washed succes-
sively with anhydrous t-BuOH and THF prior to use, at a range
of aw values (aw = 0–0.95). The possible hydrolysis of Z-Phe-Phe-
OH was assumed to be similar to Z-Phe-Phe-NH2. After 7 days
of incubation, no Z-Phe-OH was detected. Also in later reactions
in which Z-Phe-Phe-NH2 was formed, no product hydrolysis was
apparent in time. Hydrolysis of the dipeptide (Fig. 1, reaction 3)
thus does not take place in our system. This is in line with the
work from Li et al. in which no hydrolysis of Z-Asp-Val-NH2 was
observed by native Alcalase in acetonitrile containing 10% buffer
[28]. Also Hou et al. did not observe hydrolysis of Bz-Arg-Gly-NH2
by Alcalase in acetonitrile containing 10% buffer [29]. Although
Jönsson et al. did observe hydrolysis of Ac-Phe-Ala-NH2 (by immo-
bilized ␣-chymotrypsin in acetonitrile containing 10% water), the
Except for the reactions used to investigate dipeptide synthesis
˚
at aw values near 0, 75 mg 3 A molecular sieves, 8–12 mesh beads,
were used. For the reactions used to investigate dipeptide synthesis
˚
at aw values near 0, different amounts of 3 A molecular sieve pow-
der were added to the reaction mixture instead of the coarse beads.
Due to the small volumes used in our system, the amount and there-
fore the adsorptive capacity of powder can be controlled better (by
weighing off a specific amount of powder) than that of beads. We
assume the molecular sieve powder to have the same adsorptive
capacity for water molecules as the molecular sieve beads because,
similarly to the beads, it was sold as a drying agent. Furthermore,
the powder has the same elemental composition as the beads.
To study the effect of pH on dipeptide synthesis, the following
buffers were used: 50 mM Tris (hydroxylmethyl) aminomethane
– hydrochloric acid buffer (pH 7–9), 50 mM sodium carbonate –
sodium bicarbonate buffer (pH 9.5–10.5), and 50 mM sodium bicar-
bonate buffer – sodium hydroxide (pH 11).
2.5. Substrate and dipeptide (product) hydrolysis
To investigate whether hydrolysis of the substrate, Z-Phe-OCam,
takes place in our system, 300 ␮l THF containing 11 ␮mol Z-Phe-
OCam was incubated at 25 ◦C with 1.7 mg of Alcalase CLEA-OM,
that was not washed with anhydrous t-BuOH and THF prior to use.
A sample, for HPLC analysis, was taken after 24 h of incubation time.
To test spontaneous substrate hydrolysis, 150 ␮l THF containing
15 ␮mol Phe-NH2 and 150 ␮l THF containing 11 ␮mol Z-Phe-OCam

46
P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 43–49
0.95, only a minimal amount of dipeptide (Z-Phe-Phe-NH2) was
synthesized. The S/H ratio at this aw is 0.01.
If the hydrolysis of the substrate should be minimal because the
substrate is, for example, very expensive, the aw should thus be low
(aw ꢁ 0.2). To prevent the formation of a hydrolysis product (Z-Phe-
OH), the presence of molecular sieves was found to be necessary, i.e.
the aw must be extremely low to prevent hydrolysis. In the absence
of both molecular sieves and additional water, hydrolysis was still
observed. This means that there is still enough water left on the
enzyme formulation that can compete with the dipeptide synthesis
reaction. The successive washing steps with anhydrous t-BuOH and
THF thus do not remove all the water from the enzyme formulation.
The loss of water from the enzyme formulation to the bulk solvent
means that the two pertinent phases (i.e. the THF and the enzyme
formulation) were not at equilibrium. The aw of the bulk solvent
was set by the addition of an appropriate amount of water to the
THF. During the 24 h incubation period for peptide synthesis, the
water was then likely redistributing between the CLEA-OM and the
bulk solvent.
