From the Department of Applied Microbiology and Gene
Technology, TNO Nutrition and Food Research Institute, Utrechtseweg 48, 3704 HE Zeist, The Netherlands and the § Laboratory of
Organic Chemistry and Catalysis, Delft University of Technology,
Julianalaan 136, 2628 BL Delft, The Netherlands
Received for publication, November 22, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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The Caldariomyces fumago
chloroperoxidase was successfully expressed in Aspergillus
niger. The recombinant enzyme was produced in the culture medium
as an active protein and could be purified by a three-step purification
procedure. The catalytic behavior of recombinant chloroperoxidase
(rCPO) was studied and compared with that of native CPO. The specific
chlorination activity (47 units/nmol) of rCPO and its pH optimum (pH
2.75) were very similar to those of native CPO. rCPO catalyzes the
oxidation of various substrates in comparable yields and selectivities
to native CPO. Indole was oxidized to 2-oxindole with 99% selectivity
and thioanisole to the corresponding R-sulfoxide
(enantiomeric excess >98%). Incorporation of 18O
from labeled H218O2 into the
oxidized products was 100% in both cases.
Chloroperoxidase (CPO1;
EC 1.11.1.10) is a heavily glycosylated monomeric hemoprotein, with
a sugar content of 18% of its molecular mass of 42 kDa (1). The
chloroperoxidase is secreted by the filamentous fungus
Caldariomyces fumago and was first purified and described in
1966 by Morris and Hager (2). In vivo, CPO catalyzes
oxidative chlorination. In vitro, in the absence of Cl Site-directed mutagenesis has proved to be a powerful tool in exploring
structure-function relationships in classical peroxidases (23); in
particular, horseradish peroxidase has been studied in great detail by
Morishima and co-workers (33-36) and by Smith and co-workers (37-40).
Furthermore, Ortiz de Montellano and co-workers (41-44) have used
site-specific mutagenesis to engineer horseradish peroxidase with
oxygen transfer catalytic properties, suitable for enantioselective
sulfoxidation and epoxidation reactions. The use of such an approach
for the C. fumago CPO could help in revealing the structural
basis of the unique properties of this enzyme and to explore further possibilities.
For site-directed mutagenesis studies, an efficient expression system
for the cpo gene is required. As CPO is a protein with several post-translational modifications, i.e. N-
and O-glycosylation, disulfide bridge formation, cleavage of
N-terminal and C-terminal sequences, and prosthetic group incorporation
(1), prokaryotic hosts appear not suitable for synthesizing the active
protein. Indeed, Zong et al. (45), reporting the expression
of cpo in Escherichia coli, showed that the
non-glycosylated enzyme was secreted into the periplasm in its apoform
and only after a tedious high pressure-assisted reconstitution process
could limited amounts of the active holoenzyme be recovered. Therefore,
other, eukaryotic, expression systems have been considered. Expression
of the cpo using the baculovirus system resulted in the
production of extracellular inactive CPO, which could not be
reconstituted to active protein (46). Similarly, attempts to produce
CPO in Saccharomyces cerevisiae and Pichia
pastoris have been unsuccessful (46, 47). Recently, the genetic
transformation of C. fumago and the expression of mutant
forms of CPO in the parental host have been reported (48). However,
this system has the inconvenience of the presence of native CPO
background, which hampers the screening for recombinant CPO producing
strains, and has failed in providing specific CPO mutant
proteins.2
We have explored the possibility of producing CPO in another
filamentous fungal expression host, namely Aspergillus
niger. Filamentous fungi are capable of secreting large amounts of
proteins in the extracellular medium. Since versatile DNA transfer and gene expression systems are available for these organisms, the necessary tools are available for the production of recombinant proteins. Furthermore, A. niger has no detectable
extracellular peroxidases, and therefore, in contrast to the C. fumago system, no interference of endogenous oxidising activities
when screening for CPO producing transformants. To date several reports
on the expression of fungal metalloproteins in filamentous fungi have been published (49-52). However, although production of active recombinant enzymes was found in most cases, yield levels were still
far from those obtained for less complex fungal proteins, making the
secretion of metalloproteins an intriguing subject of study. Here, we
describe the expression of the C. fumago cpo gene in
A. niger. Fully active recombinant CPO was produced and purified. Its catalytic properties were compared with those of the
native CPO from C. fumago.
