Characterization of different commercial soybean peroxidase preparations and use of the enzyme for N-demethylation of methyl N-methylanthranilate to produce the food flavor methylanthranilate

Article abstract of DOI:10.1021/jf9909656

The potential of different peroxidase preparations for the N- demethylation of methyl N-methylanthranilate to produce the food flavor methylanthranilate (MA) was investigated. All tested peroxidase preparations were able to catalyze the N-dealkylation. The tested soybean preparations vary widely with respect to their heme content. Furthermore, the operational stability of purified soybean peroxidase (SP) is at least 25-fold lower than that of horseradish peroxidase and only 5-fold higher than that of microperoxidase 8. Thus, the presence of a large protein chain around a porphyrin cofactor in a peroxidase is, by itself, insufficient to explain the observed differences in operational stability. Despite its relatively low operational stability, SP proved to be the most efficient biocatalyst for the production of MA with high yield and purity, especially observed at the high temperature and low pH values at which SP appeared to be optimally active.

Full text of DOI:10.1021/jf9909656

J. Agric. Food Chem. 2000, 48, 1949−1954
1949
Ch a r a cter iza tion of Differ en t Com m er cia l Soybea n P er oxid a se
P r ep a r a tion s a n d Use of th e En zym e for N-Dem eth yla tion of Meth yl
N-Meth yla n th r a n ila te To P r od u ce th e F ood F la vor
Meth yla n th r a n ila te
Marjon J . H. Van Haandel,† Florence C. E. Sarabe`r,† Marelle G. Boersma,† Colja Laane,†
Yvette Fleming,† Hugo Weenen,‡ and Ivonne M. C. M. Rietjens*,†
Department of Biomolecular Sciences, Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3,
6703 HA Wageningen, The Netherlands, and Quest International,
Huizerstraatweg 28, 1411 GP Naarden, The Netherlands
The potential of different peroxidase preparations for the N-demethylation of methyl N-methyl-
anthranilate to produce the food flavor methylanthranilate (MA) was investigated. All tested
peroxidase preparations were able to catalyze the N-dealkylation. The tested soybean preparations
vary widely with respect to their heme content. Furthermore, the operational stability of purified
soybean peroxidase (SP) is at least 25-fold lower than that of horseradish peroxidase and only 5-fold
higher than that of microperoxidase 8. Thus, the presence of a large protein chain around a porphyrin
cofactor in a peroxidase is, by itself, insufficient to explain the observed differences in operational
stability. Despite its relatively low operational stability, SP proved to be the most efficient biocatalyst
for the production of MA with high yield and purity, especially observed at the high temperature
and low pH values at which SP appeared to be optimally active.
Keyw or d s: Soybean peroxidase; methyl N-methylanthranilate; food flavor; N-demethylation
INTRODUCTION
Heme-based enzymes such as the peroxidases and the
mammalian cytochromes P450 are among the most
versatile enzymes in biochemistry, able to catalyze the
conversion of a wide range of substrates in many
different types of reactions. Among these are aromatic
and aliphatic hydroxylations, heteroatom dealkylations,
epoxidations, (de)halogenations, and heteroatom oxy-
genations (Dawson, 1988; Griffin, 1991; Ortiz de Mon-
tellano, 1992). From a biotechnological point of view the
use of peroxidases is favored over that of cytochromes
P450 because peroxidases can be more easily obtained
and use H2O2 as a cheap and clean oxidant to create
the so-called high-valent-iron-oxo-porphyrin intermedi-
ates required for catalysis (Chance, 1952; Dolphin et al.,
1971; Groves et al., 1981; Hoffman et al., 1979; Low et
al., 1996; Palcic et al., 1980; Thomas et al., 1970). Many
different peroxidases are known (Diehn, 1993; Dunford
and Stillman, 1976; Patterson and Poulos, 1995; Schull-
er et al., 1996; Sessa and Anderson, 1981). Of all these
peroxidases horseradish peroxidase (HRP) is the one
best studied from mechanistic and catalytic points of
view (Dunford, 1991; J ones and Suggett, 1968a,b; Ro-
driguez-Lopez et al., 1996a,b; Van Haandel et al., 1998).
