3.
Conesa, A.; Weelink, G.; van den Hondel, C.; Punt, P.
C-terminal propeptide of the Caldariomyces fumago chloroperoxidase: an intramolecular chaperone? Journal Article
In: FEBS LETTERS, vol. 503, no. 2-3, pp. 117-120, 2001, ISSN: 0014-5793.
@article{ISI:000170641000001,
title = {C-terminal propeptide of the Caldariomyces fumago chloroperoxidase: an
intramolecular chaperone?},
author = { A. Conesa and G. Weelink and C. van den Hondel and P. Punt},
url = {http://dx.doi.org/10.1016/S0014-5793(01)02698-9},
doi = {10.1016/S0014-5793(01)02698-9},
issn = {0014-5793},
year = {2001},
date = {2001-08-01},
journal = {FEBS LETTERS},
volume = {503},
number = {2-3},
pages = {117-120},
abstract = {The Caldariomyces fumago chloroperoxidase (CPO) is synthesised as a
372-aa precursor which undergoes two proteolytic processing events.
removal of a 21-aa N-terminal signal peptide and of a 52-aa C-terminal
propeptide. The Aspergillus niger expression system developed for CPO
was used to get insight into the function of this C-terminal propeptide.
A. niger transformants expressing a CPO protein from which the
C-terminal propeptide was deleted failed in producing any extracellular
CPO activity, although the CPO polypeptide was synthesised. Expression
of the full-length gene in an A. niger strain lacking the KEX2-like
protease PclA also resulted in the production of CPO cross-reactive
material into the culture medium, but no CPO activity. Based on these
results, a function of the C-terminal propeptide in CPO maturation is
indicated. (C) 2001 Federation of European Biochemical Societies.
Published by Elsevier Science B.V. All rights reserved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The Caldariomyces fumago chloroperoxidase (CPO) is synthesised as a
372-aa precursor which undergoes two proteolytic processing events.
removal of a 21-aa N-terminal signal peptide and of a 52-aa C-terminal
propeptide. The Aspergillus niger expression system developed for CPO
was used to get insight into the function of this C-terminal propeptide.
A. niger transformants expressing a CPO protein from which the
C-terminal propeptide was deleted failed in producing any extracellular
CPO activity, although the CPO polypeptide was synthesised. Expression
of the full-length gene in an A. niger strain lacking the KEX2-like
protease PclA also resulted in the production of CPO cross-reactive
material into the culture medium, but no CPO activity. Based on these
results, a function of the C-terminal propeptide in CPO maturation is
indicated. (C) 2001 Federation of European Biochemical Societies.
Published by Elsevier Science B.V. All rights reserved.
372-aa precursor which undergoes two proteolytic processing events.
removal of a 21-aa N-terminal signal peptide and of a 52-aa C-terminal
propeptide. The Aspergillus niger expression system developed for CPO
was used to get insight into the function of this C-terminal propeptide.
A. niger transformants expressing a CPO protein from which the
C-terminal propeptide was deleted failed in producing any extracellular
CPO activity, although the CPO polypeptide was synthesised. Expression
of the full-length gene in an A. niger strain lacking the KEX2-like
protease PclA also resulted in the production of CPO cross-reactive
material into the culture medium, but no CPO activity. Based on these
results, a function of the C-terminal propeptide in CPO maturation is
indicated. (C) 2001 Federation of European Biochemical Societies.
Published by Elsevier Science B.V. All rights reserved.
2.
Conesa, A.; van de Velde, F.; van Rantwijk, F.; Sheldon, R.; van den Hondel, C.; Punt, P.
Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme Journal Article
In: JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 21, pp. 17635-17640, 2001, ISSN: 0021-9258.