In order to compare the above results with a system in which
CLEA-OM is equilibrated to the same aw as the bulk solvent, the
experimental set-up was modified. Before incubating the enzyme
and substrates for 24 h, CLEA-OM was incubated with 1.5 ml of THF
at a range of aw values. The water transferred from or to the enzyme
was so minimal that it did not measurably change the aw of the bulk
THF. After 24 h, the THF was removed and THF containing the sub-
strates was added. The aw of the THF containing the substrates was
set 4 h before incubation with the enzyme. As was observed in the
system in which CLEA-OM was used immediately, the highest cat-
alytic activity was achieved at the highest aw value tested (0.95)
(Fig. 2). Nevertheless, in comparison to the system in which CLEA-
OM was used immediately, a lower catalytic activity was achieved
at aw values between 0 and 0.4. The relative rate remained approx-
imately constant at 52% in the aw range of 0.2 to 0.4. Above an aw
value of 0.4, the relative rate increased with aw. As was observed in
the system in which CLEA-OM was used immediately, the S/H ratio
in the equilibrated system also decreased rapidly with increasing
aw values and the hydrolysis reaction remained to dominate above
an aw value of about 0.2 (Fig. 2).
The main difference between the system in which CLEA-OM
was used immediately and the equilibrated system, is the relatively
high catalytic activities observed in the system in which CLEA-OM
was used immediately at the lower aw (aw < 0.4) values. These high
enzymatic activities, as compared to those observed with the equi-
librated system, indicate that it might be quite interesting to use
this ‘hydration gradient’ (and therefore non-equilibrium situation)
between the enzyme formulation and the bulk solvent in dipep-
tide synthesis. Indeed, initially relatively high catalytic activities
and high S/H ratios can be achieved using a relatively dry bulk sol-
vent and a hydrated enzyme formulation. In line with this thought,
Basso et al. developed an innovative method to maintain the hydra-
tion state of the enzyme while preventing hydrolytic side reactions.
They adsorbed hydrated thermolysin onto Celite rods and per-
formed an enzymatic dipeptide synthesis in toluene at an aw value
of 0.7–0.75 [39].
Fig. 2. Effect of aw on the total catalytic activity (ꢀ) and on the synthesis/hydrolysis
(S/H) ratio (᭹) of Alcalase CLEA-OM immediately, after washing with anhydrous t-
BuOH and THF, added to the reaction mixture and on the total catalytic activity (ꢁ)
and on the S/H ratio (ꢀ) of Alcalase CLEA-OM equilibrated (24 h) before addition to
the reaction mixture. The total conversion of Z-Phe-OCam at a certain aw is relative
to the highest conversion obtained.
substrate ester, Ac-Phe-OEt, was a considerably better substrate for
hydrolysis than the dipeptide [19].
Apparently, Z-Phe-OCam is a much better substrate in the
hydrolytic reaction for the enzyme than the current dipeptide;
Z-Phe-Phe-NH2 hydrolysis can thus be neglected. In the present
system the incubation time is thus not a critical parameter in the
optimization of the dipeptide synthesis because once the dipeptide
is formed, it will not be hydrolyzed.
3.2. Effect of aw on dipeptide synthesis
The coupling of Z-Phe-OCam and Phe-NH2, catalyzed by Alcalase
CLEA-OM, was investigated at a range of aw values. The enzyme
formulation was washed successively with anhydrous t-BuOH and
THF before the substrates, and molecular sieve beads or an appro-
priate amount of water were added. The enzyme and substrates
were incubated for 24 h. In the presence of molecular sieves the
aw of the reaction mixture can be safely assumed to be the lowest
attained and approaching zero, aw ∼ 0.
