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1.
Biotechnol Lett ; 26(23): 1815-9, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15672220

ABSTRACT

Chloroperoxidase from Caldariomyces fumago catalyses the oxidation of 1,2-dihydronaphthalene to (1R,2R)-(+)-dihydroxytetrahydronaphthalene in homogenous citrate buffer/ionic liquid mixtures, using t-butyl hydroperoxide as O2 donor. It tolerates up to 30 (v/v) 1,3-dimethylimidazolium methylsulfate or 1-butyl-3-methylimidazolium methylsulfate. The enzyme activity in these ionic liquid co-solvent systems is retained for 24 h, but it falls to 3 h using non-ionic organic solvents such as t-BuOH or acetone.


Subject(s)
Ascomycota/enzymology , Chloride Peroxidase/biosynthesis , Chloride Peroxidase/chemistry , Citrates/chemistry , Naphthalenes/chemistry , Solvents/chemistry , tert-Butylhydroperoxide/chemistry , Chloride Peroxidase/analysis , Enzyme Activation , Ions , Kinetics , Solutions
2.
FEBS Lett ; 503(2-3): 117-20, 2001 Aug 17.
Article in English | MEDLINE | ID: mdl-11513866

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.


Subject(s)
Ascomycota/enzymology , Chloride Peroxidase/chemistry , Amino Acid Sequence , Ascomycota/genetics , Aspergillus niger/genetics , Base Sequence , Chloride Peroxidase/biosynthesis , Chloride Peroxidase/genetics , DNA Primers/genetics , Enzyme Precursors/biosynthesis , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Gene Expression , Molecular Chaperones/biosynthesis , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Sequence Data , Protein Conformation
3.
J Biol Chem ; 276(21): 17635-40, 2001 May 25.
Article in English | MEDLINE | ID: mdl-11278701

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 (18)O from labeled H(2)18O(2) into the oxidized products was 100% in both cases.


Subject(s)
Chloride Peroxidase/genetics , Fungal Proteins/genetics , Aspergillus niger/enzymology , Aspergillus niger/genetics , Catalysis , Chloride Peroxidase/biosynthesis , Fungal Proteins/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Substrate Specificity
4.
Biochemistry ; 39(5): 1133-41, 2000 Feb 08.
Article in English | MEDLINE | ID: mdl-10653660

ABSTRACT

The vanadate cofactor in vanadium chloroperoxidase has been studied using UV-VIS absorption spectroscopy. A band is present in the near-UV that is red-shifted as compared to free vanadate and shifts in both position and intensity upon change in pH. Mutation of vanadate binding residues has a clear effect on the spectrum. Substrate-induced spectral effects allow direct measurement of separate kinetics steps for the first time for vanadium haloperoxidases. A peroxo intermediate is formed upon addition of H(2)O(2), which causes a decrease in the absorption spectrum at 315 nm, as well as an increase at 384 nm. This peroxo form is very stable at pH 8.3, whereas it is less stable at pH 5.0, which is the optimal pH for activity. Upon addition of halides to the peroxo form, the native spectrum is re-formed as a result of halide oxidation. Stopped-flow experiments show that H(2)O(2) binding and Cl(-) oxidation occur on the millisecond to second time scale. These data suggest that the oxidation of Cl(-) to HOCl occurs in at least two steps. In the presence of H(2)O(2), the affinity for the vanadate cofactor was found to be much higher than previously reported for vanadate in the absence of H(2)O(2). This is attributed to the uptake of pervanadate by the apo-enzyme. Human glucose-6-phosphatase, which is evolutionarily related to vanadium chloroperoxidase, is also likely to have a higher affinity for pervanadate than vanadate. This could explain the enhanced insulin mimetic effect of pervanadate as compared to vanadate.


Subject(s)
Chloride Peroxidase/chemistry , Chloride Peroxidase/metabolism , Vanadates/metabolism , Vanadium/metabolism , Apoenzymes/chemistry , Apoenzymes/metabolism , Ascomycota/enzymology , Binding Sites/genetics , Chloride Peroxidase/biosynthesis , Chloride Peroxidase/genetics , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , Kinetics , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrophotometry, Ultraviolet/methods , Substrate Specificity/genetics , Vanadates/chemistry
5.
Microbiology (Reading) ; 142 ( Pt 8): 2129-35, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8760926

ABSTRACT

The non-haem chloroperoxidase gene (cpoF) from the pyrrolnitrin producer Pseudomonas fluorescens BL914 was cloned using an oligonucleotide derived from part of the N-terminal amino acid sequence of chloroperoxidase (CPO-P) from Pseudomonas pyrrocina as a probe. Based on the overexpression of cpoF in Escherichia coli and the stability of CPO-F against higher temperatures and proteases, the enzyme was purified to homogeneity. Partial characterization of the enzyme showed that it belongs to the class of bacterial non-haem CPOs. To investigate the role of CPO-F in pyrrolnitrin biosynthesis, the cpoF gene was inactivated by insertion of a kanamycin cassette. Exchange of the chromosomal cpoF gene against the disrupted copy had no influence on pyrrolnitrin production demonstrating that CPO-F was not involved in pyrrolnitrin biosynthesis.


