Dipeptide synthesis in near-anhydrous organic media: Long-term stability and reusability of immobilized Alcalase

Article abstract of DOI:10.1016/j.molcatb.2013.03.014

The long-term stability and re-use of Alcalase covalently immobilized onto macroporous acrylic beads (Cov) in tetrahydrofuran (THF) were investigated. Cov can be used to synthesize dipeptides under near-anhydrous conditions in THF. Cov was incubated with and without molecular sieves (beads or powder) in THF, in order to investigate whether its stability is affected by the presence of molecular sieves. After different incubation periods, the enzyme activity was determined in an aqueous environment. In addition, Cov was repeatedly recycled to examine its reusability. Without molecular sieve beads, Cov hardly inactivated in THF. With molecular sieve beads, Cov lost activity over time. Incubated Cov samples were rotated on a blood rotator, entailing mechanical forces between Cov and the molecular sieve beads. Mechanical damage of Cov by the molecular sieve beads was found to be the main reason for the instability of Cov. During reuse, intermediate rehydration of Cov also caused a small but significant activity loss.

Full text of DOI:10.1016/j.molcatb.2013.03.014

Journal of Molecular Catalysis B: Enzymatic 93 (2013) 23–27
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Dipeptide synthesis in near-anhydrous organic media: Long-term stability and
reusability of immobilized Alcalase
P. Vossenberga,∗, H.H. Beeftinka, T. Nuijensb, P.J.L.M. Quaedfliegb, 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:
The long-term stability and re-use of Alcalase covalently immobilized onto macroporous acrylic beads
(Cov) in tetrahydrofuran (THF) were investigated. Cov can be used to synthesize dipeptides under near-
anhydrous conditions in THF. Cov was incubated with and without molecular sieves (beads or powder)
in THF, in order to investigate whether its stability is affected by the presence of molecular sieves. After
different incubation periods, the enzyme activity was determined in an aqueous environment. In addi-
tion, Cov was repeatedly recycled to examine its reusability. Without molecular sieve beads, Cov hardly
inactivated in THF. With molecular sieve beads, Cov lost activity over time. Incubated Cov samples were
rotated on a blood rotator, entailing mechanical forces between Cov and the molecular sieve beads.
Mechanical damage of Cov by the molecular sieve beads was found to be the main reason for the insta-
bility of Cov. During reuse, intermediate rehydration of Cov also caused a small but significant activity
loss.
Received 24 January 2013
Received in revised form 19 March 2013
Accepted 23 March 2013
Available online xxx
Keywords:
Immobilized protease
Operational stability
Reusability
Molecular sieves
Near-anhydrous
Mechanical damage
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
media on a large scale was found to be hydrated (water satu-
rated) Alcalase covalently immobilized onto macroporous acrylic
Alcalase (also referred to as Subtilisin A and Subtilisin Carslberg)
is a protease that may be used in the chemo-enzymatic synthesis of
peptides. Peptides play an important role in the fields of health care,
nutrition and cosmetics [1–5]. Alcalase can for instance catalyze
the coupling of an ␣-amino amide and a chemically synthesized
activated N-protected amino acid, e.g. an N-protected amino acid
carbamoylmethyl (Cam) ester [6–12].
beads (in this paper abbreviated as Cov) [12]. This formulation
has a reasonable activity with respect to dipeptide synthesis in
near-anhydrous organic media, and, from a practical point of view,
a reasonable size (150–300 ␮m in diameter [13]) and a uniform
spherical shape. In addition, Cov features good short-term opera-
tional stability in THF as it could be reused at least twice without
significant activity loss with respect to dipeptide synthesis [12],
under the proviso that the enzyme was rehydrated in-between
subsequent dipeptide synthesis reactions under near-anhydrous
conditions.
The present study focuses on the long-term stability and
extended reuse of hydrated Cov in THF. In this manuscript no dipep-
tide synthesis data are presented but the conditions used to study
the long-term stability and extended reuse of hydrated Cov in THF
are identical to the reaction conditions used in previous work to
synthesize dipeptides [11,12]. We, therefore, expect that the results
from this manuscript can be directly translated to dipeptide syn-
thesis when carried out for longer periods of time.
