Microwave radiation accelerates trypsin-catalyzed peptide hydrolysis at constant bulk temperature

Article abstract of DOI:10.1016/j.tetlet.2015.09.003

The influence of microwave radiation on trypsin activity was explored using a CEM CoolMate apparatus at a constant bulk temperature. Digestion of Nα(±)-benzoyl-d/l-arginine-4-nitroanilide hydrochloride, azocasein and casein catalyzed by trypsin from the b

Full text of DOI:10.1016/j.tetlet.2015.09.003

Tetrahedron Letters 56 (2015) 5804–5807
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Microwave radiation accelerates trypsin-catalyzed peptide hydrolysis
at constant bulk temperature
⇑
Sina Atrin Mazinani, Benjamin DeLong, Hongbin Yan
Department of Chemistry, Brock University, 500 Glenridge Ave., St. Catharines, Ontario L2S 3A1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
The influence of microwave radiation on trypsin activity was explored using a CEM CoolMate apparatus
Received 2 August 2015
Revised 22 August 2015
Accepted 2 September 2015
Available online 8 September 2015
at a constant bulk temperature. Digestion of Na( )-benzoyl-D/L-arginine-4-nitroanilide hydrochloride,
azocasein and casein catalyzed by trypsin from the bovine pancreas was found to be accelerated when
the reaction mixture was exposed to microwave radiation, while the bulk temperature was maintained
constant through external cooling. Trypsin activity was found to be increased significantly when the reac-
tion mixture was irradiated with microwave at a constant temperature. Cyclic dichroism measurement of
trypsin exposed to microwave radiation suggests that there are changes in the secondary structure of
trypsin exposed to microwave and conventional heating, however, these changes are presumably due
to self-cleavages of trypsin.
Keywords:
Microwave heating
Conventional heating
Trypsin
Ó 2015 Elsevier Ltd. All rights reserved.
Casein
Enzyme activity
Microwave heating has become an increasingly popular heating
method in a wide range of applications over the past few decades.
In organic synthesis, microwave heating has been shown to be very
useful, often leading to faster reactions, higher yields and better
selectivity.1–6 One of the areas that has attracted heated debate
relates to whether ‘microwave specific effects’ exist.7–12 Some
recent publications suggest that such effects are possible, which
could presumably be attributed to ‘selective heating’ of reactants
with high absorption cross sections.13,14 We are interested in the
effect of microwave irradiation on reactions that involve biomole-
cules such as enzymes, particularly when the reaction mixture is
maintained at a constant bulk temperature while exposed to low
power microwave, as these experiments could provide some
insight into the influence of microwave exposure on biological sys-
tems, where the macroscopic temperature is mediated by bulk
surroundings.
While some work has demonstrated that microwave radiation
affects protein/enzyme structures,15–18 bulk literature evidence
has suggested that enzymatic activity can be affected when
enzymes are exposed to microwave. The literature in this area
prior to 2007 is summarized in a review.19 This area of research
was further demonstrated in more recent work.20–32 It is worth
noting that among the challenges in establishing reproducible
results in these observations, monitoring and regulation of the
reaction temperature accurately has been the most difficult
one.33 In this respect, Kappe and co-workers34 showed that there
was no difference in reactivity and enantioselectivity in the kinetic
resolution of rac-1-phenylethanol catalyzed by immobilized
lipases under conventional or microwave heating, when the tem-
peratures were maintained the same. In this work, we further
explored the influence of microwave exposure on trypsin activity
while the bulk reaction temperature is kept constant through
external cooling.
Towards this goal, trypsin from the bovine pancreas was chosen
as the enzyme and a dipeptide, Na( )-benzoyl-D/L-arginine 4-ni-
troanilide hydrochloride (BAPNA), as the substrate (Scheme 1).
BAPNA is readily hydrolyzed by trypsin to give N-benzoyl arginine
and p-nitroanaline. As the latter can be quantified readily by a col-
orimetric assay, this system provides an easy approach to study the
effect of microwave irradiation on enzyme activity.
In order to maintain the reaction temperature while the
reaction mixture is exposed to microwave, the CEM CoolMate
NO2
O
NH2
NO2
H
N
H
N
COO
N
H
Trypsin
Microwave
O
O
NH
NH
NH2
H2N
NH2
( )-benzoyl-
H2N
⇑
Corresponding author. Tel.: +1 905 688 5550x3545.
E-mail address: tyan@brocku.ca (H. Yan).
Scheme 1. Hydrolysis of N
a
D
/
L
-arginine 4-nitroanilide hydrochloride
by trypsin.
http://dx.doi.org/10.1016/j.tetlet.2015.09.003
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