Strains--
Escherichia coli DH5 Reagents--
Native chloroperoxidase from Caldariomyces
fumago was obtained from Chirazyme Labs (Urbana, IL) and used
without further purification. The enzyme solution contained 11.4 mg/ml
CPO with Rz 1.23 (Rz = purity standard = A400/A280 = 1.44 for pure
enzyme) and an activity of 22.8 kilounits/ml (standard
monochlorodimedone (MCD) assay as described by Morris and Hager (Ref.
2)). o-Anisidine was purchased from Fluka and hemin from
Sigma. Gel filtration low molecular weight calibration kit was
purchased from Amersham Pharmacia Biotech. Indole, 5-bromoindole,
5-chloroindole, 5-methoxyindole, thioanisole, ethyl phenyl sulfide, and
methyl p-methoxyphenyl sulfide, were purchased from Aldrich.
The corresponding sulfoxides were prepared by chemical oxidation
according to Drabowicz et al. (53). 18O-Labeled
hydrogen peroxide (H218O2; 90%
18O) was obtained from Campro Scientific.
Analysis and Equipment--
UV measurements were performed on a
Cary 3 spectrophotometer from Varian. A Megafuge 2.0R from Heraeus
Instruments was used for centrifugation. A Metrohm Dosimat 665 was used
for continuous addition of H2O2.
Enzyme purification was performed with a Waters Delta Prep 4000 HPLC
system equipped with an Amersham Pharmacia Biotech fast flow column
(d = 5 cm; 750-ml DEAE-Sepharose) and a Waters fraction collector.
Gel filtration chromatography was done using a Superose 12 HPLC column
(Amersham Pharmacia Biotech, 10 × 300 mm) with a Waters 590 programmable HPLC pump with detection on a Waters 486 tunable absorbance detector at 280 or 400 nm with Waters
Millennium32 software. Fractions were collected using a
Waters fraction collector.
Samples for analyzing the enantioselective oxidation of sulfides were
quenched with sodium sulfite, diluted with a hexane/isopropyl alcohol
mixture of 75:25 (v:v), and dried over Na2SO4.
After centrifugation, the samples were analyzed on chiral HPLC using a
Chiralcel OD column (Daicel Chemical Industries, Ltd., 250 × 4.6 mm), eluent flow 0.6 ml min
Samples for analyzing indole oxidation were quenched with a saturated
sodium sulfite solution and diluted with methanol. After centrifugation, the samples were analyzed with reversed phase HPLC
using a custom-packed Symmetry C18 cartridge (Waters
Radial-Pak, 8 × 100 mm, 7 µm) contained in a Waters RCM 8 × 10 compression unit, with simultaneous detection on a Waters 410 differential refractometer and a Waters 486 tunable absorbance detector
at 254 nm with Waters Millennium32 software.
tert-Butyl alcohol was used as internal standard. A methanol/water mixture of 70:30 (v/v) at 1.0 ml
min
GS-MS analysis was performed on a CP SIL5CB MS column (25 m × 0.25 mm) and a VG 70-SE mass spectrometer.
Chloroperoxidase activity was determined by the standard chlorination
method as described by Morris and Hager (2), defining 1 unit of
chloroperoxidase activity as the amount enzyme that catalyzes the
formation of 1 µmol of dichlorodimedone in 1 min. Total protein
content was determined by the method of Bradford using bovine serum
albumin as a standard. To obtain the ferrous-CO complex of CPO (48),
the Fe(III) was first reduced to Fe(II) with dithionite and then
incubated for 2 min with CO. The reactivity of the recombinant enzyme
to the Ellmann reagent for free SH-groups was carried out as described
(54).
Construction of cpo Expression Vector--
pCf6, a
plasmid containing cpo genomic clone, was a gift from Dr.