Fig. 2 shows the effect of aw on the total catalytic activity (syn-
thesis plus hydrolysis). The aw value on the x-axis is a recalculated
aw value according to the THF water content determined with
Karl–Fischer titration after peptide synthesis. The relative conver-
sions in Fig. 2 (left y-axis) were calculated by normalization with
respect to the conversion at aw = 0.95 with CLEA-OM (washed with
anhydrous t-BuOH and THF, immediately added to the reaction
mixture). At an aw value of 0.95, 6.6 mmol Z-Phe-OCam was con-
verted per gram of CLEA-OM in 24 h. Next to achieving a high
catalytic activity at high aw values, a high catalytic activity was
also achieved at very low aw (aw < 0.1) values. The amount of spon-
taneous hydrolysis of Z-Phe-OCam at high aw (aw > 0.8) values,
mentioned above, was negligible compared to the total amount of
hydrolysis at these aw values.
3.3. Effect of pH on dipeptide synthesis
Fig. 2 also shows the effect of aw on the synthesis/hydrolysis
(S/H) ratio. The hydrolysis reaction consists only of the hydrolysis
of the activated amino acid (Fig. 1, reaction 2). At extremely low aw
values, the S/H ratio is theoretically infinite (‘inf’) as no hydrolysis of
the substrate was measured. The S/H ratio decreases rapidly with
increasing aw values. Based on the measured S/H ratio values at
different aw values, it can be concluded that from an aw value of
about 0.2, the S/H ratio becomes less than 1. The hydrolysis reaction
thus dominates above an aw value of about 0.2. At an aw value of
The pH of the buffer used to wash Alcalase CLEA-OM was var-
ied from 7 to 11 to examine whether this had an effect on the
subsequent coupling in near dry conditions (Fig. 3). The following
buffers were used: 50 mM Tris (hydroxylmethyl) aminomethane
– hydrochloric acid buffer (pH 7–9), 50 mM sodium carbonate
– sodium bicarbonate buffer (pH 9.5–10.5), and 50 mM sodium
bicarbonate buffer – sodium hydroxide (pH 11). Miyazawa et al.
observed a large effect of the pH of the buffer solution from which

P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 43–49
47
Fig. 5. Effect of different concentrations of molecular sieve powder on the relative
composition of the reaction mixture after 3 h of incubation at 25 ◦C using Alcalase
CLEA-OM.
Fig. 3. Effect of the pH of the washing buffer solution on the relative composition
of the reaction mixture after 24 h of incubation at 25 ◦C using Alcalase CLEA-OM.
lyophilized enzymes were prepared on the peptide yield, with an
optimum at pH 10.7 [13]. In addition, Li et al. observed a significant
effect of the pH of the 10% (v/v) buffer solution that was added to
acetonitrile on the peptide yield, with an optimum at pH 10 [28].
Alcalase CLEA-OM was hydrated with buffer and subsequently
washed with anhydrous t-BuOH and THF. The enzyme and sub-
strates were incubated for 24 h in the presence of molecular sieve
beads.
between pH 8.0 and 10.5 coincides partly with the pH optimum of
Alcalase activity in aqueous conditions, which is between pH 8 and
9 [40]. In addition, only deprotonated Phe-NH2 can react to form
the dipeptide and this form of Phe-NH2 is available at alkaline pH
[7,41].
In order to investigate whether a pH optimum also exists when
buffer is added directly to the reaction medium, the effect of the
buffer pH on the reactions catalyzed by Alcalase CLEA-OM at an
aw of 0.6 was studied (Fig. 4). At an aw of 0.6 both synthesis and
hydrolysis reactions occur. Alcalase CLEA-OM was hydrated with
buffer, that had the same pH and concentration as the buffer added
to the reaction mixture, and subsequently washed with anhydrous
t-BuOH and THF. The same buffers were used as above, when study-
ing the effect of the washing buffer pH on the subsequent coupling
in near dry conditions. The enzyme and substrates were incubated
for 24 hours at an aw of 0.6. To achieve an aw of 0.6, 5.9 ␮l of buffer
was added to 300 ␮l of THF containing 1.7 mg CLEA-OM.