Subject(s)
Chloride Peroxidase/metabolism , Pseudomonas fluorescens/enzymology , Pyrrolnitrin/biosynthesis , Chloride Peroxidase/biosynthesis , Chloride Peroxidase/isolation & purification , Chromatography, Ion Exchange , Cloning, Molecular , Conjugation, Genetic , Escherichia coli , Kinetics , Mutagenesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping , Sequence Deletion
6.
Gene ; 130(1): 131-5, 1993 Aug 16.
Article in English | MEDLINE | ID: mdl-8344520

ABSTRACT

The nucleotide sequence of a 1.5-kb fragment of Pseudomonas pyrrocinia DNA containing the chloroperoxidase(CPO)-encoding gene (cpo) and its flanking regions was determined. The cpo codes for a protein of 278 amino acids (aa). The mature enzyme contains no N-terminal methionine, so that the CPO monomer consists of 277 aa with a calculated M(r) of 30,304. Expression studies showed that the cpo from P. pyrrocinia is functionally expressed in Escherichia coli and Streptomyces lividans. Based on the overproduction of the CPO in E. coli, a novel and simple purification procedure was developed allowing the isolation of about 800-fold more CPO per gram of cells than was originally isolated from P. pyrrocinia. Comparison with the aa sequence of the bromoperoxidase BPO-A2 from S. aureofaciens ATCC10762 revealed an identity of 38%.


Subject(s)
Chloride Peroxidase/genetics , Chloride Peroxidase/isolation & purification , Genes, Bacterial , Pseudomonas/enzymology , Pseudomonas/genetics , Amino Acid Sequence , Base Sequence , Chloride Peroxidase/biosynthesis , Chloride Peroxidase/chemistry , Cloning, Molecular , DNA, Bacterial/analysis , Escherichia coli/genetics , Gene Expression , Genetic Vectors , Molecular Sequence Data , Peroxidases/chemistry , Peroxidases/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Sequence Alignment , Streptomyces aureofaciens/genetics
7.
J Bacteriol ; 170(2): 1007-11, 1988 Feb.
Article in English | MEDLINE | ID: mdl-2828306

ABSTRACT

The chloroperoxidase gene from the filamentous fungus Caldariomyces fumago has been isolated within a 16.3-kilobase insert in the vector lambda EMBL3. The DNA sequence of the gene and its immediate flanking regions has been determined, and the start site of transcription has been mapped by primer extension.


Subject(s)
Chloride Peroxidase/genetics , Genes, Fungal , Mitosporic Fungi/genetics , Peroxidases/genetics , Base Sequence , Chloride Peroxidase/biosynthesis , Cloning, Molecular , DNA Restriction Enzymes , DNA, Fungal/genetics , Gene Expression Regulation , Mitosporic Fungi/enzymology , Molecular Sequence Data , Nucleic Acid Hybridization , Protein Biosynthesis , Software , Transcription, Genetic
8.
J Biol Chem ; 261(32): 15058-61, 1986 Nov 15.
Article in English | MEDLINE | ID: mdl-3771564

ABSTRACT

The fungus Caldariomyces fumago can be induced to secrete the heme protein chloroperoxidase at levels of 500 mg/liter. Chloroperoxidase synthesis is controlled at the mRNA level. Glucose strongly represses production of chloroperoxidase mRNA and protein, whereas fructose induces both to high levels. Chloroperoxidase has been partially sequenced by automated Edman degradation of tryptic peptides. Based on this amino acid sequence data, a 2-fold degenerate, 29-base oligonucleotide (29-mer) complementary to chloroperoxidase mRNA was synthesized. Polyadenylated RNA, purified from C. fumago, was used as substrate for cDNA synthesis using the 29-mer as primer. cDNAs were made double-stranded and cloned into plasmid pBR322 by conventional methods. Screening the resultant cDNA bank by colony hybridization with the 29-mer as probe showed that 18% of the clones contained the 29-mer sequence. Dideoxy sequencing of one clone (pMA340) identified it as part of the coding region for chloroperoxidase by comparison with known amino acid sequences. In addition, the amino-terminal coding region of clone pMA340 reveals a putative signal peptide for chloroperoxidase. Clone pMA340 was then used in Northern analysis of chloroperoxidase mRNA levels under conditions which induce and repress enzyme secretion.


Subject(s)
Chloride Peroxidase/genetics , Fructose/pharmacology , Genes, Fungal/drug effects , Genes/drug effects , Glucose/pharmacology , Mitosporic Fungi/genetics , Peroxidases/genetics , RNA, Messenger/genetics , Transcription, Genetic/drug effects , Amino Acid Sequence , Base Sequence , Chloride Peroxidase/biosynthesis , Enzyme Repression , Mitosporic Fungi/enzymology
9.
Can J Microbiol ; 27(12): 1298-305, 1981 Dec.
Article in English | MEDLINE | ID: mdl-7332880

ABSTRACT

Ten strains of Caldariomyces fumago and related fungi were found to produce extracellular chloroperoxidase when grown on a glucose - malt extract medium. High enzyme levels and pigment production were observed for C. fumago ATCC 16373 and C. fumago CMI 89362. Removal of malt extract from the medium and the replacement of glucose by fructose as the carbon source provided a defined medium which, by comparison with the complex medium, produced the following results with both fungal strains. Chloroperoxidase was produced to similar levels, with maximum production after 6 days rather than 12 days of growth; pigmentation of the medium was reduced by 90% and the pH of the medium remained constant, thus stabilizing enzyme activity. Addition or urea or proline as a nitrogen supplement to nitrate enhanced enzyme production by strain CMI 89362. Comparison of the two strains indicated that CMI 89352 produced higher levels of chloroperoxidase than ATCC 16373.


Subject(s)
Chloride Peroxidase/biosynthesis , Mitosporic Fungi/enzymology , Peroxidases/biosynthesis , Amino Acids/pharmacology , Carbohydrates/pharmacology , Culture Media , Mitosporic Fungi/growth & development , Nitrates/pharmacology , Phosphates/pharmacology , Pigments, Biological/biosynthesis , Urea/pharmacology
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