However, biotechnological application of HRP is seri-
ously hampered by the fact that the enzyme is relatively
expensive and has a limited thermostability (McEldoon
and Dordick, 1996). In contrast to HRP, soybean per-
F igu r e 1. N-Dealkylation of MNMA to MA, the model
reaction of the present study.
oxidase (SP) has been reported to show better temper-
ature behavior (McEldoon et al., 1995; McEldoon and
Dordick, 1996; Toiguchi et al., 1989). Furthermore, SP
is more economical for use in biotechnological applica-
tions because it can be obtained relatively easily from
soybean hulls, a major byproduct of the food industry.
Finally, microperoxidase 8 (MP8), being a heme-based
minienzyme with a potentially wide substrate specificity
and able to function at relatively high temperatures as
well (Aron et al., 1986; Cunningham et al., 1991; Osman
et al., 1996), may be another peroxidase of industrial
interest.
On the basis of these considerations, the objective of
the present study was to investigate the biocatalytic
potential of SP, HRP, and MP8 for the production of
fine chemicals. Literature data report on a wide range
of soybean preparations and isoenzymes (Diehn et al.,
1993; McEldoon et al., 1995; Gijzen et al., 1993; Sessa
and Anderson, 1981) and, therefore, we compared
different commercially available samples with respect
to their heme content and number of heme-containing
proteins, to determine the best source for SP purifica-
tion. As a model reaction the N-demethylation of methyl
N-methylanthranilate (MNMA), leading to the produc-
tion of methylanthranilate (MA) (Figure 1), was used.
This model reaction was chosen for the following
* Author to whom correspondence should be addressed
(telephone 31-317-482868; fax 31-317-484801; e-mail
ivonne.rietjens@p450.bc.wau.nl).
† Wageningen University.
‡ Quest International.
10.1021/jf9909656 CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/19/2000

1950 J. Agric. Food Chem., Vol. 48, No. 5, 2000
Van Haandel et al.
preincubated for 2 min at 70 °C for SP and at 37 °C for HRP
and MP8 (unless indicated otherwise). The reaction was
started by the addition of H2O2 (2.5 mM final concentration).
The incubation was carried out for 1 min (unless indicated
otherwise) and stopped by freezing the sample into liquid
nitrogen. Samples were stored in liquid nitrogen until analyzed
and defrozen by centrifugation in an Eppendorf centrifuge (5
min; 13000g; 4 °C) prior to analysis by HPLC.
reason: MNMA from citrus leaves is readily available
and a relatively cheap source, whereas MA is more
expensive than MNMA. Therefore, the investigated
reaction provides an industrially relevant route for the
natural production of an important topnote flavor in
Concord grape.
MATERIALS AND METHODS
HP LC. HPLC chromatography of SP samples solubilized
as described above and of solutions of purified SP, HRP, and
MP8 was performed on a Bakerbond wide-pore butyl RP-7116-
00 column (4.6 × 250 mm) (J . T. Baker Research Products,
Deventer, The Netherlands). Elution was performed at a flow
of 1 mL/min using a linear gradient from 0.1% trifluoroacetic
acid in 100% nano pure to 0.1% trifluoroacetic acid in 50%
water plus 50% acetonitrile in 50 min. Detection was per-
formed at 210-450 nm using a photodiode array detector
(Waters 996). HPLC analysis of the incubations for N-dem-
ethylation of MNMA was performed on a reversed-phase
Lichrosphere RP8 column (4.6 × 150 mm) (Alltech, Breda, The
Netherlands) using a linear gradient from 0 to 80% methanol
in water in 26 min at a flow of 1.0 mL/min. Detection was at
332 nm using a diode array detector (Waters 996). Products
were identified and quantified using commercially available
standards. The concentration of MA was quantified using a
calibration curve consisting of the HPLC peak area plotted
against the concentration of the compound in the injected
sample.
Deter m in a tion of Hem e a n d P r otein Con ten t. For the
sample containing purified SP, the heme concentration was
determined by using the pyridine-chromogen method (Aron
et al., 1986). The heme content of solubilized SP samples was
generally low compared to the other proteins present. This
hampered the heme determination by the pyridine-chromogen
method. Heme determination for these samples was done using
HPLC and a calibration curve made with SP from Enzymol.
Protein was measured according to the method of Bradford
(Sigma) using bovine serum albumin (Boehringer Mannheim,
Germany) as the standard.