In previous work we studied the effect of water activity (aw
)
on the rate of dipeptide synthesis [11]. It was found that a care-
fully chosen amount of molecular sieves was required to prevent
hydrolysis, but still allow enzymatic activity; a too large amount
of molecular sieves was found to dehydrate and inactivate the
enzyme [11]. Furthermore, we compared different Alcalase formu-
lations with respect to their dipeptide synthesis capability in neat
organic solvent in the presence of molecular sieves (i.e. under near-
anhydrous conditions at very low water activity). Hydration prior
to drying (with anhydrous tert-butanol and anhydrous tetrahydro-
furan (THF)) of the Alcalase formulations resulted in a significant
increase in rate of the subsequent dipeptide synthesis. The most
promising enzyme formulation for dipeptide synthesis in organic
The stability of cross-linked subtilisin crystals and native subtil-
isin Carlsberg in polar solvents has been studied before [14–16].
This type of data is, however, not available for the covalently
immobilized Alcalase formulation (i.e. Cov) we are interested in.
Ultimately a process design for peptide synthesis in organic media
requires knowledge of and data on the operational stability of the
∗
Corresponding author. Tel.: +31 317 482954.
E-mail address: petravossenberg@gmx.de (P. Vossenberg).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.03.014

24
P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 23–27
enzyme formulation that was found capable of synthesizing these
peptides. Extending the operational stability of an enzyme formu-
lation by minimizing the rate of inactivation requires a mechanistic
view on the causes of instability of the enzyme formulation.
The aim of our study was to investigate whether hydrated Cov
is stable in dry THF and whether its stability is affected by the
presence of molecular sieves. For this, Cov was incubated with and
without molecular sieves (beads or powder) in THF and its aque-
ous activity was subsequently determined. In addition, Cov was
repeatedly recycled in order to examine its reusability.
in time a separate Eppendorf tube was incubated, so all time points
in the figures represent independent experiments.
2.4. Long-term stability of native Alcalase
Native Alcalase (2 mg) was incubated in 1 ml of anhydrous THF
at 25 ◦C in 2 ml Eppendorf safe-lock tubes placed on a blood rotator
spinning at 40 rpm. After different incubation periods (0–42 days),
the aqueous activity of the complete content of the Eppendorf tubes
(native Alcalase and THF) was analyzed (Section 2.5). For each time
point a separate Eppendorf tube was incubated.
2. Materials and methods
2.5. Aqueous Alcalase activity
2.1. Enzymes
The aqueous activity of Alcalase was assayed by monitoring the
hydrolysis of 25% (v/v) ethyl lactate at 40 ◦C and pH 6.8 (10 ml of
100 mM sodium phosphate buffer pH 6.8, 20 ml of Milli-Q, and
10 ml of ethyl lactate). The resulting lactic acid was titrated with
0.1 mol l−1 sodium hydroxide using pH-stat equipment (719 Stat
Titrino Metrohm; Herisau, Switzerland). The pH-stat equipment
was connected to a computer that logged the consumption of
sodium hydroxide every 2 s. The method was based on a protocol
obtained from ChiralVision [20]. The blank consumption of sodium
hydroxide was monitored for 10 min. Subsequently the Alcalase
formulation was added. The aqueous Alcalase activity is defined by
the rate of sodium hydroxide consumption (corrected for the blank
consumption of sodium hydroxide). The rate was based on 100 data
points, thus in an interval of 200 s in total.
Alcalase® covalently immobilized onto macroporous acrylic
beads (Cov; ChiralVision, Leiden, The Netherlands) and lyophilized
native protease from Bacillus licheniformis (also referred to as
Alcalase®, Subtilisin A, and Subtilisin Carslberg; Sigma–Aldrich,
Zwijndrecht, The Netherlands) were used. Cov contains the enzyme
Alcalase from Novozymes Corporation [17], which is covalently
immobilized onto Immobeads 150 (cross-linked copolymer of
methacrylate carrying oxirane groups).
2.2. Chemicals
All chemicals used were reagent or analytical grade. t-BuOH
˚
and THF were dried over 3 A molecular sieves, 8–12 mesh beads
(Sigma–Aldrich), for ≥1 day prior to use. The molecular sieves were
dried at 200 ◦C and t-BuOH was pre-heated to a liquid (40 ◦C) prior
to use.
2.6. Settling rate
To get a rough indication of the settling rate of Cov and of molec-
ular sieve beads and powder, they were added separately to 10 ml
of THF in a 10 ml test tube of 9 cm in height [21]. After addition
of Cov (40 mg), molecular sieve beads (800 mg), or molecular sieve
powder (800 mg) to the THF, the mixture was shaken and subse-
quently allowed to settle. The settling time was measured and from
this the settling rate was calculated [21].
