Efficiency of 2.45 and 5.80 GHz microwave irradiation for a hydrolysis reaction by thermostable β-Glucosidase HT1

Article abstract of DOI:10.1080/09168451.2014.891931

Microwave irradiation at different frequencies gave unique results for the hydrolyses of glycosyl bonds by β-Glucosidase HT1. With the observed relative complex permittivity data for the reaction buffer, 2.45 GHz microwave radiation affected both waters and ions, while 5.80 GHz only affected waters. We, here, propose that would be one of the unique "microwave nonthermal effects".

Full text of DOI:10.1080/09168451.2014.891931

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Efficiency of 2.45 and 5.80ꢀGHz microwave
irradiation for a hydrolysis reaction by thermostable
β-Glucosidase HT1
a
b
a
a
Izuru Nagashima , Jun-ichi Sugiyama , Tomomi Sakuta , Masahide Sasaki & Hiroki
a
Shimizu
a
Bioproduction Research Institute, National Institute of Advanced Industrial Science and
Technology (AIST), Sapporo, Japan
b
Nanosystem Research Institute, National Institute of Advanced Industrial Science and
Technology (AIST), Tsukuba, Japan
Published online: 29 Apr 2014.
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To cite this article: Izuru Nagashima, Jun-ichi Sugiyama, Tomomi Sakuta, Masahide Sasaki & Hiroki Shimizu (2014)
Efficiency of 2.45 and 5.80ꢀGHz microwave irradiation for a hydrolysis reaction by thermostable β-Glucosidase HT1,
Bioscience, Biotechnology, and Biochemistry, 78:5, 758-760, DOI: 10.1080/09168451.2014.891931
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Bioscience, Biotechnology, and Biochemistry, 2014
Vol. 78, No. 5, 758–760
Note
Efficiency of 2.45 and 5.80 GHz microwave irradiation for a hydrolysis
reaction by thermostable β-Glucosidase HT1
1
2
1
1
Izuru Nagashima , Jun-ichi Sugiyama , Tomomi Sakuta , Masahide Sasaki and
1,
Hiroki Shimizu *
1
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),
2
Sapporo, Japan; Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology
(
AIST), Tsukuba, Japan
Received October 22, 2013; accepted December 25, 2013
http://dx.doi.org/10.1080/09168451.2014.891931
Microwave irradiation at different frequencies
gave unique results for the hydrolyses of glycosyl
bonds by β-Glucosidase HT1. With the observed rel-
ative complex permittivity data for the reaction buf-
fer, 2.45 GHz microwave radiation affected both
waters and ions, while 5.80 GHz only affected
waters. We, here, propose that would be one of the
unique “microwave nonthermal effects”.
electrical incubator EC-40R (AS ONE Corp.) for
conventional heating, commercially available MWS-
1000 (EYELA, Tokyo Rikakikai Co., Ltd.) for the
2.45 GHz microwave, and an in-house build up
machine based on ATMW500B-5.8 G (Amil Co., Ltd.)
1
2)
for the 5.80 GHz microwave (Fig. 1).
First, the reactions were heated conventionally. We
found that 60 °C gave the best results, which corre-
sponded with the supplier’s information that the opti-
mum temperature is 60 °C. The reaction proceeded
even at lower temperatures smoothly, but enzyme inac-
tivation occurred within 20 min at 70 °C and the reac-
tion was stopped in only 69% yield.
Key words: microwave; enzyme; glycosidase; gluco-
sidase; hydrolysis
It has become common in recent years to use micro-
wave for chemical reactions. Although microwave is
used mainly as an effective heating tool, the authors
reported some results that would be caused by micro-
Then, we performed the same reactions but with
heating by 2.45 GHz microwave using MWS-1000
(EYELA), which could strictly control the reaction
temperature. The power was set as 100 W. Practically,
the irradiation power became initially 100 W but sooner
was automatically attenuated 0–40 W (40, 50, and
60 °C) or 50–70 W (70 °C) to keep set temperature. A
total of 50 °C gave the best performance for the reac-
tion completed within 20 min, in contrast to the 30 min
required by conventional heating. Even at 40 °C, the
reaction was completed within 30 min, but it was not
finished until 60 min in the incubator. When the reac-
tion was performed at 60 °C, the enzyme became inac-
tive and the reaction was stopped by 90% in 20 min.
Thus, although an optimum temperature for the enzyme
is reported at 60 °C, 60 °C by 2.45 GHz microwave
inactivated the enzyme. From these results, we con-
cluded that 2.45 GHz microwave affected more effec-
tively than conventional heating, lowering the optimum
temperature, and accelerating the reaction.
1
–5)
wave nonthermal effects.
Meanwhile, a few reports
claimed that microwave gives only thermal effects
6
,7)
caused by a rapid heating
and a controversy has
arisen this year between Dr Kappe and Dr Dudley