Ch em ica ls. MNMA (ex. citrus leaves), MA (ex. grapes),
N-methylanthranilate, and anthranilate were obtained from
Quest International (Naarden, The Netherlands). H2O2 (30%
in water) was from Merck (Darmstadt, Germany). HRP and
cytochrome c from horse heart were obtained from Boehringer
(Mannheim, Germany). MP8 was prepared by proteolytic
digestion of horse heart cytochrome c essentially as described
before (Aron et al., 1986; Kraehenbuhl et al., 1974). Dried
soybeans were obtained from Mervo Products (Hengelo, The
Netherlands). Soybean flakes consisting of brown flakes made
of soybean hull were obtained from Quest International.
Soybean pellets consisting of brown dried pellets from soybean
hull and soybean extract were obtained from ADUMIN (Tel
Aviv, Israel). Soybean flour was a pale yellow flour-like powder
commercially obtained as provaflor soybean flour from Cargill
(Amsterdam, The Netherlands). SP was either purified or
obtained from Enzymol International (Columbus, OH). HPLC
analysis proved both purified SP preparations to be identical.
Solu biliza tion of th e Differ en t Soybea n Sa m p les. The
different soybean samples were preincubated at x% (w/v) (x )
1 for extract and flour, 17 for pellets, and 10 for flakes) in 0.1
M potassium phosphate (pH 7.6) for 1 h at 0 °C and then
homogenized by mixing during 2 min using a vortex. The
mixture obtained was centrifuged using an Eppendorf centri-
fuge for 10 min at 4 °C (13000g). The supernatant thus
obtained was used for further characterization of the heme
content, measurement of peroxidase activity, and/or purifica-
tion of SP. For the dry beans a different procedure was
followed. This sample was preincubated at 20% (w/v) in 0.1 M
potassium phosphate (pH 7.6) for 2.5 h and then homogenized
during 15 min at maximum speed using a blender (Braun,
Frankfurt, Germany). The mixture obtained was centrifuged
for 10 min at 4 °C (16000g). The supernatant obtained was
filtered using glasswool and then centrifuged for 20 min at 4
°C (27000g). The supernatant thus obtained was used for
further characterization of the heme content and measurement
of peroxidase activity. On the basis of the solubilization
protocol used, the studied soybean fractions contain especially
soluble forms of peroxidases.
P er oxid a se Activity w ith Gu a ia col. Peroxidase activity
was detected on the basis of oxidation of guaiacol as described
by Gillikin and Graham (1991). The assay was performed in
50 mM potassium acetate (pH 5.5) at 25 °C and contained
(final concentrations) 8 mM guaiacol, 0.5 mM H2O2, and 2 nM
peroxidase. The reaction was initiated by the addition of H2O2.
RESULTS
H em e P r ot ein Con t en t of Differ en t Soyb ea n
Sa m p les. Figure 2a presents the HPLC chromatogram
of soybean extract with detection at 280 nm for protein
content andsmore importantlysat 395 nm for heme-
containing proteins. The HPLC chromatogram of soy-
bean extract contains one major heme-containing peak
with retention time of 49.2 min. Table 1 summarizes
the results from this and similar HPLC analyses of
different types of commercially available soybean prepa-
rations. The different samples vary considerably with
respect to their heme protein compositions, although
especially the peak at 49.2 min and, to a minor extent,
the peak at 55.1 min are present in most of the samples.
The results obtained indicate either the presence of
different heme-containing proteins or the presence of
different degradation or glycosylated products of one
major heme protein in the different commercial samples.
In addition to the heme protein composition, the
amount of heme protein present in the various soybean
preparations could be derived from these HPLC data.
Because direct measurement of heme content by spec-
troscopic methods was hampered by the high amount
of other proteins present in the samples, the heme
content was derived from the HPLC data using a
P u r ifica tion of SP . Soybean extract was used as the
starting material and prepared as described above only using
20 mM Tris-HCl (pH 7.4). Purification was performed by a
single affinity chromatography step on concavalin A-Sepharose
(Pharmacia Biotech, Uppsala, Sweden), performed essentially
as described previously (Gillikin and Graham, 1991; Sessa and
Anderson, 1981; Toiguchi et al., 1989). The column was
equilibrated with 20 mM Tris-HCl (pH 7.4) containing 0.5 M
NaCl, 1 mM CaCl2, and 1 mM MnCl2 and eluted with a
gradient of 0-100% 0.5 M methyl R-D-mannopyranoside
(Sigma, St. Louis, MO) in the same buffer. Fractions were
analyzed for absorption at A280nm and A395nm and for peroxidase
activity using guaiacol as the substrate. Peroxidase-containing
fractions were pooled, desalted, and concentrated using an
Amicon 8010 filter apparatus with a YM-10 filter and dialyzed
overnight against 20 mM Tris-HCl (pH 7.4). The purity of the
final sample was analyzed by HPLC and SDS-PAGE (Scha¨g-
ger and von J agow, 1987), and its specific activity was
determined as described below.