2.3. Long-term stability of Cov
Before use, Cov (40 mg) was washed with successively 1 ml of
Milli-Q, to initially completely hydrate the enzyme, and 1 ml of each
anhydrous t-BuOH and THF, to remove the excess water. A wash-
ing step involved adding washing liquid (Milli-Q, t-BuOH, or THF) to
Cov, shaking the sample for 10 s, centrifuging the sample for 2 min
at 10,000 rpm in order to facilitate the separation of the washing
liquid and the enzyme formulation, and removing the washing liq-
uid manually using a pipette. This method was analogous to the
procedure used to produce propanol-rinsed enzyme preparations
[18,19].
2.7. Effect of reuse on the aqueous activity of Cov
Hydrated Cov (40 mg) was incubated in 1 ml of anhydrous THF
at 25 ◦C in 2 ml Eppendorf safe-lock tubes placed on a blood rota-
tor spinning at 40 rpm. Hydrated Cov was obtained by washing
with successively 1 ml of each Milli-Q, anhydrous t-BuOH, and
anhydrous THF. After 24 h, Cov was rehydrated by washing with
successively 1 ml of each Milli-Q, anhydrous t-BuOH, and anhy-
drous THF. After a different number of incubation/washing steps,
with a total of 8 recycles, the aqueous activity of Cov was analyzed
(Section 2.5). This was done in duplo. The above procedure was also
executed for:
Cov (40 mg) was incubated in 1 ml of anhydrous THF at 25 ◦C in
2 ml Eppendorf safe-lock tubes placed on a blood rotator spinning
at 40 rpm:
(1) without molecular sieves;
˚
(2) with 10 mg of dry 3 A molecular sieve beads per mg Cov (in
duplo);
˚
(3) with 10 mg of dry 3 A molecular sieve powder (Sigma–Aldrich)
(1) Cov (re)hydrated with 1 ml of 50 mM Tris buffer (pH 8) con-
taining 20 mM calcium chloride instead of Milli-Q, in order to
investigate the possible reversible loss of essential calcium ions
during washing.
(2) Cov washed with Milli-Q, anhydrous t-BuOH, and THF, but
after the washing step with Milli-Q, Cov was centrifuged for
10 instead of 2 min at 10,000 rpm, in order to further facilitate
the separation of Milli-Q and Cov.
per mg Cov (in duplo);
(4) with 10 mg pre-hydrated molecular sieve beads per mg Cov.
The molecular sieve beads were pre-hydrated by adding them
to a vial with Milli-Q for 10 min and subsequently removing the
excess water using tissue paper;
(5) with 17.5 mg pre-hydrated molecular sieve beads per mg Cov.
(3) Cov washed with Milli-Q and two times 1 ml of anhydrous THF.
The washing step with anhydrous t-BuOH was thus omitted.
(4) Cov washed with two times 1 ml of anhydrous THF. The wash-
ing steps with Milli-Q and anhydrous t-BuOH were thus both
omitted.
Cov was also incubated with 10 mg of dry molecular sieve beads
per mg Cov without spinning on a blood rotator (in duplo).
After different incubation periods (0–32 days), the aqueous
activity of the full content of the Eppendorf tubes (Cov, THF, and
molecular sieves) was analyzed (Section 2.5). For each data point

P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 23–27
25
Carlsberg when incubated in tert-amyl alcohol and in acetonitrile
[15].
In contrast to Cov incubated in THF without molecular sieve
beads, Cov incubated with molecular sieve beads, while spinning
on a blood rotator, is unstable: after 30 days of incubation, the
remaining aqueous activity was about 55% (Fig. 1A).
One can think of different reasons for the inactivation of Cov
in the presence of molecular sieve beads in THF. It is possible that
(1) Cov is irreversibly dehydrated and thereby inactivated when
molecular sieves are present to serve as a high-capacity water
sink. It is also possible that (2) the shear, abrasion, and collisions
(mechanical forces) between the small and light Immobeads of
the Cov formulation (diameter: 150–300 ␮m [13]), and the large,
heavy, and rough molecular sieve beads (diameter: 1.7–2.4 mm
[22]), may lead to the inactivation of enzyme molecules on the sur-
face of the Immobeads. This is similar to the way cells are disrupted
in a bead mill, in which glass beads are used to create high shearing
forces and collisions [23].