about whether microwave does, in fact, have a unique
8
,9)
nonthermal effect.
We thought that investigating the
efficiency of different microwave frequencies could
reveal a solution to this dispute. Glucosidase, which
hydrolyzes glycosyl bonds, is one of the most common
enzymes in the medicinal and biomass research fields.
β-Glucosidase HT1 is a commercially available glycosi-
1
0)
dase and its optimum temperature is relatively high,
at 60 °C in 50 mM sodium acetate buffer (pH 5.0).
Here, we approached the microwave nonthermal effects
with controlling the reaction temperature by conven-
tional method (incubator) and by using 2.45 and 5.80
GHz microwave irradiations for this β-Glucosidase
HT1 reaction.
Next, we tested 5.80 GHz microwave effectiveness
by using a microwave irradiation machine prepared
in-house. The oscillated power from the magnetron was
50 W and attenuated by the cold water load to
10–15 W, then irradiated intermittently to keep fixed
temperature manually. The reactions were not acceler-
ated as in the experiment with 2.45 GHz microwave,
4
-Methoxyphenyl glucopyranoside has been chosen
11)
as the substrate for this research.
(Scheme 1) The
reactions were performed at 40, 50, 60, and 70 °C in
0 mM sodium acetate buffer (pH 5.0) with 10 mM
NaCl. Three types of heating methods were applied:
2
*Corresponding author. Email: hiroki.shimizu@aist.go.jp
©
2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
.45 and 5.80 GHz microwave effect for β-Glucosidase HT1
759
Nyquist diagram in Fig. 2 for distilled water (left) and
for 50 mM sodium acetate buffer (pH 5.0) (right),
where the horizontal and the vertical axes corresponded
to the real and the imaginary parts, respectively. Dielec-
tric property within a complex plane was presented
semicircular by Equation (1).
Â
Ã
2
2
0
00
r
e ðxÞ ꢀ ferð0Þ þ erð1Þg=2 þfe ðxÞg
r
2
¼
½fe ð0Þ ꢀ e ð1Þg=2ꢁ
(1)
r
r
The higher the conductivity, the further the actual
diagram departs from the semicircle at the lower fre-
Scheme 1. Hydrolysis by β-Glucosidase HT1.
14)
quency and showed rectilinear.
In the case of dis-
tilled water (Fig. 2, left), the approximate curve was
semicircular in all the measured bands, which suggests
that pure water behaved only as a dielectric under irra-
diation microwaves. On the other hand, the result from
the buffer solution experiments showed the rectilinear
locus as an electric conductor from 200 MHz to
and the initial rates of the reactions were similar to that
of conventional heating. In addition, the enzyme activi-
ties were lost by 20 and 10 min at 60 and 70 °C,
respectively. From these results, it was apparent that
5
.80 GHz microwave did not improve this reaction.
Although we got fruitful results using 2.45 GHz
2
2
.45 GHz and the semicircular locus as dielectric from
.45 to 14 GHz (Fig. 2, right). When the impressed
microwave irradiation which accelerated the glucosi-
dase reaction, 5.80 GHz microwave did not cause any
improvement. To determine how and on which mole-
cules microwave worked, we examined the absorption
property of the microwave for water and the buffer
voltage of the electromagnetic wave was constant, the
electromagnetic energy was lost and the irradiated
material was heated proportionally with the imaginary
part, which is the vertical axis of the figure. The dielec-
tric was heated based on the vibration phase delay of
the dipole to the vibration of the impressed electric
1
3)
solution. Relative complex permittivity at room tem-
perature was measured from 200 MHz to 14 GHz by
the reflection probe method. The result is shown as the
Fig. 1. Results of hydrolyses.
Notes: Reactions were heated by incubator (A), 2.45 GHz (B), and 5.80 GHz (C) microwaves at 40 °C (○), 50 °C (●), 60 °C (4), 70 °C (▲),
and 80 °C (×). In the case of 2.45 GHz microwave experiment, the irradiation power was controlled automatically, so we set the staring time (t = 0
min) when the reaction was started, and was immediately irradiated the microwave to take the set temperature. Meanwhile in the case of
5.80 GHz microwave experiment, attenuation should be performed manually after the microwave irradiation was started, so we set the start time
(t = 0 min) when the reaction reached the target temperature. Therefore, some reactions were already proceeded to some extent even at t = 0 min.
Fig. 2. Relative complex permittivity.
Notes: They measured by the reflection probe method at room temperature at 200 MHz (●), 2.45 GHz (○), 5.80 GHz (4), and 14.0 GHz (▲) for
water (left) and for 50 mM sodium acetate buffer (pH 5.0). Bold lines are observed results and broken lines are the approximate lines.