In cu ba tion s w ith MNMA. Incubations with MNMA were
performed in (final concentrations) 0.1 M potassium acetate
(pH 4) for SP or in 0.1 M potassium phosphate (pH 7.6) for
HRP and MP8, containing 5 µM heme protein and 1 mM
MNMA added as 1% (v/v) of a 0.1 M stock solution in dimethyl
sulfoxide. To some incubations was added ascorbic acid at a
final concentration of 3 mM. The incubation mixture was

SP-Catalyzed N-Demethylation of MNMA
J. Agric. Food Chem., Vol. 48, No. 5, 2000 1951
Ta ble 1. Com p osition of Hem e-Con ta in in g P r otein s in
Va r iou s Com m er cia l Soybea n P r ep a r a tion s As
Deter m in ed by HP LC An a lysis w ith Detection a t 395 n m a
heme content, retention time % of total
sample
(obtained from)
nmol of heme/
g of sample
of peaks at
395 nm, min at 395 nm
peak area
soybean extract
(ADUMIN)
1291
43.8
44.9
49.2
55.1
2.8
8.0
80.7
4.5
∑ others
4.0
soybean flakes
(QUEST)
2
6
28.2
49.2
55.1
18.8
28.8
52.4
∑ others
0.0
soybean pellets
(ADUMIN)
3.0
49.2
55.1
72.0
25.6
2.4
∑ others
0.0
soybean flour
(Cargill)
75
8
33.3
55.1
53.6
46.4
∑ others
0.0
dried soybeans
(Mervo Products)
33.3
49.2
24.0
76.0
∑ others
0.0
a
Only those peaks with an area >2% of the total intensity are
specified.
SDS-PAGE, the purified SP showed one prominent
protein band of 37 kDa.
In flu en ce of p H a n d Tem p er a tu r e. The pH opti-
mum was determined for the conversion of MNMA to
MA by purified SP and for the peroxidase activity of SP
assayed with guaiacol. In both cases SP has a pH
optimum at 4. At pH 4 the temperature dependence was
also investigated. Reaction rates increased with increas-
ing temperature up to an optimum at 70 °C. Blank
incubations without H2O2 or without SP showed no
product formation at all pH values and temperatures
tested. On the basis of these results all subsequent
incubations with SP were performed at 70 °C and pH
4.
N-Dem eth yla tion of MNMA As Ca ta lyzed by
Differ en t P er oxid a ses. Figure 3 presents the HPLC
chromatograms of an incubation of MNMA with purified
SP and, for comparison, with similar concentrations of
HRP and MP8. All assays were performed at the pH
optimum of the respective enzymes (Dunford, 1991;
Osman et al., 1996). The temperature used was 37 °C
for HRP because of its thermoinstability (McEldoon and
Dordick, 1996) and 70 °C for MP8 and SP. Formation
of MA is observed in all cases. Formation of N-methyl-
anthranilate (Figure 3, arrow 2) or of anthranilate
(Figure 3, arrow 1) is not observed.
The results obtained with the different peroxidase
samples vary not only with respect to the amount of MA
formed but also with respect to the amount and nature
of the side products observed. From control experiments
we can state that this difference in secondary product
composition was not linked to the different pH and
temperature conditions used for each enzyme. Compari-
son of the chromatograms in Figure 3 reveals that SP
proved to be the most efficient biocatalyst for the
N-demethylation of MNMA to MA. Measurement of the
MNMA demethylation by SP at 37 °C results in only
F igu r e 2. HPLC chromatogram of (a) soybean extract and
(b) purified SP from soybean extract. Detection was at 280 and
395 nm.
calibration curve made on the basis of commercially
available SP. Table 1 presents the heme contents of the
different samples. Marked differences are observed. The
soybean extract especially contains a relatively large
amount of heme proteins, suggesting that the extraction
procedure was directed at selective enrichment of
soluble and/or heme proteins. Also of interest is that
both soybean flakes as well as soybean pellets contain
about the same amount of heme as the solubilized
fraction from dried soybeans, whereas the soybean flour
was relatively rich in heme content.