Hager (University of Illinois, Urbana-Champaign, IL). Primers CLP15E/A
(5'-GGAATTCACATGTTCTCCAAGGTCC-3') and CLP1CTERM3
(5'-CGCGCGGATCCAAGCTTAAAGGTTGCGGG-3') were used to amplify the
DNA sequence encoding the full-length CPO precursor (GenBankTM
accession no. AJ300448) from pCf6 and introduce suitable cloning sites.
The resulting PCR product was EcoRI/BamHI-digested and cloned into pUC19 to
render pCPO3. The amplified cpo fragment was checked by
sequence analysis, excised from pCPO3 as an
AflIII/HindIII fragment and cloned into the
pAN52-10Not Aspergillus expression vector (55) at the
NcoI/HindIII cloning sites, which resulted in
pCPO3.I. In vector pCPO3.I, the CPO coding sequence is placed under
control of the A. niger glucoamylase promoter and A. nidulans trpC terminator. Finally, the A. nidulans AmdS selection marker (56) was introduced in
pCPO3.I at a unique NotI site to obtain the cpo
expression vector pCPO3.I-AmdS (Fig. 1).
Transformation Procedures--
Fungal co-transformation was
carried out as described (57), using pCPO3.I-AmdS and pAB4-1 (58)
plasmids. Transformants were selected on fructose minimal medium plates
without uridine and containing acetamide as sole nitrogen source.
Transformants were selected for multicopy integration of the expression
cassettes on acrylamide plates (59) and for extracellular peroxidase
activity on o-anisidine plates as described (52) using
0.05% H2O2 in 0.1 M sodium
phosphate buffer, pH 2.7, as developing buffer.
Molecular and Protein Methods--
Molecular methods were
carried out essentially as described (60). Total fungal RNA was
isolated using the RNAzolTM kit from Cinna/Biotecx. For Northern
analysis experiments, a 1-kilobase StyI fragment from pCF6
containing most of the cpo coding region was used as a
probe. SDS-PAGE was performed with a Bio-Rad MiniprotII system using
the Tris-glycine method and 10% polyacrylamide gels. N-terminal
determination of recombinant CPO (rCPO) was performed by Edman
degradation after SDS-PAGE of the purified protein and blotting onto a
polyvinylidene difluoride membrane. For deglycosylation experiments,
proteins were treated with endoglycosidase H (EndoH; New England
Biolabs) following manufacturer's instructions.
Polyclonal Antisera--
For preparation of polyclonal
antibodies, CPO from C. fumago IMI 089362 was purified
according to van Deurzen et al. (61). A 3-mg aliquot of the
purified CPO was treated with acetone plus 0.3% HCl to remove the heme
group (62), and both holo- and apochloroperoxidase were used for rabbit
immunization. Immunizations were performed in duplicate using
100 µg of protein in Freund's complete
adjuvant-H2O2 (1:1). Boosters were administered
after 2 and 16 weeks after immunization using 100 µg of protein in
Freund's incomplete adjuvant. Rabbits were bled 1 week after the last
booster, and optimal sera dilution was determined by enzyme-linked
immunosorbent assay.
Production and Purification of rCPO--
Fungal culturing was
carried out in 2-liter Erlenmeyer flasks containing 500 ml of
Aspergillus minimal growth medium (63) with 5% maltodextrin
and supplemented with 0.5% casein amino acids and 500 mg/liter hemin.
Cultures were inoculated with 5 × 108 conidia and
grown for 48 h at 30 °C or 22 °C in a rotary shaker revolving at 300 rpm. Medium samples were obtained by filtering the
fungal cultures through a Miracloth.
To the filtered medium (1300 ml) cold acetone (1000 ml; 45% v/v;
Oxidation of Sulfides--
For sulfide oxidation reactions, 50 µmol of sulfide was dissolved at room temperature in 1.0 ml solvent
(0.2 M phosphate buffer, pH 5.2). 24 units of
chloroperoxidase were added to the reaction mixture and stirred for 5 min. The reaction was started by the continuous addition of
H2O2 (0.15 M) at a rate of 1eq/2 h
to a total of 1.1 eq. H2O2. The reaction was
quenched after 2.5 h by the addition of an excess of
Na2SO3. The reaction mixture was homogenized by
the addition of isopropyl alcohol (400 µl) and analyzed by chiral
HPLC.