760
I. Nagashima et al.
field, and the calorific power was the largest in the spe-
cific frequency. In contrast, the electric conductor was
heated based on the Joule’s heat by monopole migra-
tion to the impressed electric field, and the higher fre-
quency showed lower heating. Therefore, the 5.80 GHz
microwave was able to heat dipole water molecules by
dielectric heating but did not heat monopole ions such
as sodium cations and acetate anions because the fre-
quency was too rapid and the Coulomb force of the
impressed electric field was ignored. On the other hand,
the relative complex permittivity at 2.45 GHz showed
ranges in both dielectric and conductive. Hence, the
heating was not only dielectric but also Joule’s heating
of ions; the 2.45 GHz microwave could affect both
dipole water and monopole ions in the buffer solution.
For hydrolysis of 4-Methoxyphenyl glucopyranoside
by β-Glucosidase HT1, 2.45 GHz microwave heating
gave advantages, the reaction became faster, and the
optimum temperature decreased to 50 °C, but 5.80 GHz
microwave heating gave no significant advantage. We
consider that this is why 2.45 and 5.80 GHz microwave
affected the reaction at the molecular level in different
ways. A possible explanation is that buffering ions
formed ion-bonds, for example, to carboxyl groups on
Glu or Asp, which are key functions for hydrolysis
reaction, or coordinated to the enzyme as the buffer sta-
bilized it. When an enzyme acted as the catalyst of a
reaction, the following steps occurred: out the coordi-
nated water and bonded ions from a reaction pocket,
occupying the substrate in the pocket, reaction and
release of the resulting product. As we discussed con-
cerning the heating mechanism, 5.80 GHz microwave
affected only the water molecules in buffer solution, but
[2] Yoshimura Y, Shimizu H, Hinou H, Nishimura S-I. A Novel
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[
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[10] Available from Thermostable Enzyme Laboratory Co., Ltd.,
Kobe, Japan.
11] Typical procedure of the reaction: the reaction was carried out in
[
1
1
.5 mL scale of 1 mM of 4-Methoxyphenyl glucopyranoside and
00 mU/mL Glucosidase HT1 in 50 mM sodium acetate buffer
(
(
pH 5.0). The reaction was heated by either incubator EC-40R
AS ONE), MWS-1000 (EYELA) for using 2.45 GHz micro-
wave or our own making machine for using 5.80 GHz. The reac-
tion was sampled (5 μL) every 10 min and stopped the reaction
by mix of 15 μL of 8 M guanidine hydrochloride solution. The
reactions were monitored by HPLC [column: Inertsil ODS-3, /
2
.45 GHz worked on both water molecules and buffer-
ing ions. Therefore, even if microwaves were not used
as a heating tool, 2.45 GHz microwave irradiation
would give some benefit for the first step of the reac-
tion, that is, taking ions out to cleave the ion bond,
decrease entropy penalty, etc. On the other hand,
4
.6 mm × 250 mm; eluate: acetonitrile with 0.1% TFA, gradient
increase from 15 to 35% over 15 min; column oven temperature:
0 °C; flow rate: 1 mL/min, detector: 220 nm] and the reaction
ratio was calculated by signal intensities of the starting material,
-methoxyphenyl glucopyranoside (Rt 7.5 min) and resulting
product, 4-methoxyphenol (Rt 14.5 min).
3
4
5
.80 GHz microwave would work only on water mole-
cules that would not produce a special advantage for the
reaction like the 2.45 GHz irradiation. Meanwhile, both
[12] The 5.80 GHz microwave irradiation equipment was applied the
system of ATMW500B-5.8 G (Amil Co., Ltd.). Microwave was
oscillated by the magnetron, attenuated by the attenuator,
matched by EH tuner to minimizing reflective power and then
irradiated to the target. Dummy load was set on the waveguide
terminal to absorb transmitted wave and the target was heated
by the progressive wave. The quartz tube (i.d. 9 mm and o.d. 10
mm), practically NMR tube, was used for the reaction vessel.
The vessel was set parallelly along the direction of the vibration
of electric field at the center of the E surface of 20 × 40 mm
waveguide. Temperature was measured directly by AMOTH
FL-2000 fiber optic thermometer (ANRITSU METER Co., Ltd.).
2
.45 and 5.80 GHz microwaves enhanced inactivation
of the enzyme. We would not propose any clear
consideration for dependency of microwave frequency
at this point.
Acknowledgments
The authors thank Mr T. Oku, Dr Y. Kashima and
Ms H. Tanaka (Thermostable Enzyme Laboratory Co.,
Ltd.) for providing the enzyme.
[
13] Relative complex permittivity was measured by open-probe
measurement system (KEYCOM Corp.) using ZVB14 vector
network analyzer (Rohde & Schwarz) calibrated by open (air),
short (copper foil) and load (Kanto Chemical Co., Inc. 11307-3B
distilled water).
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Microwave in organic synthesis. 3rd ed. Weinheim: Wiley-VCH;
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