Altogether the data indicate large differences among
the various soybean preparations. On the basis of heme
content per gram of soybean preparation, the soybean
extract shows the highest specific heme content, and,
therefore, the best starting material for purification of
SP. In addition, one heme-containing peak dominates
in the soybean extract, namely, the component with
HPLC retention time of 49.2 min, also dominant in the
least processed sample, the dried soybeans, and present
in most of the commercially available SP preparations.
P u r ifica tion of SP fr om Soybea n Extr a ct. Figure
2b presents the HPLC chromatogram (detection at 280
and 395 nm) of SP purified from soybean extract using
affinity chromatography on concavaline A-Sepharose.
Comparison of this chromatogram to the one presented
in Figure 2a indicates the significant increase in purity
of the heme-containing protein. The specific activity of
the final SP preparation was 8032 units/mg of protein
as measured with guaiacol as the substrate. The puri-
fication factor of the final SP preparation was 10. On

1952 J. Agric. Food Chem., Vol. 48, No. 5, 2000
Van Haandel et al.
F igu r e 4. Operational stability of purified SP (‚ ‚ ‚) (left
Y-axis), HRP (- - -) (right Y-axis), and MP8 (s) (right Y-axis)
in the conversion of MNMA (SP at 70 °C and pH 4; HRP and
MP8 at 37 °C and pH 7.6). The enzyme concentration was 5
µM in all cases. As the MNMA conversion by 5 µM SP was
hampered by substrate limitation, the operational stability of
SP was determined also with 0.5 µM SP (b) (right Y-axis).
of MNMA to MA by SP the amount of side products
compared to the amount of MA formed seems low
(Figure 4a). Upon complete conversion of MNMA the
yield of SP-catalyzed MA production amounted to 82%
within 10 min under the conditions specified.
Op er a tion a l Sta bility. Figure 4 presents the forma-
tion of MA from MNMA as catalyzed by the different
peroxidases in time. Due to its relatively high activity
SP had to be analyzed for operational stability at a
concentration 10 times lower than MP8 and HRP.
However, measurement of SP operational stability at
different concentrations of the enzyme (1, 0.5, and 0.2
µM) showed no significant influence of SP concentration
on its operational stability.
To describe operational stability in a quantitative
way, eq 1, describing the kinetics of inactivation as
d[P]/dt ) v ) Vmax e(-k t)
(1)
i
reported by Spee et al. (1995), can be applied. Using this
equation to analyze our data provides ki values of 0.5
( 0.1, 0.02 ( 0.004, and 2.6 ( 0.4 min-1 for SP, HRP,
and MP8, respectively. This inactivation constant, ki,
gives information about the stability of the different
peroxidases: a high ki means low operational stability.
The operational stability of SP is 5-fold higher than that
observed for MP8, but at least 25-fold lower than that
of HRP. Furthermore, the operational stability of SP in
soybean extract (before purification) was comparable to
that for purified SP (data not shown).
Ad d ition of Ascor ba te. Previous studies reported
the mechanism of peroxidase-catalyzed heteroatom
dealkylation to be either cytochrome P450-like or per-
oxidase-like (Kedderis and Hollenberg, 1983; Walker-
Griffin et al., 1978). To investigate the mechanism of
the SP-catalyzed N-demethylation of MNMA, experi-
ments were performed in the presence of ascorbate,
known to effectively block peroxidase-type of reaction
chemistry but not MP8-catalyzed cytochrome P450 type
conversion of aniline to p-aminophenol (Osman et al.,
1996). Peroxidase-type of reaction chemistry is defined
as H2O2-dependent initial one-electron oxidation of the
substrate (Dunford et al., 1976).
F igu r e 3. HPLC chromatograms of the incubation of MNMA
with H2O2 and (a) SP purified from soybean extract, (b) HRP,
and (c) MP8. The concentration of the enzymes was 5 µM. The
incubations were performed during 1 min at 70 °C and pH 4
for SP, at 37 °C and pH 7.6 for HRP, and at 70 °C and pH 7.6
for MP8. The substrate MNMA is eluted after 29 min, whereas
the product MA is eluted after 24 min. Arrows 1 and 2 indicate
where anthranilate and N-methylanthranilate, respectively,
should elute.