@article{ISI:000168866500004,
title = {Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus
niger and characterization of the recombinant enzyme},
author = { A. Conesa and F. van de Velde and F. van Rantwijk and R. Sheldon and C. van den Hondel and P. Punt},
url = {http://dx.doi.org/10.1074/jbc.M010571200},
doi = {10.1074/jbc.M010571200},
issn = {0021-9258},
year = {2001},
date = {2001-05-01},
journal = {JOURNAL OF BIOLOGICAL CHEMISTRY},
volume = {276},
number = {21},
pages = {17635-17640},
abstract = {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 O-18 from labeled
(H2O2)-O-18 into the oxidized products was 100% in both cases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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 O-18 from labeled
(H2O2)-O-18 into the oxidized products was 100% in both cases.
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 O-18 from labeled
(H2O2)-O-18 into the oxidized products was 100% in both cases.
1.
Conesa, A.; van den Hondel, C.; Punt, P.
Studies on the production of fungal peroxidases in Aspergillus niger Journal Article
In: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 66, no. 7, pp. 3016-3023, 2000, ISSN: 0099-2240.
@article{ISI:000088057600044,
title = {Studies on the production of fungal peroxidases in Aspergillus niger},
author = { A. Conesa and C. van den Hondel and P. Punt},
url = {http://dx.doi.org/10.1128/AEM.66.7.3016-3023.2000},
doi = {10.1128/AEM.66.7.3016-3023.2000},
issn = {0099-2240},
year = {2000},
date = {2000-07-01},
journal = {APPLIED AND ENVIRONMENTAL MICROBIOLOGY},
volume = {66},
number = {7},
pages = {3016-3023},
abstract = {To get insight into the limiting factors existing for the efficient
production of fungal peroxidase in filamentous fungi, the expression of
the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and
manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been
studied. For this purpose, a protease-deficient A. niger strain and
different expression cassettes have been used. Northern blotting
experiments indicated high steady-state mRNA levels for the recombinant
genes. Manganese peroxidase was secreted into the culture medium as an
active protein. The recombinant protein showed specific activity and a
spectrum profile similar to those of the native enzyme, was correctly
processed at its N terminus, and had a slightly lower mobility on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, Recombinant MnP
production could be increased up to 100 mg/liter upon hemoglobin
supplementation of the culture medium. Lignin peroxidase was also
secreted into the extracellular medium, although the protein was not
active, presumably due to incorrect processing of the secreted enzyme.
Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase
gene did not result in improved production yields.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
To get insight into the limiting factors existing for the efficient
production of fungal peroxidase in filamentous fungi, the expression of
the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and
manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been
studied. For this purpose, a protease-deficient A. niger strain and
different expression cassettes have been used. Northern blotting
experiments indicated high steady-state mRNA levels for the recombinant
genes. Manganese peroxidase was secreted into the culture medium as an
active protein. The recombinant protein showed specific activity and a
spectrum profile similar to those of the native enzyme, was correctly
processed at its N terminus, and had a slightly lower mobility on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, Recombinant MnP
production could be increased up to 100 mg/liter upon hemoglobin
supplementation of the culture medium. Lignin peroxidase was also
secreted into the extracellular medium, although the protein was not
active, presumably due to incorrect processing of the secreted enzyme.
Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase
gene did not result in improved production yields.
production of fungal peroxidase in filamentous fungi, the expression of
the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and
manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been
studied. For this purpose, a protease-deficient A. niger strain and
different expression cassettes have been used. Northern blotting
experiments indicated high steady-state mRNA levels for the recombinant
genes. Manganese peroxidase was secreted into the culture medium as an
active protein. The recombinant protein showed specific activity and a
spectrum profile similar to those of the native enzyme, was correctly
processed at its N terminus, and had a slightly lower mobility on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, Recombinant MnP
production could be increased up to 100 mg/liter upon hemoglobin
supplementation of the culture medium. Lignin peroxidase was also
secreted into the extracellular medium, although the protein was not
active, presumably due to incorrect processing of the secreted enzyme.
Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase
gene did not result in improved production yields.