Microwave activation of enzymatic catalysis

Article abstract of DOI:10.1021/ja802404g

Microwave irradiation can be used to regulate biocatalysis. Herein, the utilization of hyperthermophilic enzymes in a microwave reactor is reported. While these enzymes are inactive at low temperatures, they can be activated with microwave irradiation. To

Full text of DOI:10.1021/ja802404g

Published on Web 07/10/2008
Microwave Activation of Enzymatic Catalysis
†
‡
‡
,†
Douglas D. Young, Jason Nichols, Robert M. Kelly, and Alexander Deiters*
Departments of Chemistry, and Chemical and Biomolecular Engineering, North Carolina State UniVersity,
Raleigh, North Carolina 27695
Received April 2, 2008; E-mail: alex_deiters@ncsu.edu
Microwave irradiation has become an effective tool in synthetic
organic chemistry, dramatically increasing reaction rates and
Scheme 1. Colorimetric CelB Assay: The Enzymatic Hydrolysis of
Produces Glucose (2) and Nitrophenol (3)
1
1–3
yields. In microwave-assisted chemistry, the concept of “specific”
microwave effects is controversial in large part because these effects
4
,5
are difficult to determine. In general, specific effects triggered
by microwave irradiation enable reactions, which cannot proceed
by thermal heating alone. These effects likely arise from direct
coupling of molecules with the microwave field, independent of
3
the reaction temperature. Generally, microwave irradiation induces
2 3
a 1 M Na CO solution, and the absorbance of the alkoxide of 3
molecular rotation arising from dipole alignment with the external,
was measured at 405 nm (Figure 1, note the logarithmic activity
scale). No significant substrate hydrolysis was observed under
microwave irradiation without Pfu CelB addition. Furthermore,
under identical thermal conditions (-20 to 40 °C) (see Figure 2),
5
,6
oscillating electric field. As a result, microwaves significantly
impact molecules with high dipole moments. Proteins and peptides
have significant dipole moments, and thus may be especially
7
,8
-11
-1
-1
susceptible to microwave irradiation. Microwave induction of
no significant enzymatic activity (<10
mol min µg ) was
9
enzymatic reactions has been considered; however, whether
detected in the absence of microwave irradiation (0 W). However,
a greater than 4 orders of magnitude increase in enzymatic activity
of Pfu CelB was achieved with 300 W of microwave irradiation
specific microwave effects are important in biocatalysis is not
10
clear. These effects are difficult to measure because rapid heating
of aqueous solutions under high-power microwave irradiation can
-
6
-1
-1
(2.3 × 10 mol min µg ). Thus, a substantial microwave effect
1
1
1,5
on enzymatic catalysis was observed. This illustrates for the first
result in protein denaturation and inactivation. Moreover, the
intrinsic catalytic ability of enzymes at a relatively low temperature
can obscure the potential rate enhancement through microwave
irradiation. There have been attempts to conduct enzymatic reactions
under microwave irradiation in nonaqueous solvents, but this has
yielded inconclusive results because of greatly reduced enzymatic
time that microwaves can trigger high biocatalytic rates of a
hyperthermophilic enzyme at bulk solution temperatures far below
(∆T ) 70 °C or more) its thermal optimum.
1
0,12
activity.
Specific microwave effects in enzymatic catalysis could be
observed if, at high levels of microwave irradiation, minimal
catalytic activity arises from thermal heating of the bulk solvent.
This can be achieved in an aqueous environment, if care is taken
to (1) effectively cool the reaction mixture during irradiation using
a jacketed reaction vessel and cryogenic cooling, while precisely
measuring the temperature using a fiber optics probe (CEM
Coolmate), and (2) use hyperthermophilic enzymes which have
minimal catalytic activity at temperatures below 40 °C and denature
at much higher temperatures than their mesophilic counterparts.
Here, a ꢀ-glucosidase (CelB) from the hyperthermophilic archaeon,
Pyrococcus furiosus, was examined for biocatalytic function under
microwave irradiation; this enzyme is optimally active at ∼110 °C.
P. furiosus CelB cleaves exoglycosidic linkages in both natural (e.g.,
Figure 1. Effect on microwave irradiation on Pfu CelB, Pdu CelB, Tm
GalA, and SsoP1 CE activity. All experiments were conducted in triplicate.
1
3
cellobiose) and synthetic substrates (e.g., 1, Scheme 1).
Enzymatic assays were conducted by cooling the nitrophenoate
substrate 1 in reaction buffer (50 mM NaOAc, pH 5.5, 10% DMSO)
to -20 °C, followed by the addition of heat-pretreated Escherichia
coli cell extract containing recombinant CelB. Microwave irradiation
(
(
300 W) was applied until the reaction temperature reached 40 °C
all temperatures were measured using a fiber optics temperature
sensor); coolant at -60 °C was simultaneously circulated through
the jacketed reaction vessel. The reaction was then quenched with
†
Figure 2. Temperature profiles (measured with a fiber optics probe) of
the microwave mediated (300 W) and the thermal reactions.
Department of Chemistry.
Department of Chemical and Biomolecular Engineering.
‡
1
0048 9 J. AM. CHEM. SOC. 2008, 130, 10048–10049
10.1021/ja802404g CCC: $40.75  2008 American Chemical Society

C O M M U N I C A T I O N S
temperatures, the mesophilic analogue Pdu CelB also did not
denature, as determined by CD (Supporting Information).
The results reported here illustrate for the first time the intrinsic
effect of microwave irradiation on biocatalysis. Furthermore, they
indicate that hyperthermophilic enzymes can be activated at
temperatures far below their optimum, presumably by microwave-
induced conformational flexibility. This finding offers the prospect
of using hyperthermophilic enzymes at ambient temperatures to
catalyze reactions with thermally labile substrates and products.
Furthermore, microwaves could be used to regulate biocatalytic rates
at very low temperatures for enzymes from less thermophilic
sources. Both of these possibilities are being considered.
Acknowledgment. We acknowledge support by North Carolina
State University and CEM Corporation. D.D.Y. was supported by
a U.S. DoEd GAANN Fellowship and an ACS Medicinal Chemistry
Graduate Fellowship. R.M.K. acknowledges support from the U.S.
NSF. We thank P. Woodward and J. Notey for experimental
support.
Figure 3. Pfu CelB enzymatic activity dependence on microwave power.
All experiments were conducted in triplicate, 5 mM substrate concentration.
In all cases, the temperature increased from -20 to 40 °C.
Note that no microwave biocatalytic activation was observed in
the case of a mesophilic homologue of CelB from Prunus dulcis
(
Pdu CelB) (Figure 1); the enzyme activity that was observed was
Supporting Information Available: Experimental protocols for
enzymatic assays, and circular dichroism measurements. This material
is available free of charge via the Internet at http://pubs.acs.org.
likely related to thermal stimulation during heating between -20
to 40 °C.
To further probe the efficacy of microwave activation, two other
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(
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JA802404G
J. AM. CHEM. SOC. 9 VOL. 130, NO. 31, 2008 10049