Enzymatic activity and thermal stability of metallo proteins in hydrated ionic liquids

Article abstract of DOI:10.1002/bip.21526

Hydrated choline dihydrogen phosphate (Hy[ch][dhp]) containing 30 wt% water was investigated as a novel protein solvent. The Hy[ch][dhp] dissolved some metallo proteins (cytochrome c, peroxidase, ascorbate oxidase, azurin, pseudoazurin and fructose dehydrogenase) without any modification. These proteins retained the surroundings of the active site after dissolution in Hy[ch][dhp]. Some metallo proteins were found to retain their activity in the Hy[ch][dhp].

Full text of DOI:10.1002/bip.21526

Enzymatic Activity and Thermal Stability of Metallo Proteins in
Enzymatic Activity and Thermal Stability of Metallo Proteins in Hydrated
Hydrated Ionic Liquids
Ionic Liquids
Kyoko Fujita, Hiroyuki Ohno
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo184-8588, Japan
Received 30 April 2010; revised 23 June 2010; accepted 14 July 2010
Published online 27 July 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21526
chemistry, enzymatic catalysis,5 and green chemistry, exploiting
their excellent stability and unusual solvent properties. A num-
ber of enzymes retain their catalytic activity in certain IL.6,7 Li-
ABSTRACT:
Hydrated choline dihydrogen phosphate (Hy[ch][dhp])
pases, in particular, maintain their activity in hydrophobic and
containing 30 wt% water was investigated as a novel
anhydrous ILs. Previously published papers mentioned that
protein solvent. The Hy[ch][dhp] dissolved some metallo
their selectivity and operational stability are often better than
proteins (cytochrome c, peroxidase, ascorbate oxidase,
azurin, pseudoazurin and fructose dehydrogenase)
without any modification. These proteins retained the
surroundings of the active site after dissolution in
Hy[ch][dhp]. Some metallo proteins were found to retain
these in traditional volatile solvents.8 In most reports of hydro-
phobic ILs, the enzymes are not dissolved but are merely in a
dispersed state and are therefore regarded as a heterogeneous
catalyst. Some hydrophilic ILs accelerate the dissolution of pro-
tein, but in these cases the protein secondary structure has been
lost after dissolution.9,10 Such structural changes generally cause
loss of enzymatic activity.11 To induce solubility while main-
taining the activity of enzymes in ILs, polyether structure and
related additives have been employed.12,13 These structures
were incorporated into the protein surface as polyethylene ox-
ide (PEO) modification and on the component ions of ILs.
This PEO-modification is effective, but is difficult to prepare; it
is also difficult to separate the PEO-modified proteins from
unreacted PEO chains. Thus, there is a strong interest in pre-
paring ILs having suitable affinity with proteins.
#
their activity in the Hy[ch][dhp]. 2010 Wiley
Periodicals, Inc. Biopolymers 93: 1093–1099, 2010.
Keywords: ionic liquids; enzymatic activity; metallo
proteins
This article was originally published online as an accepted
preprint. The ‘‘Published Online’’ date corresponds to the
preprint version. You can request a copy of the preprint by
emailing the Biopolymers editorial office at biopolymers@wiley.
com
We have studied hydrated ILs as novel solvents for pro-
teins.8,14 The hydrated ILs maintain basic properties of pure
ILs, but a small amount of water considerably improved
protein solubility. Solubility varies with the component
ions. Our previous work showed that hydrated ILs, compris-
ing appropriate anions which have oxo acid residues, dis-
solved cyt c quite well. Furthermore, the structure and activ-
ity of cyt c differed according to the component ions. We
investigated the effect of these ions on the structure and the
activity of dissolved cyt c.14 We expected kosmotropicity to
be a factor influencing the properties of dissolved cyt c.