S. A. Mazinani et al. / Tetrahedron Letters 56 (2015) 5804–5807
5805
reaction vessel circumvented by a jacket where a microwave-
transparent fluid, such as perfluoropolyether Galden HT 110,
circulates. The coolant is pre-cooled in a reservoir by either dry
ice or liquid nitrogen. An important feature of this setup is that
the reaction temperature is monitored in situ with an optic fiber
temperature probe, which provides feedbacks to the system so that
the microwave output can be adjusted in a real-time fashion to
maintain a pre-determined temperature in the reaction mixture.
Note that in all the experiments described in this work, stirring
was set at ‘high’ to ensure homogeneity of reaction mixtures.
When BAPNA was subjected to trypsin digestion, it became
clear that hydrolysis was accelerated by microwave radiation,
while the bulk reaction temperature was kept constant by simulta-
neous cooling. Figure 2 shows the progress of two reactions carried
out at 25 °C, as measured in the absorbance of product formed over
time, one in the absence of microwave (control) and another
exposed to up to 20 W microwave radiation while the temperature
optic fibre probe to CoolMate
reaction vessel
cooling jacket
back to CoolMate
CEM Discover
Microwave Reactor
from CoolMate
Stirring bar
Figure 1. The CEM CoolMate Microwave System that is used in this study.
Microwave System (Fig. 1) was used. This microwave reactor oper-
ates at a frequency of 2.45 GHz, and provides the ability to keep
constant bulk reaction temperatures while the reaction mixture
is exposed to microwave irradiation. This system features a
Figure 2. Digestion of BAPNA (1.0 mM) by trypsin (5.0 lM). Solid line: reaction at 25 °C in the absence of microwave; dashed line: reaction mixture was exposed to
microwave radiation of varying power of up to 20 W, while the bulk temperature was kept at 25 °C through external cooling by a CoolMate system. The flow rate of the
CoolMate system was kept constant.
Figure 3. Plot of changes in absorbance at 440 nm of samples taken 5 min after reactions initiate, versus initial azocasein concentrations. Both control (reactions carried out
in the absence of microwave) and microwave (exposed to microwave radiation of varying power of up to 20 W) reactions were carried out at 22 °C. The flow rate of the
CoolMate system was kept constant.

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S. A. Mazinani et al. / Tetrahedron Letters 56 (2015) 5804–5807
was maintained at 25 °C. Indeed, the increase in the rate of reaction
through microwave radiation was observed when the reactions
were carried out under other conditions, such as 37 °C and various
trypsin concentrations (data not shown); however, in order to
derive a quantitative comparison of enzyme properties, it became
clear that BAPNA is not a suitable substrate due to its limited sol-
ubility and, as a consequence, low absorbance of the product
formed.
We next examined the effect of microwave radiation on the rate
of digestion of azocasein by trypsin.35 Similar trends, that is, accel-
eration of the rate of hydrolysis by microwave, were observed.
Despite the challenges in generating a standard curve that corre-
lates absorbance with concentration of product formed, which
led to difficulty in obtaining quantitative comparison, a plot of
changes in absorbance at 440 nm of samples taken from the reac-
tion mixture 5 min after the reaction started at a range of initial
azocasein concentrations (Fig. 3) clearly indicated an increase in
the reaction rate, specially at higher initial azocasein concentra-
tions. The results suggest that while trypsin is presumably satu-
rated at low concentrations of azocasein, at higher azocasein
concentrations, digestion of the substrate by trypsin is accelerated
by microwave irradiation while the bulk temperature is kept the
same as in the control.
In order to derive a quantitative comparison of trypsin kinetics,
casein was subsequently used as trypsin substrate as a calibration
curve can be readily determined.36 Due to the constraint on the
volume of the microwave reaction vessel, Km and Vmax values were
not determined; instead, the trypsin activity (or ‘apparent’ activity
in the presence of microwave) was determined at a range of casein
concentrations.
As is shown in Figure 4, compared with untreated control, the
trypsin activity is increased when it was exposed to microwave
irradiation while the bulk temperature was maintained constant
at 22 °C. The increased activity is particularly profound at high
Figure 4. Trypsin activity determined at different casein concentrations. Data are triplicate for both control and microwave experiments. Microwave reactions were carried
out with exposure to a constant 10 W microwave while the temperature was maintained at 22 °C. Definition of a unit: the amount in micromoles of -tyrosine equivalents
released from casein per minute. Flow rates of the CoolMate system were manually adjusted so that a constant bulk temperature can be maintained.
L
Figure 5. CD spectra of trypsin: freshly prepared and untreated (red solid line), heated at 37 °C in an oil bath (black dashed line) for 40 min, and heated by up to 20 W
microwave for 40 min while the bulk temperature was maintained at 37 °C (black solid line).

S. A. Mazinani et al. / Tetrahedron Letters 56 (2015) 5804–5807
5807
casein concentrations where trypsin is fully saturated. Note that in
References and notes
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shots of the temperature and microwave power profiles), the bulk
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system, a control experiment was carried out where the casein
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ing, however, without simultaneous cooling by the CoolMate sys-
tem. It is quite clear from Figure 3S in the Supplementary
Material that the temperature as measured by an optic fiber probe
rose from 20 °C to 40 °C in 4 min.
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Acknowledgments
This research was supported by the Natural Science and
Engineering Research Council of Canada. The authors also wish to
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Supplementary data
Supplementary data (experimental procedures for this work
and representative documentation of temperature and microwave
power profiles) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.tetlet.2015.09.003.