To investigate whether Cov is irreversibly dehydrated and
inactivated by the molecular sieves (option 1 for inactivation), it
was incubated with pre-hydrated molecular sieve beads. Ten mg of
pre-hydrated molecular sieve beads per mg Cov caused the same
loss of activity as 10 mg of dry molecular sieve beads per mg Cov
(Fig. 1B). Irreversible dehydration is therefore not a reason for Cov
inactivation by molecular sieve beads. The use of molecular sieve
powder should then also have led to inactivation by irreversible
dehydration. This was not the case: Cov that was incubated with
molecular sieve powder was found to be rather stable: 90% of the
initial activity was found to remain after 32 days (Fig. 1A). In addi-
tion, a larger amount of pre-hydrated molecular sieve beads per
unit amount of Cov (17.5 mg/mg) led to a faster inactivation than a
lower amount (10 mg/mg) (Fig. 1B). To our knowledge, irreversible
dehydration due to the presence of molecular sieves has not been
reported elsewhere either. Reversible dehydration due to the pres-
ence of molecular sieves, however, has been reported [24–26].
The effect of mechanical forces on Cov activity (option 2 for inac-
tivation) was studied by incubating Cov and molecular sieve beads
without spinning and therefore without shear and collisions. In
addition, Cov was incubated with an equivalent amount of molec-
ular sieve powder, while spinning on a blood rotator. Powder will
lead to less shear and less impact per collision than beads due to
the small size of the powder particles (3–5 ␮m in diameter [22]).
The Cov activity that remained after 32 days with molecular sieve
beads without spinning was around 90% (data not shown). Cov with
molecular sieve powder was found to be rather stable (Fig. 1A).
Apparently, mechanical forces are an important cause for the inac-
tivation of Cov in the presence of molecular sieve beads in this
particular system.
Fig. 1. Long-term stability of Cov in THF. (A) Cov without molecular sieve beads
(᭹), with dry molecular sieve beads (10 mg beads per mg Cov) (ꢀ), and with dry
molecular sieve powder (10 mg powder per mg Cov) (ꢀ). B: Cov with dry molecular
sieve beads (10 mg beads per mg Cov) (ꢀ), with pre-hydrated molecular sieve beads
(10 mg beads per mg Cov) (ꢁ), and with more pre-hydrated molecular sieve beads
(17.5 mg beads per mg Cov) (ꢁ). 95% confidence intervals are shown.
For each number of washes a separate Eppendorf tube was incu-
bated.
3. Results and discussion
The effect of shear on enzyme activity has been reported earlier.
Some research groups have observed an effect [27–35], whereas
others have not [36–39]. The effect of mechanical forces on enzyme
activity caused by the presence of molecular sieve beads has, to our
knowledge, never been studied in detail. Bovara et al. used the com-
bination of molecular sieves and immobilized lipase (adsorbed on
Hyflo Super Cel, which is a celite support), and reused the immo-
bilized lipase several times (by filtering off the enzyme and adding
new substrates). They ascribed a reduction in enzyme activity to
leakage of enzyme from the support due to mechanical shear by
molecular sieves [40]. In our case, the entire contents of the incu-
bation vials were added to the aqueous activity assay; enzyme loss
from the support to the bulk liquid can thus in itself not account
for activity losses. Native Alcalase is also not less stable in THF than
immobilized Alcalase (data not shown).
3.1. Long-term stability of Cov
To investigate whether hydrated Cov is stable in dry THF and
whether its stability is affected by the presence of water-capturing
molecular sieves, Cov was incubated with and without molecular
sieves (beads or powder) in anhydrous THF at 25 ◦C and subse-
quently its activity was determined in aqueous conditions. This
aqueous measurement will compensate for reversible inactivation
due to dehydration, if any.
Fig. 1 shows the remaining aqueous activity (activity compared
to initial activity) of Cov incubated with and without molecular
sieve beads in THF spinning on a blood rotator. Without molec-
ular sieve beads, Cov hardly inactivates in THF: after 30 days of
incubation the remaining aqueous activity was about 90% (Fig. 1A).
This is in agreement with the results of Schulze and Klibanov, who
reported almost no loss of aqueous activity of native subtilisin
In the end, the best way to minimize the inactivation of Cov
is to omit the use of molecular sieve beads. Molecular sieves are,
however, needed in dipeptide synthesis to prevent hydrolytic side

26
P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 23–27
reactions [11]. Molecular sieve powder instead of molecular sieve
beads could be used in dipeptide synthesis because Cov with molec-
ular sieve powder was found to be rather stable. Molecular sieve
beads will, however, be preferred over powder if the molecular
sieves need to be separated from Cov. This separation is required in
case of recycling of Cov, involving rehydration of Cov and regen-
eration of the molecular sieves. The molecular sieve beads are
significantly larger (diameter: 1.7–2.4 mm [22]) than the Cov par-
ticles (diameter: 150–300 ␮m [13]) and settle very fast (about 150
times faster than Cov; the settling rate is about 9 cm s−1 in THF). The
molecular sieve powder, however, is very fine (3–5 ␮m in diameter
[22]) and settles slowly (the settling rate is about 75 ␮m s−1 in THF,
which is about 8 times slower than that of Cov).