Kosmotropicity (closely related to the Hofmeister series) is
well established in the aqueous phase. In general in the
aqueous phase, a pair of strong kosmotropic anions, and a
chaotrope cation, stabilize proteins, whereas strong kosmo-
tropic cations destabilize proteins.15–17 Even in ILs, there
have recently been many reports about the effect of kosmotro-
INTRODUCTION
onic liquids (ILs) are remarkable materials with very dif-
ferent properties from molecular liquids.1,2 ILs are capa-
ble of having negligible vapor pressure and high thermal,
chemical, and electrochemical stability.3 They also have
tunable properties including polarity, hydrophobicity,
I
and solvent miscibility.4 ILs have been applied in electro-
Correspondence to: Hiroyuki Ohno; e-mail: ohnoh@cc.tuat.ac.jp
Contract grant sponsor: Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan
Contract grant numbers: 21225007, 17073005
Contract grant sponsor: Tokyo University of Agriculture and Technology
C
VV
2010 Wiley Periodicals, Inc.
Biopolymers Volume 93 / Number 12
1093

1094
Fujita and Ohno
picity on physical properties such as two phase behavior,18 and
on the activity of biomolecules.19 In our previous report we
found that the effect of component ions on cyt c was corre-
lated with kosmotropicity.14 In particular, hydrated choline
dihydrogen phosphate (Hy[ch][dhp]), which is one of the best
combinations of chaotropic cation and kosmotropic anion, is
an excellent solvent for cyt c. We believe that kosmotropicity
affects protein structure in hydrated ILs. Cyt c underwent no
structural change and retained its activity in Hy[ch][dhp],
even after 18 months stored at room temperature.
FIGURE 1 Photographs of dissolved metallo proteins in
Hy[ch][dhp]. (a) cyt c, (b) HRP, (c) Az, (d) paz, and (e) ASOD.
Protein Solubility
This work examines the value of Hy[ch][dhp] as a solvent
for proteins. Some studies of proteins dissolved in ILs used
mainly cyt c8 and lysozyme.20,21 Here, the effect on the struc-
ture and activity of various metallo proteins have been investi-
gated in Hy[ch][dhp]. The effect of dissolution on the structure
and activity are important when protein and other biomole-
cules are considered in vitro. Metallo proteins were chosen for
this study because they are well understood physicochemically
and they have many different functions in vivo, including enzy-
matic reaction, transport and storage of useful materials.22 Fur-
thermore, metallo proteins have been widely studied including
electrochemically expecting bioapplications, such as biocata-
lytic reaction, biosensors, and biofuel cells. We have been ana-
lyzing the electron transfer reactions of metallo proteins in a
nonaqueous phase.12 In the present study, we evaluate the
Hy[ch][dhp] as a reaction medium for metallo proteins.
Metallo proteins were mixed with [ch][dhp] containing 30
wt% water (about five water molecules per ion pair), and
they were found to comprise two groups, soluble and insolu-
ble. The proteins cyt c, HRP, Az, paz, and ASOD dissolved
and gave clear homogeneous solutions; the original color is
shown in Figure 1. These proteins were soluble in
Hy[ch][dhp] with concentration over 1 mM. In contrast, he-
moglobin and myoglobin scarcely dissolved at all in
Hy[ch][dhp] and formed a dispersion in the vial. Their solu-
bility was calculated to be below 0.5 lM in the clear layer
with UV-vis spectroscopy after centrifugation. Solubility was
still low when the water content was as high as 40 wt%. Fur-
thermore, when myoglobin was dissolved in buffer first and
then mixed with Hy[ch][dhp], the myoglobin still did not
dissolve in the IL. The lyophilized condition of the sample
did not affect the solubility. Cyt c and myoglobin therefore
have very different solubilities despite their similar proper-
ties. Table I shows basic properties of the proteins studied
here, molecular weight, number of amino acids, isoelectric
point (PI), and secondary structure. However, these do not
correlate with the solubility in Hy[ch][dhp], so that other
factors must determine this; an investigation is in progress.