17% of the amount of MA formed at 70 °C (data not
shown), whereas measurement of the MNMA demethy-
lation by MP8 at 37 °C results in 63% of the amount of
MA formed at 70 °C (data not shown). This illustrates
that the relatively high potential of SP as compared to
MP8 and especially HRP is related to the ability of the
enzyme to function at relatively high temperature.
Another important observation is that upon conversion
Upon addition of 3 mM ascorbate to the incubations
of the present study, the production of MA by SP as well

SP-Catalyzed N-Demethylation of MNMA
J. Agric. Food Chem., Vol. 48, No. 5, 2000 1953
as the peroxidase activity assayed with guaiacol ap-
peared to be fully inhibited.
the inactivation mechanism could be intramolecular.
Because HRP (ki ) 0.02 ( 0.004 min-1) showed an
operational stability at least 25 times higher than that
observed for SP and 130 times higher than that ob-
served for MP8, the present results with SP indicate
that the presence of a large protein chain around the
porphyrin cofactor in a peroxidase is, by itself, insuf-
ficient to explain the observed differences in operational
stability.
DISCUSSION
The present work describes the possibilities and
limitations for using commercially available SP prepa-
rations and other peroxidases for the production of the
food flavor MA from MNMA.
SP was isolated from soybean extract, purified, and
characterized. The peroxidase-catalyzed conversion of
guaiacol and of MNMA to MA at low pH is in line with
early reports of peroxidase-catalyzed conversions ob-
served at even lower pH values, which supported the
notion that SP is resistant to acidic conditions and holds
onto its heme more tightly than does HRP (McEldoon
et al., 1995). Comparison of the pH optimum observed
in the present study to pH optima reported in the
literature (McEldoon et al., 1995; Schmitz et al., 1997;
Sessa and Anderson, 1981; Toiguchi et al., 1989) sug-
gests that the actual pH optimum observed for SP may
vary between 2 and 6 depending to some extent on the
substrate and assay conditions used. Alternatively, this
might be due to the fact that SP used in the different
studies was obtained from different soybean sources,
resulting in purification of different isoenzymes. The
temperature optimum of 70 °C for the conversion of
MNMA to MA by SP and for the conversion of guaiacol
by SP is in line with results reported by others (McEl-
doon et al., 1995; McEldoon and Dordick, 1996; Toiguchi
et al., 1989), who found temperature optima of 40-60
°C for other assays and SP inactivation to occur above
80 °C. The observation in the present study that the
pH and temperature effects on demethylation of MNMA
are similar to the pH and temperature effects on
peroxidase activity assayed with guaiacol corroborates
the conclusion that the demethylation proceeds by a
peroxidase-type of reaction mechanism.
Because MNMA from citrus leaves is a relatively
cheap source and MA is more expensive than MNMA,
the investigated reaction provides a route for the
natural production of the Concord grape flavor. The
observation that ascorbate fully inhibits the SP-cata-
lyzed N-demethylation of MNMA also corroborates the
hypothesis that the N-dealkylation proceeds by a per-
oxidase-type of reaction mechanism and not by a cyto-
chrome P450-type of reaction mechanism (Hollenberg
et al., 1985; Kedderis and Hollenberg, 1983; Miwa et
al., 1983; Nakamura et al., 1992; Pandey et al., 1989).
The observation of peroxidase-catalyzed N-demethyla-
tion is in line with literature data, which report per-
oxidase-catalyzed N-dealkylations of other N-alkylated
substrates (Kedderis and Hollenberg, 1983; Walker-
Griffin et al., 1978).
Despite its relatively low operational stability, SP
proved to be the most efficient biocatalyst for the
production of MA with high yield and purity. This
potential of SP to catalyze the N-demethylation of
MNMA to MA more efficiently than HRP and MP8 was
especially observed at high temperature and low pH
values, at which SP appeared to be optimally active.
ABBREVIATIONS USED
SP, soybean peroxidase; MP8, microperoxidase 8;
HRP, horseradish peroxidase; MNMA, methyl N-me-
thylanthranilate; MA, methylanthranilate.
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Received for review August 27, 1999. Revised manuscript
received February 17, 2000. Accepted February 24, 2000. We
gratefully acknowledge financial support from IOP (Grant
IKA94045).
Osman, A. M.; Koerts, J .; Boersma, M. G.; Boeren, S.; Veeger,
C.; Rietjens, I. M. C. M. Microperoxidase/H2O2-catalyzed
J F9909656