The y-axis of Fig. 3 (as well as the y-axis of Figs. 4 and 5) is labeled
‘relative composition.’ The relative composition of, for example, Z-
Phe-Phe-NH2 was calculated as follows:
[ZPhePheNH2]t
[ZPheOCam]0
relative composition ZPhePheNH2 (%) =
× 100
where [ZPhePheNH2]t is the concentration of Z-Phe-Phe-NH2 at the
time of measurement and [ZPheOCam]0 is the initial concentration
of Z-Phe-OCam, both in mol ml−1
.
The pH of the buffer used to wash Alcalase CLEA-OM did have
an effect on the subsequent coupling (Fig. 3). A broad pH optimum
exists ranging from pH 8.0 to 10.5. Likely the small amounts of
buffer that were present in the reaction mixture and surrounding
the enzyme have an effect on the synthetic activity of the enzyme.
These residual amounts of buffer were, however, so small that no
hydrolysis was observed. This enhancement in synthetic activity
In a monophasic organic solvent, pH is undefined [42]. The
enzyme is a polyelectrolyte with a certain ionization state. The
addition of 5.9 ␮l buffer to the reaction mixture (to achieve an
aw of 0.6) means that about 35 ␮g of buffer salts were added. It
is doubtful whether this small amount of buffer salts can alter the
ionization state of 1.7 mg CLEA-OM. The washing step with 1 ml
of buffer before incubation, however, may have a more significant
effect on the ionization state of the enzyme and therefore on the
synthesis and hydrolysis reactions that occur (as was seen when
studying the effect of the washing buffer pH on the subsequent
coupling in near dry conditions), as the volume used is much larger.
The rates of dipeptide synthesis and substrate hydrolysis do not
seem to be significantly affected by the pH of the buffer solution
that was added to the reaction mixture (Fig. 4). No optimum was
observed. The pH of the buffer at an aw of 0.6 neither affects the
synthetic nor the hydrolytic activity of the enzymes significantly at
the optimum pH range.
3.4. Dipeptide synthesis at very low aw values
As stated before, the aw of the reaction system must be
extremely low to prevent hydrolysis. Nevertheless, the system
should not be completely dry as enzymes need some water to
maintain their catalytically active conformation [6–8]. We believe
that an optimum in solely dipeptide synthesis can be achieved
when the dipeptide synthesis is carried out near an aw value of
the bulk solvent of 0. In order to achieve aw values near 0, molecu-
lar sieve powder, of which the concentration was varied, instead of
Fig. 4. Effect of the pH of the buffer solution that was added to achieve an aw of 0.6
on the relative composition of the reaction mixture after 24 h of incubation at 25 ◦
using Alcalase CLEA-OM.
C

48
P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 43–49
molecular sieve beads were used. Due to the small volumes used
in our system, the amount and therefore the adsorptive capacity of
powder can be controlled better (by weighing off a specific amount
of powder) than that of beads.
The effect of the concentration of molecular sieves present in the
reaction medium on the coupling of Z-Phe-OCam and H-Phe-NH2
was investigated (Fig. 5). Alcalase CLEA-OM was first hydrated with
Milli-Q and subsequently washed with anhydrous t-BuOH and THF.
The enzyme and substrates were incubated for 3 h in the presence
of different amounts of molecular sieve powder.
With Karl–Fischer titration it was not possible to accurately
determine the water content of these samples as the water con-
tent is too low. Therefore the specific aw of the reaction systems
containing different amounts of molecular sieve powder could not
be calculated.
An optimum in dipeptide synthesis was achieved when the
dipeptide synthesis was carried out in the presence of about
7.5–20 mg ml−1 of molecular sieve powder (Fig. 5). When only a
small amount of molecular sieve powder (<3 mg ml−1) was added
to the reaction mixture hydrolysis could not be prevented. When
a relatively large amount of molecular sieve powder was added to
the reaction mixture (>35 mg ml−1) the rate of dipeptide synthesis
was significantly reduced. Apparently, at this point, the activity of
Alcalase CLEA-OM was reduced due to dehydration.