RESULTS AND DISCUSSION
Hy[ch][dhp] was prepared with a small amount of water. It
is plausible that these mixed water molecules hydrate the
component ions so that they are bound water, not free water
as in the buffer solution. When Hy[ch][dhp] prepared with
different amounts of water was heated to 1008C, the water
content became constant, at approximately two water mole- Protein Features After Dissolving in Hy[ch][dhp]
cules per ion pair (14 wt%). We suggest that all water mole- The conditions of the active site were important in determin-
cules are strongly bound to the component ions of IL, and ing the original characteristics of proteins. The environment
there are no free water molecules, when Hy[ch][dhp] is pre- of the active site of the dissolved proteins, cyt c, HRP, Az,
pared with less than 14 wt% water. However, Hy[ch][dhp] and ASOD in the IL was analyzed by resonance Raman (RR)
with 14 wt% water has high viscosity, and it has difficult to spectroscopy. The RR spectra of metallo proteins have been
use as a solvent. Addition of water considerably decreased extensively studied. RR spectra are sensitive to the coordina-
the viscosity. The viscosity of Hy[ch][dhp] with 30 wt% tion state and spin state of the active site. All spectra show
water is much less than with 20 wt% water (further details of the original features based on the native structure of the
the relation between water content and viscosity will be active site (see Figure 2). These spectra were compared with
stated below). A large excess of water might lead to a concen- those in buffer solution. For ASOD, the spectrum in
trated salt solution which is deleterious to proteins.8,23,24 We Hy[ch][dhp] was compared with the difference spectrum in
therefore used Hy[ch][dhp] with 30 wt% water as a solvent buffer solution, because it was difficult to observe a clear
in this work. In this solution, there are about five water mol- spectrum of ASOD in buffer (see Figure 3). We have previ-
ecules per ion pair. This Hy[ch][dhp] was examined as sol- ously reported the similarity of the RR spectra for cyt c in
vents for a series of metal proteins.
Hy[ch][dhp] and in buffer.14 HRP is a heme containing
Biopolymers

Metallo Proteins in Hydrated Ionic Liquids
1095
Table I Basic Properties of Dissolved Proteins in Hy[ch][dhp]
Protein
Molecular Weight
Hydrophobic Residue
pI
Helical (%)
Beta Sheet (%)
Cytochrome c from horse heart
Hemoglobin from human
Myoglobin from bovin heart
12,400
64,500
16,700
44,000
14,000
13,100
140,000
44/104
316/574
77/153
10
6.8
7.3
7.5
–
40
76
74
52
12
16
13
1
0
0
Peroxidase from horseradish
152/306
65/129
71/123
1
Azurin II from Alcaligenes Xylosoxidans
Pseudoazurin from Achrom bactor cycloclastes
Ascorbate oxidase from Cucurbita
46
35
36
8.4
5–6
283/552
enzyme which catalyses the oxidation of various organic the RR mode near 260 cm21 comprising the Cu-N(His imid-
compounds with hydrogen peroxide.25,26 Figure 2a shows the azole) stretching, and the modes near 400 cm21 comprising
ferric state of HRP in both buffer solution and Hy[ch][dhp]. the Cu-S(Cys) remain the same as in the native state. ASOD
These spectra imply that the iron axial ligand does not is one of the multi copper proteins which catalyse the oxida-
undergo change in Hy[ch][dhp]. Az is a type I copper con- tion of ascorbic acid (with oxygen).29 Its spectrum in
taining protein; this family is called blue-copper proteins.27,28 Hy[ch][dhp] showed that bands assigned to the Cu-N (pep-
As shown in Figure 2b, the RR modes near 400 cm21 in blue tide or amide side chain) and Cu-S (cystein) vibrations are
copper proteins have typically been assigned to Cu ligand maintained in Hy[ch][dhp] as shown in Figure 3. These find-
stretching motions. In Hy[ch][dhp], the spectra suggest that ings show that these dissolved proteins maintained the native
surroundings of the active site even in Hy[ch][dhp].
Next, the secondary and tertiary structures of proteins
were investigated by circular dichroism (CD) measurements.
Cyt c was used as the target protein for this analysis, because
the relation between the structure and CD spectrum has been
studied in detail before.30,31 For comparison, cyt c was dis-
solved in buffer, Hy[ch][dhp], and similarly hydrated N-
butyl-N-methyl pyrrolidinium dihydrogen phosphate
([C4mpyr][dhp]). In the far UV region (250–250 nm), the
CD spectrum is an effective probe for protein secondary
structure such as a-helix and b-sheet. In the case of cyt c, this
FIGURE 2 RR spectra of dissolved (a) HRP and (b) Az in Hy[ch][dhp] FIGURE 3 RR spectrum of dissolved ASOD in Hy[ch][dhp] and
difference spectrum in buffer.
and buffer.
Biopolymers

1096
Fujita and Ohno
hand, the CD spectrum of cyt c dissolved in Hy[ch][dhp]
was almost identical to that in buffer, so that cyt c is expected
to maintain the tertiary structure. Hy[ch][dhp] showed
excellent affinity with some metallo proteins in which pro-
teins dissolved well, and the active site and tertiary structure
maintain the native state. It should be noted here that
[ch][dhp] has a special ionic combination which has been
chosen by natural selection as the head groups of lipid
bilayers in living organisms.33 This similarity may have cer-
tain reason for the stabilisation of proteins.