Oxidation of thioanisole with
H218O2 was performed at 0.5-ml
scale. Oxidation was started with the stepwise addition of
H218O2 (1.0%; 5 µl/min to a
total of 95 µl). 5 min after the last addition, the reaction mixture
was extracted with dichloromethane and the reaction products were
analyzed with GC-MS.
Oxidation of Substituted Indoles--
Oxidation of substituted
indoles were performed at room temperature in 1.0-ml aliquots
containing 10 µmol of indole derivative dissolved in
tert-butyl alcohol, 0.2 M phosphate buffer, pH
5.2 (50:50, v/v). 8 units of chloroperoxidase were added to the
reaction mixture, stirred for 5 min, and the reaction was started by
the continuous addition of H2O2 (0.15 M) at a rate of 1 eq/h, to a total of 1.1 eq of
H2O2. The reactions were monitored by removing aliquots and analyzing by HPLC.
The oxidation of indole with H218O2
was performed at 0.5-ml scale. Oxidation was started with the stepwise
addition of H218O2 (0.4%; 5 µl/min to a total of 55 µl). 5 minutes after the last addition, the
reaction mixture was extracted with dichloromethane and the reaction
products were analyzed with GC-MS.
Isolation of A. niger Transformants Producing rCPO--
In a
co-transformation experiment, A. niger strain MGG029 was
transformed with a mixture of plasmids pCPO3.I-AmdS and pAB4-1. Several uridine prototrophic, acetamide utilizing transformants were
obtained and were transferred to both acrylamide and
o-anisidine containing plates. Efficient growth and
esporulation on acrylamide plates reflects multicopy integration of the
transforming vector (59), and colored halo formation on
o-anisidine plates indicates extracellular peroxidase
activity (52). Four transformants growing vigorously on acrylamide and
developing an intense halo with the o-anisidine test were
selected. These four strains were cultured on maltose minimal medium
for 48 h and analyzed for cpo mRNA synthesis by
Northern blotting and extracellular CPO production by Western analysis
(data not shown). From this analysis, the best producing transformant,
strain [MGG029]pCPO3.I#5, was selected for production and
purification of rCPO.
Production and Purification of rCPO--
Extracellular production
of rCPO could be readily detected in shake-flask cultures of strain
[MGG029]pCPO3.I#5 without the need of extra heme supplementation.
However, rCPO production levels could be increased by 10-fold upon
hemin addition to the culture medium at a concentration of 500 mg/liter. An additional 5-fold increase was achieved by switching the
culturing temperature from 30 °C to 22 °C. Under these conditions
up to 10 mg/liter rCPO could be produced from strain
[MGG029]pCPO3.I#5.
rCPO was purified to electrophoretic homogeneity by acetone
precipitation and column chromatography as reported for CPO from C. fumago (47, 61). The figures corresponding to the
purification of rCPO are given in Table
I.
Molecular Characterization of rCPO--
Fig.
2 shows the UV spectra of purified rCPO
and native CPO (nCPO, commercial preparation with
Rz 1.23). As it can be seen, the ratio between
A400 (indicating heme-containing protein) and A280 (indicating total protein), or
Rz value, is lower for rCPO (0.54) in comparison
to nCPO. Homogeneous CPO from C. fumago has a
Rz of 1.44. This suggests that rCPO is only
partly (~40%) occupied with heme. Similarly to nCPO, the absorption
spectrum of the ferrous-CO complex of rCPO showed a Soret peak at 450 nm (data not shown), indicating the correct formation of the heme
thiolate ligand with Cys29 (64).
To further characterize rCPO, we compared the behavior on SDS-PAGE of
the native and recombinant proteins. Two major protein bands could be
detected in the nCPO preparate, possibly corresponding to isozymes A
and B. These two CPO forms have the same amino acid composition and
specific activity, but they differ in the carbohydrate composition (1).
rCPO migrated as a single band at a position that is 5-10 kDa,
respectively, higher than the native isozymes. As we suspected that
this difference in size was due to a overglycosylation of the
recombinant enzyme, we treated both rCPO and nCPO with EndoH to remove
N-linked glycans. As previously reported (1), EndoH
digestion of nCPO produced two species of reduced molecular weight.