4. Conclusions
In the present system the incubation time is not a critical param-
eter in the optimization of the dipeptide synthesis because once the
dipeptide is formed, it will not be hydrolyzed. Only hydrolysis of
the substrate, Z-Phe-OCam, takes place.
The rate of peptide synthesis could not be increased by increas-
ing aw values without significantly increasing the rate of hydrolysis,
i.e. without significantly decreasing the synthesis/hydrolysis (S/H)
ratio. At extremely low aw values, i.e. in the presence of an excess
amount of molecular sieves, the S/H ratio is theoretically infinite as
no hydrolysis product was measured. Above an aw value of about
0.2, the hydrolysis reaction dominates.
The pH of the buffer used to wash Alcalase CLEA-OM has an effect
on the coupling. A broad pH optimum exists ranging from pH 8.0
to 10.5. The pH of the buffer that is added directly to the reaction
medium, to achieve an aw of 0.6, neither affects the synthetic nor
the hydrolytic activity of the enzyme.
An appropriate amount of molecular sieves can prevent hydrol-
ysis of the substrate and still allow enzymatic activity. Nevertheless,
if Alcalase CLEA-OM is pre-incubated with molecular sieves for 24 h
before adding substrates, minimal activity is observed. The pre-
incubation with molecular sieve powder thus seems to have dried
the enzyme to such an extent that it had a negative effect on its
activity. The use of molecular sieves over longer periods of time
should therefore be carefully considered as they may dehydrate
and thereby inactivate the enzyme in time.
The above investigation of the effect of the concentration of
molecular sieves present in the reaction medium on the coupling of
Z-Phe-OCam and H-Phe-NH2 using Alcalase CLEA-OM was carried
out in triplicate. Fig. 5 is an illustrative example of the three series
done. The results all show the same trend.
Acknowledgements
If it is assumed that molecular sieves are a perfect sink or at
least have a very high partition coefficient for water, the enzyme
formulation and the solvent may lose water in time. This would
lead to inactive enzyme formulations as enzymes are known to
require some essential water to maintain their catalytic active con-
formation [6–8]. An attempt was made to dry the Alcalase CLEA-OM
formulation even further than with anhydrous t-BuOH and THF
alone by storing it with molecular sieve beads for 18 hours before
use in an enzymatic reaction. Indeed, this extremely dry Alcalase
CLEA-OM formulation was found totally incapable of dipeptide syn-
thesis. To have an indication whether the enzyme formulation loses
water in time in the presence of THF and molecular sieves, and
thereby inactivates, non-washed Alcalase CLEA-OM was incubated
in THF with different amounts of molecular sieve powder for 24 h
before adding the substrates. The Alcalase CLEA-OM pre-incubated
with 6-17 mg ml−1 molecular sieve powder converted on average
2.4% of the substrate to dipeptide and 0.9% to Z-Phe-OH in 3 h. The
Alcalase CLEA-OM pre-incubated with 28–635 mg ml−1 molecular
sieve powder converted on average 1.6% of the substrate to dipep-
tide in 3 h and no hydrolysis was observed. Pre-incubation with
a small amount of molecular sieve powder thus caused a some-
what higher dipeptide synthesis rate but hydrolysis could not be
prevented. In any case, the conversion achieved by the Alcalase
CLEA-OM formulation pre-incubated with molecular sieve powder
is significantly lower than the >40% conversion achieved by the
Alcalase CLEA-OM formulation to which molecular sieve powder
was added at the same time as the substrates (Fig. 5). The pre-
incubation with molecular sieve powder thus seems to have dried
the enzyme to such an extent that only a minimal activity remained.