Enzymatic Activity
We have reported that cyt c retains its activity after dissolving
in Hy[ch][dhp].14 In this case the activity was investigated af-
ter dissolving in buffer, so that the cyt c was diluted. Con-
versely, some proteins, such as cellobiose dehydrogenase,
exhibited activity in the dissolved state in Hy[ch][dhp].34 In
this work we made enzymatic assays of metallo proteins dis-
solved in Hy[ch][dhp], HRP, and ASOD.
HRP dissolved in Hy[ch][dhp] was added to the mixed
solution of pyrogallol and hydrogen peroxide. Figure 5 shows
the absorption spectra before and after mixing of HRP in
Hy[ch][dhp]. After mixing of HRP, the absorbance at 420
nm was increased by generation of purpurogallin, indicating
the progress of the enzymatic reaction. No spectral change
was obtained in the absence of peroxidase addition. This
means that the HRP was active and capable of performing
the enzymatic reaction after dissolving in Hy[ch][dhp].
For the investigation of ASOD activity, ASOD and ascor-
bic acid (AA) were dissolved in Hy[ch][dhp], respectively
and the spectral change was detected upon mixing of the two
FIGURE 4 CD spectrum of dissolved cyt c in (a) [C4mpyr][dhp],
(b) Hy[ch][dhp], and (c) buffer.
region gives information about a-helix content. Features of
the CD spectra of cyt c were almost exactly the same in buffer
and in Hy[ch][dhp]. The band intensities at 222 and 208
nm, used for calculation of the a-helix content, were also
similar. Consequently the a-helix content did not change
after dissolution in Hy[ch][dhp]. In the case of
[C4mpyr][dhp], the cation has an aromatic ring structure, so
the spectrum cannot be observed although cyt c dissolved
well in it. In the near UV (250–350 nm) and Soret (350–450
nm) regions, the CD spectrum reflects changes in the tertiary
structure of cyt c related to the environment of aromatic side
chains and the heme pocket. The spectra show significant
changes in [C4mpyr][dhp] (see Figure 4). Similar spectra
were observed in the unfolded state and when dissolved in
organic solvents and ILs. This spectral change indicates that
the distance and orientation of try-59 and phenylalanine are
different in Hy[C4mpyr][dhp]. In this state, one axial ligand
Met-80 must be significantly weakened. This result agrees
well with our previous finding that CT band absorption in
UV-vis spectroscopy disappeared after dissolution in most
hydrated ILs.32 These spectral results together indicate that
the secondary structure remains unchanged but the tertiary
FIGURE 5 Spectral change of mixed solutions of pyrogallol and
hydrogen peroxide in buffer and Hy[ch][dhp], before and after
structure is loosened in most hydrated ILs. On the other addition of HRP.
Biopolymers

Metallo Proteins in Hydrated Ionic Liquids
1097
mal stability of other proteins in Hy[ch][dhp], FDH was
chosen; it is commercially available and its thermal stability
is widely known.35 In this study, the FDH dissolved in
buffer and in Hy[ch][dhp] were treated at 20–708C for 15
min, and these underwent enzymatic assay after dilution
with buffer. Figure 7 shows the residual activity in aqueous
solution after incubation in buffer, in Hy[ch][dhp] with 30
wt% water, and in Hy[ch][dhp] with 80 wt% water. The
activity at 208C was defined as 100% residual activity. In
both buffer solution and Hy[ch][dhp], the activity
decreased gradually with increasing incubation tempera-
ture. The decreasing curve was totally different, however.