Nevertheless, in a stirred batch reactor, mechanical damage of
Cov by molecular sieve beads may be minimal as the particles will
not tumble over one another along a wall.
3.2. Effect of reuse on the aqueous activity of Cov
To examine Cov reusability, it was repeatedly recycled
with intermediate rehydration consisting of 2–3 washing steps.
(Re)hydrated Cov was incubated in THF (without substrates) at
25 ◦C while spinning on a blood rotator. After 24 h, Cov was rehy-
drated by washing with Milli-Q, anhydrous t-BuOH, and anhydrous
THF, successively. Previously, this intermediate rehydration step
was found necessary when reusing Cov in dipeptide synthesis (12).
After each incubation/washing step, with a total of 8 recycles, the
aqueous activity of Cov was determined. The aqueous activity was
determined instead of the dipeptide synthesis capability of Cov to
prevent the use of molecular sieves during the activity measure-
ment.
Cov loses aqueous enzyme activity during each rehydration
cycle (Fig. 2). Different options to prevent this loss of enzyme activ-
ity were examined. Cov was rehydrated with Tris buffer (pH 8)
containing 20 mM CaCl2 instead of Milli-Q (Fig. 2), which did not
prevent the activity loss of Cov. The loss of enzyme activity can
therefore not be explained by the reversible loss of essential cal-
cium ions [41–43] during washing with Milli-Q. In addition, the
loss of activity did not change by prolonging the centrifugation time
after washing with Milli-Q, in order to possibly recover small Cov
beads (Fig. 2). When replacing the t-BuOH washing step with a THF
washing step (Fig. 2B), the rate of activity loss of Cov is significantly
reduced; beyond 4 washes there is no overlap in the 95% confi-
dence intervals of Cov washed with Milli-Q, t-BuOH and THF, and
of Cov washed with Milli-Q and 2× THF. It thus seems that wash-
ing with t-BuOH partially inactivates the enzyme, either due to the
nature of the solvent or due to the temperature (at 40 ◦C t-BuOH
is a liquid) at which the solvent is used. When replacing the wash-
ing steps with t-BuOH and Milli-Q both by a washing step with THF
(Fig. 2C), there is no significant further reduction in the activity loss
of Cov compared to the activity loss of Cov washed with Milli-Q and
2× THF. It thus seems that some enzyme particles are lost during
washing, although the Alcalase in the Cov preparation is said to
be covalently immobilized. Washing with Milli-Q and THF cannot
easily be circumvented, as a significant activity loss with respect
to dipeptide synthesis was observed, when Cov was reused with
molecular sieves without intermediate rehydration [12].
Fig. 2. Effect of reuse on the aqueous activity of Cov incubated in THF. Cov washed
with Milli-Q, t-BuOH and THF (᭹); Cov washed with Tris buffer containing CaCl2,
t-BuOH and THF (ꢀ); Cov washed with Milli-Q, t-BuOH and THF, longer centrifu-
gation after Milli-Q wash step (ꢀ); Cov washed with Milli-Q and 2× THF (ꢁ); Cov
washed with 2× THF (ꢁ). (A) All data; (B + C) two series from (A) with 95% confidence
intervals.
4. Conclusions
Without molecular sieves, Cov hardly inactivated in THF. With
molecular sieve beads, while spinning on a blood rotator, Cov lost
activity over time. Mechanical forces between Cov and the molecu-
lar sieve beads were found to be the main reason for its instability.
In order to reuse Cov for dipeptide synthesis in the presence of

P. Vossenberg et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 23–27
27
molecular sieves, it needs to be rehydrated in between the batches.
Nevertheless, each intermediate rehydration step also caused some
enzyme activity loss.
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Conflict of interest
[20] ChiralVision, 2009, http://www.chiralvision.com/pdf/protease ELU activity
assay.pd
The authors report no conflict of interest. The authors alone are
responsible for the content and writing of the paper.
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Acknowledgements
This work is part of the IBOS-2 research project “chemo-
enzymatic peptide synthesis,” which is financially supported by the
NWO-ACTS (The Netherlands) and DSM (Geleen, The Netherlands).
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