This result suggests that although the rate of catalysis in the sys-
tems to which molecular sieves are added at the same time as the
substrates is very high in the first hours of incubation, due to the
initially hydrated and thus active enzyme, the rate of catalysis will
be minimal after 24 h of incubation, due to the significant dehy-
dration of the enzyme by the molecular sieves. The exact rate of
this dehydration and its effect on the enzyme activity are subject
to further study.
This work is part of the IBOS-2 research project “chemo-
enzymatic peptide synthesis,” which is financially supported by the
NWO-ACTS (The Netherlands). We thank Dr. ir. Peter J.L.M. Quaed-
flieg from DSM Innovative Synthesis (Geleen, The Netherlands) for
the valuable discussions.
References
[1] F. Guzmán, S. Barberis, A. Illanes, Electronic Journal of Biotechnology 10 (2007)
279–314.
[2] T. Nuijens, C. Cusan, A.C.H.M. Schepers, P.J.L.M. Quaedflieg, Journal of Peptide
Science 16 (2010) 44–47.
[3] P.J.L.M. Quaedflieg, T. Nuijens, C. Cusan, A.C.H.M. Schepers, World Patent Appli-
cation 2010/057961 (A1) (2010).
[4] T. Nuijens, C. Cusan, A.C.H.M. Schepers, J.A.W. Kruijtzer, D.T.S. Rijkers, R.M.J.
Liskamp, P.J.L.M. Quaedflieg, Journal of Molecular Catalysis B: Enzymatic 71
(2010) 79–84.
[5] P. Vossenberg, H.H. Beeftink, M.A. Cohen Stuart, J. Tramper, Biocatalysis and
Biotransformation (2011), doi:10.3109/10242422.2011.631213.
[6] H. Gaertner, A. Puigserver, European Journal of Biochemistry 181 (1989)
207–213.
[7] F. Bordusa, Chemical Reviews 102 (2002) 4817–4867.
[8] P. Adlercreutz, European Journal of Biochemistry 199 (1991) 609–614.
[9] A. Zaks, A.M. Klibanov, The Journal of Biological Chemistry 263 (1988)
3194–3201.
[10] J. Partridge, G.A. Hutcheon, B.D. Moore, P.J. Halling, Journal of the American
Chemical Society 118 (1996) 12873–12877.
[11] G. Ljunger, P. Adlercreutz, B. Mattiasson, Enzyme and Microbial Technology 16
(1994) 751–755.
[12] P.J. Halling, Biotechnology Techniques 6 (1992) 271–276.
[13] T. Miyazawa, M. Hiramatsu, T. Murashima, T. Yamada, Letters in Peptide Science
9 (2002) 173–177.
[14] A. Ferjancic, A. Puigserver, H. Gaertner, Applied Microbiology and Biotechnol-
ogy 32 (1990) 651–657.
[15] A.V. Belyaeva, A.V. Bacheva, E.S. Oksenoit, E.N. Lysogorskaya, V.I. Lozinskii, I.Y.
Filippova, Russian Journal of Bioorganic Chemistry 31 (2005) 529–534.
[16] I.Y. Filippova, E.N. Lysogorskaya, Russian Journal of Bioorganic Chemistry 29
(2003) 496–501.
[17] K. Oyama, S. Nishimura, Y. Nonaka, K.-i. Kihara, T. Hashimoto, Journal of Organic
Chemistry 46 (1981) 5242–5244.
[18] H. Kise, A. Hayakawa, Enzyme and Microbial Technology 13 (1991) 584–588.
[19] Å. Jönsson, P. Adlercreutz, B. Mattiasson, Biotechnology and Bioengineering 46
(1995) 429–436.

P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 43–49
49
[20] M.V. Sergeeva, V.M. Paradkar, J.S. Dordick, Enzyme and Microbial Technology
20 (1997) 623–628.
[21] P. Clapés, P. Adlercreutz, B. Mattiasson, Journal of Biotechnology 15 (1990)
323–338.