In buffer solution, activity was started to decrease at 308C
and had almost vanished at 508C. In Hy[ch][dhp], activity
decreased from incubation at 408C and no activity was
observed at 708C. In consequence, FDH that had been dis-
solved in Hy[ch][dhp] showed greater thermal stability at
around 208C than that in buffer solution. When FDH was
incubated in Hy[ch][dhp] containing excess water (80
wt%), no improvement of thermal stability was observed
(Figure 7c). This shows that when [ch][dhp] is present as
solute in aqueous solutions, there is no significant differ-
ence from the buffer solution. This might be the same
effect of thermal stability observed in cyt c in previous
work.8 The alleviation of degradation of proteins at higher
temperatures in Hy[ch][dhp] suggests that the chemical
FIGURE 6 Absorbance change at 245 nm of AA before and after
addition of ASOD.
liquids. Figure 6 shows the change in absorbance at 245 nm
of AA in Hy[ch][dhp] from before to after addition of
ASOD. When ASOD was mixed with AA at the arrow point,
the absorbance at 245 nm based on the absorption of AA
decreased with progress of the enzymatic reaction to produce
the dehydroascorbate. This result shows that ASOD retains
its activity after dissolving in Hy[ch][dhp], and the enzy-
matic reaction takes place in it. Although enzymatic assay of
HRP and ASOD were detected in Hy[ch][dhp] that was used
as a reaction solvent, the reaction rate was slow compared
with that in buffer solution. These catalytic reactions pro-
gress under diffusion control. The viscosity is proposed to be
one reason for this slowing. The viscosity of Hy[ch][dhp]
with 30 wt% water was 30 cP at room temperature, whereas
that of aqueous solution was 0.9 cP. The viscosity of
Hy[ch][dhp] with 20 wt% water was 440 cp. In the
Hy[ch][dhp] with 20 wt% water, both reactions for HRP and
ASOD were much slower than those in Hy[ch][dhp] with 30
wt% water. However, the viscosity is considered to be one of
factors for reaction rate, there are a few other possible factors
to affect the enzymes such as pH, salt concentration, and
hydration state. However, the change of apparent rate cannot
fully be explained by the changes at active site of enzymes,
because there are no changes in RR spectra.
Thermal Stability of FDH
We have reported the improvement of the thermal stability
FIGURE 7 Residual activity of FDH dissolved and treated in (a)
[ch][dhp] with 30 wt% water, (b) buffer and (c) [ch][dhp] with 80
of cyt c dissolved in Hy[ch][dhp].8 To investigate the ther- wt% water.
Biopolymers

1098
Fujita and Ohno
and structural steps involved are greatly diminished per- ther investigation is in order. This new medium should
haps due to the low water content. Viscosity is expected as expand the condition for many functional proteins. Many
other possible factor, that the increase in the thermal sta- scientific applications should be developed with the hydrated
bility of some proteins has been induced by increasing so- ILs.
lution viscosity though the lowering thermal motion of
these proteins. In view of the thermal stability of FDH in
MATERIALS AND METHODS
Hy[ch][dhp], long-term stability is also likely to be
increased. Investigation of the long term stability of FDH
in Hy[ch][dhp] is under way.
Materials
Cytochrome c (cyt c) from horse heart and peroxidase from horse-
radish (HRP) were purchased from Sigma-Aldrich and used without
further purification. Ascorbate oxidase from Cucurbita (ASOD) was
purchased from Wako and used without further purification.
Azurin II from Alcaligenes Xylosoxidans (Az) was expressed and
These observations suggest that complex forces are
exerted on proteins dissolved in Hy[ch][dhp]. For example,
the affinity of proteins and component ions and/or water
which is interacting with ions is one possible force. The
amount of water, and the water state provides another. purified as described previously.37 Pseudoazurin was isolated from
Achromobacter cyclastes IAM 1013 (Paz) as described previously.38
Gluconobacter sp. D-Fructose dehydrogenase (FDH) was purchased
These factors might have a complex outcome on the solu-
bility, activity and thermal stability. Recently the effect of
from Toyobo enzymes and used without further purification.
the water state has been studied as part of the concept of
[ch][dhp] was synthesized according to the method14 of choline
bromide solution was treated on an ion exchange resin (Amberlite
molecular crowding.36 Biomolecules occupy a significant
fraction of the cellular volume, leading to a crowded inter-
cellular environment. Thus, proteins and other polymers
exist at high concentration. The water state, water activity
and the behavior of biomolecules are then different from
diluted conditions. Hydrated ILs might generate a pseudo
IRN77) and mixed with phosphoric acid solution. The solvent
evaporated and the product was dried in vacuo. The resulting
1
[ch][dhp] was identified using H NMR, DSC, elemental analysis
and electrospray mass spectrometry.
environment for molecular crowding. Further experiments CD Measurement
CD spectra were recorded with a JASCO J-720 at 258C. A square
will study the water in relation to protein stability and
whether the water condition is controllable by the water
content and the IL involved.
quartz cuvette (path length: 1 mm) was used for CD measurement.