Both deglycosylated rCPO and nCPO shifted to a similar position on
SDS-PAGE (Fig. 3), indicating that the
differences in size could indeed be attributed to overglycosylation of
the recombinant enzyme. Furthermore, similarly to nCPO, the recombinant enzyme was not reactive to the Ellmann reagent (54), indicating a
correct formation of the single disulfide bridge present in chloroperoxidase (65).
To analyze whether the CPO signal sequence was correctly processed in
A. niger, the purified extracellular rCPO was submitted to
sequencing of its N terminus. However, no amino acid sequence could be
recovered from this analysis, suggesting that the recombinant enzyme
was blocked at its N terminus.
Catalytic Properties--
To analyze whether the recombinant CPO
was fully active some of its catalytic properties were measured. The
specific chlorination activity (MCD assay as described by Morris and
Hager (Ref. 2)) was determined. The specific chlorination activity of
purified rCPO was 47 units/nmol of heme. The pH optimum for the
chlorination of monochlorodimedone was measured for rCPO and native
CPO. rCPO and nCPO showed the same pH profile with a pH optimum at pH
2.75.
The enantioselective sulfoxidation of thioanisole and derivatives (see
Scheme 1) was used to monitor the
enantioselective properties of the enzyme. Although, as shown in Table
II, results obtained in 1-ml scale
experiments differed slightly from the results published for 50-ml
scale experiments, similar to the native CPO, recombinant CPO produced
predominantly the R-sulfoxide in up to 99%
enantiomeric excess. Experiments with labeled
H218O2 showed 100% incorporation
of 18O into thioanisole sulfoxide for both nCPO and rCPO
(data not shown).
The regioselectivity of rCPO was studied by means of the oxidation of
indole and derivatives (see Scheme 2). As
shown in Table III, the conversions
obtained with rCPO was slightly lower than those obtained with native
CPO. However, both rCPO and native CPO yield the corresponding
2-oxindoles in virtually quantitative yield. Experiments with labeled
H218O2 showed 100% incorporation
of 18O into 2-oxindole for both nCPO and rCPO (data not
shown).
Chloroperoxidase from the filamentous fungus Caldariomyces
fumago is an enzyme of unique versatility as a catalyst for
synthetically useful oxygen transfer reactions. Structurally, the
enzyme shares characteristics of the P450 cytochromes and the heme
peroxidases. These features make CPO a very attractive example for
function-structure relationship studies of oxidative enzymes. To make
this possible, an efficient recombinant expression system for the
cpo gene is required. Recently, we reported the expression
of two fungal heme-containing peroxidases in the filamentous fungus
A. niger (52). Production of the recombinant proteins was
achieved by placing the peroxidase coding sequences under control of
efficient Aspergillus expression signals. Using a similar
approach, the C. fumago cpo gene has been efficiently
expressed in A. niger and the recombinant enzyme was
secreted into the culture medium as an active protein.
The production of rCPO could be increased by heme addition to the
culture medium. Similar results have been obtained in previous studies
by our and other groups on the expression of fungal peroxidases in
Aspergillus species (50, 52, 66). However, our results show
that despite heme supplementation, rCPO was only partially (40%)
incorporated with heme. This is in contrast to our observations on the
production of Phanerochaete chrysosporium manganese
peroxidase in A. niger (52), where the recombinant enzyme
could be produced with the same heme content as the native protein. A
possible reason for this different behavior may be the different nature
of heme attachment in the manganese peroxidase (axial ligand histidine) and CPO (axial ligand cysteine) protein.
EndoH treatment and SDS-PAGE analysis revealed a higher molecular
weight of rCPO in comparison to nCPO as a result of overglycosylation of the recombinant enzyme. Overglycosylation has been reported for the
expression of other heterologous proteins in Aspergillus spp. (52, 67). In these reports, it was shown that the excess of
glycosyl groups did not have a major effect on the properties of the
recombinant enzymes. Our results on the characterization of the
recombinant chloroperoxidase indicate that this is also the case for
rCPO. This is in agreement with the observation that CPO isozymes,
differing in glycosylation pattern, maintain the same specific activity
(1). Furthermore, Zong et al. (45) in their studies on the
expression of chloroperoxidase in E. coli showed that
glycosylation is not an essential requirement for the activity of this enzyme.