[22] P. Clapés, G. Valencia, P. Adlercreutz, Enzyme and Microbial Technology 14
(1992) 575–580.
[23] Y. Kimura, T. Yoshida, K. Muraya, K. Nakanishi, R. Matsuno, Agricultural and
Biological Chemistry 54 (1990) 1433–1440.
[24] M. Capellas, M.D. Benaiges, G. Caminal, G. González, J. López-Santín, P. Clapés,
Biocatalysis and Biotransformation 13 (1996) 165–178.
[25] J.U. Klein, A. Prykhodzka, V. Cerovsky, Journal of Peptide Science 6 (2000)
541–549.
[26] S.-y. Okazaki, M. Goto, S. Furusaki, Enzyme and Microbial Technology 26 (2000)
159–164.
[27] S.-T. Chen, S.-Y. Chen, K.-T. Wang, Journal of Organic Chemistry 57 (1992)
6960–6965.
[28] S.-J. Li, J.-A. Wang, L. Xu, X.-Z. Zhang, J. Li, D. Suo, Preparative Biochemistry and
Biotechnology 38 (2008) 334–347.
[33] F.A. Gothard, M.F.C. Clobanu, D.G. Breban, C.I. Bucur, G.V. Sorescu, Indus-
trial and Engineering Chemistry Process Design and Development 15 (1976)
333–337.
[34] P.J. Halling, Enzyme and Microbial Technology 16 (1994) 178–206.
[35] G.A. Hutcheon, P.J. Halling, B.D. Moore, Measurement and control of hydra-
tion in nonaqueous biocatalysis, in: B. Rubin, E.A. Dennis (Eds.), Methods in
Enzymology, Academic Press, New York, 1997, pp. 465–472.
[36] N. Fontes, N. Harper, P.J. Halling, S. Barreiros, Biotechnology and Bioengineering
82 (2003) 802–808.
[37] J. Partridge, P.J. Halling, B.D. Moore, Chemical Communications
841–842.
7 (1998)
[38] B.D. Moore, J. Partridge, P.J. Halling, Very high activity biocatalysts for low-
water systems, in: E.N. Vulfson, P.J. Halling, H.L. Holland (Eds.), Enzymes in
Nonaqueous Solvents: Methods and Protocols, Humana Press Inc., Totowa, NJ,
2001, pp. 97–104.
[39] A. Basso, L.d. Martin, C. Ebert, L. Gardossi, P. Linda, Chemical Communications
(2000) 467–468.
[40] H. Uhlig, Microbial Proteases: Brief Overview, in: Industrial Enzymes and their
Applications, John Wiley & Sons, Inc, New York, 1998, pp. 161–179.
[41] S.M.A. Salam, K.-i. Kagawa, T. Matsubara, K. Kawashiro, Enzyme and Microbial
Technology 43 (2008) 537–543.
[42] A.J.J. Straathof, Biocatalysis in organic media using enzymes, in: A. Pandey,
C. Webb, C.R. Soccol, C. Larroche (Eds.), Enzyme Technology, Springer Sci-
ence + Business Media, Inc., Asiatech Publishers, Inc, New York, 2006, pp.
105–123.
[29] R.-Z. Hou, Y. Yang, G. Li, Y.-B. Huang, H. Wang, Y.-J. Liu, L. Xu, X.-Z. Zhang,
Biotechnology and Applied Biochemistry 44 (2006) 73–80.
[30] X.-z. Zhang, X. Wang, S. Chen, X. Fu, X. Wu, C. Li, Enzyme and Microbial Tech-
nology 19 (1996) 538–544.
[31] G. Bell, A.E.M. Janssen, P.J. Halling, Enzyme and Microbial Technology 20 (1997)
471–477.
[32] J. Gmehling, U. Onken, Vapor-Liquid Equilibrium Data Collection, DECHEMA,
Frankfurt/Main, 1977.