Solutions of 0.1 mM cyt c were prepared with phosphate buffer (pH
7.0) or Hy[ch][dhp] with 30 wt% water. These samples were
recorded in the wavelength ranges 200–250 nm and 250–470 nm.
CONCLUSIONS
Raman Spectroscopy
We have studied the effectiveness of Hy[ch][dhp] as a novel
solvent for metallo proteins. Hy[ch][dhp] dissolves some
metallo proteins, although the mechanism is not yet clear.
Proteins dissolved in Hy[ch][dhp] were shown to keep the
surroundings of the active site and secondary structure the
same as in buffer solution. The enzymatic reactions were
generally influenced by the salt concentration, because con-
centrated salt water causes the deactivation of enzymes. The
electrostatic interactions may also reduce even the activity of
those enzymes remaining in the native state. It might cause
the decrease of the interaction between enzymes and sub-
strate, and the dissociation of subunit structure. However,
various metallo proteins retained their original native form
and activity in Hy[ch][dhp]. Significant thermal stability of
FDH was also observed. The surprising results we found
could be caused by the combination of restraint of hydroly-
sis, the water circumstances due to the component ions, the
minimized mobility of molecules, and other environmental
conditions. Hy[ch][dhp] is likely to be a significant matrix
Raman spectra were obtained using a JASCO NRS-1000 spectrome-
ter with a Kaiser Optical holographic super-notch filter and a liquid
N2-cooled CCD detector. A detailed description has been given else-
where.14 Proteins were dissolved in phosphate buffer or
Hy[ch][dhp]. The protein concentrations were typically 1.0–3.0
mM, and an excitation wavelength was 568 nm. Spectra were col-
lected on samples in the bulk condition at room temperature, using
a backscattering geometry. The peak frequencies were calibrated rel-
ative to an indene standard, and are accurate to 61 cm21
.
Enzymatic Activity
Enzymatic assay of HRP,39 ASOD,40 and FDH35 was performed
according to the published test procedure. Absorption spectra and
the change in absorbance were recorded using a Shimadzu UV-
2450. For the enzymatic assay of HRP, 0.5% (w/w) hydrogen perox-
ide solution (H2O2) and 5% pyrogallol solution were prepared with
deionized water. Peroxidase enzyme solution containing 0.4–0.7
unit/ml was prepared using 100 mM potassium phosphate buffer
(pH 7.0). [ch][dhp] (675 mg) was mixed with phosphate buffer (80
lL), H2O2 solution (40 lL), and pyrogallol solution (80 lL). The
absorption spectrum was monitored before and after addition of
for proteins which realize the long shelf life in vivo, and fur- HRP solution (25 lL).
Biopolymers

Metallo Proteins in Hydrated Ionic Liquids
1099
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after addition of ASOD in Hy[ch][dhp].
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35. TOYOBO product information (D-fructose dehydrogenase
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9. De Diego, T.; Lozano, P.; Gmouh, S.; Vaultier, M.; Iborra, J. L.
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749–758.
10. Madeira Lau, R.; Sorgedrager, M. J.; Carrea, G.; Van Rantwijk,
F.; Secundo, F.; Sheldon, R. A. Green Chem 2004, 6, 483–487.
11. van Rantwijk, F.; Secundo, F.; Sheldon, R. A. Green Chem 2006,
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38. Kohzuma, T.; Yamada, M.; Deligeer; Suzuki, S. J Electroanalyti-
cal Chem 1997, 438, 49–53.
12. Ohno, H.; Suzuki, C.; Fukumoto, K.; Yoshizawa, M.; Fujita, K.
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39. SIGMA product information (peroxidase from horseradish, EC
1.11.1.7).
13. Maruyama, T.; Yamamura, H.; Kotani, T.; Kamiya, N.; Goto, M.
Org Biomol Chem 2004, 2, 1239–1244.
40. WAKO product information (ascorbate oxidase from cucurbita
sp., EC 1.10.3.3).
14. Fujita, K.; MacFarlane, D. R.; Forsyth, M.; Yoshizawa-Fujita, M.;
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Reviewing Editor: Eric J. Toone
Biopolymers