The N terminus of rCPO appeared to be blocked. This was not completely
surprising, since native CPO is known to posses a N-terminal glutamic
acid residue, which is mostly cyclized into a pyrrolidone carboxylic
acid (1). Such molecules, whose formation is induced in acidic
environments, are unreactive to the Edman's reagent. As the culture
medium of A. niger reaches a pH = 2, this may explain the N-terminal blockage of rCPO in case the A. niger-produced protein would have the native N terminus.
To further validate the A. niger production system for CPO,
we have assessed whether the structural and catalytic properties of
rCPO were comparable with these of native CPO. Experimental data showed
the correct formation of the heme thiolate ligand as well as single
disulfide bond in the recombinant CPO. The specific chlorination
activity of rCPO (47 units/nmol of heme) was in agreement with the
activities reported by Morris and Hager (Ref. 2; 70 units/nmol), Van
Deurzen et al. (Ref. 15; 53 units/nmol), and Libby et
al. (Ref. 68; 59 units/nmol). Additionally, the pH optimum for
this chlorination reaction (pH 2.75 for rCPO) coincided with that of
the native CPO and with the pH of the MCD assay as described by Morris
and Hager (2). From these results we conclude that the natural
chlorination activity of CPO is completely present in the recombinant enzyme.
Similarly, the oxygen transfer properties of CPO were not changed upon
expression of the enzyme in Aspergillus. Recombinant CPO
showed an enantioselectivity of 99% for the sulfoxidation of
thioanisole derivatives (the R-sulfoxide being predominantly formed) and a regioselectivity of 99% for the oxidation of indole derivatives to the corresponding 2-oxindoles. In aqueous buffer solutions (sulfoxidation reaction, Table II), the yields obtained with
rCPO were comparable with those obtained with native CPO. However, when
a mixture of tert-butyl alcohol and aqueous buffer (50:50
(v/v)) was used (oxidation of indoles, Table III), rCPO resulted in a
slightly lower yield than native CPO. Although the reasons for this
result are not clear, it is possible that different glycosylation of
rCPO has an influence on the stability of the enzyme in mixtures of
tert-butyl alcohol and aqueous buffer.
Regio- and enantioselective oxidation reactions catalyzed by CPO are
known to be oxygen transfer reactions in which the oxygen atom from CPO
compound I is directly transferred to the substrate molecule. For rCPO
we found 100% incorporation of 18O from labeled
H218O2 into thioanisole sulfoxide
and 2-oxindole. This is in agreement with the results of labeling
studies with native CPO, as reported for sulfoxidations (69) and
oxidation of indole (17). Hence, we conclude that both the chlorination
activity and the oxygen transfer properties of CPO are fully retained
in the recombinant enzyme.
To our knowledge, this is the first report of the production of fully
active chloroperoxidase in a heterologous expression system. We have
shown that the catalytic properties of the enzyme remained basically
unchanged, which makes of the A. niger expression system a
suitable system for mechanistic and mutagenesis studies of this unique enzyme.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, CPO catalyzes a variety of synthetically useful
(enantioselective) oxygen transfer reactions (3-5), e.g.
asymmetric epoxidation of olefins (6-8); allylic, benzylic, and
propargylic hydroxylation (9-11); asymmetric sulfoxidation (12-15);
and oxidation of indoles to the corresponding 2-oxindoles (16, 17). In
catalyzing these oxygen transfer reactions, CPO behaves more like the
P-450 cytochromes than like classical peroxidases such as the
peroxidases from horseradish roots, soybeans, and the fungus
Coprinus cinereus, which mostly catalyze one-electron
oxidations, e.g. polymerization of phenol and anilinic
compounds (18, 19). Moreover, the iron protoporphyrin in CPO is ligated
to the active site through a cysteine residue (20-22), as
characteristic of P-450 cytochromes, whereas the axial ligand in
peroxidases normally is a histidine residue (23). Interestingly, the
C. fumago CPO shows no sequence similarity to other
extracellular heme peroxidases (24-28) or to known microbial vanadium
haloperoxidases (29-31) but is most similar to the Aspergillus nidulans stcC (32), a member of the sterigmatocystin biosynthetic gene cluster, and also shows significant sequence similarity to a
Agaricus bisporus cellulolytic gene (accession number
AJ293759).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was used for
construction and propagation of vector molecules. A. niger
MGG029 (prtT, gla::fleor,
pyrG; Ref. 52) was used as recipient strain in
transformation experiments.
1, and detected on
a Waters 486 tunable absorbance detector at 220 nm with Waters
Millennium32 software. A hexane/isopropyl alcohol mixture
of 75:25 (v:v) was used as eluent. 1,2,3-Trimethoxybenzene was used as
internal standard.
1 was used as eluent for all indole derivatives.
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Fig. 1.
The cpo expression vector
pCPO3.I-AmdS.
20 °C) was slowly added at 4 °C, and after 1 h of
incubation at
20 °C, precipitated impurities were removed by
centrifugation (4400 min
1; 20 min; 0 °C).
Cold acetone was then slowly added to the supernatant (1000 ml; final
concentration 60% v/v;
20 °C), and CPO precipitation occurred
overnight at
20 °C. The supernatant was removed by decanting, and
the precipitated protein was dried for 10 min. The protein pellet was
dissolved in phosphate buffer (300 ml; 10 mM; pH 5.2), adjusted to pH 5.8 with 10 mM
H3PO4, and brought onto a DEAE-Sepharose (Amersham Pharmacia Biotech, 750 ml) fast flow column in phosphate buffer (20 mM; pH 5.8; flow 10 ml
min
1). The column was washed with phosphate
buffer (20 mM; pH 5.8; 10 ml
min
1) for 1 h. The enzyme was eluted
with a 20-200 mM phosphate buffer gradient (pH 5.8; 10 ml
min
1) during 4 h. Fractions having
peroxidase activity (MCD assay) above 0.25 units/ml were pooled,
adjusted to pH 5.2, and concentrated over a 30-kDa membrane
(Centriprep-30 concentrator, Amicon) at a speed of 1800 rpm. Further
purification was done by gel filtration on a Superose 12 HPLC column
(Amersham Pharmacia Biotech, 10 × 300 mm; phosphate buffer, pH
5.2; 200 mM; 0.5 ml min
1).
RESULTS
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ABSTRACT
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RESULTS
DISCUSSION
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Purification of rCPO
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Fig. 2.
UV spectra of rCPO (thick
line) and nCPO (thin
line).
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Fig. 3.
Western blotting analysis of rCPO and
nCPO. Proteins were detected with a CPO polyclonal
antiserum (see "Experimental Procedures"). Proteins were partially
deglycosylated by treatment with EndoH (+). The deglycosylation protein
bands are indicated by an arrow.
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Scheme 1.
Oxidation of sulfides.
R = H or OCH3; R' =
CH3 or
CH2-CH3.
Oxidation of sulfides by native and recombinant CPO
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Scheme 2.
Oxidation of substituted indoles.
R = Br,
Cl, or
OCH3.
Oxidation of substituted indoles by native and recombinant CPO
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DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Dr. Hager (University of Illinois, Urbana-Champaign, IL) for providing the cpo genomic clone, Prof. H. Duine and B. W. Groen (University of Delft, Delft, The Netherlands) for their collaboration in the purification of CPO, and G. van Duijn (TNO Nutrition and Food Research Institute, The Netherlands) for assistance in obtaining the CPO antisera.
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FOOTNOTES |
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* This work was supported in part by Dutch Innovation Oriented Program on Catalysis Grant IKA94013.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. E-mail: ppunt@voeding.tno.nl.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M010571200
2 L. P. Hager, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: CPO/nCPO, chloroperoxidase from C. fumago; rCPO, recombinant CPO expressed in A. niger; MCD, monochlorodimedone; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; GC, gas chromatography; MS, mass spectroscopy; EndoH, endoglycosidase H.
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