Novel peroxidases of Marasmius scorodonius degrade beta-carotene (original) (raw)
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Novel peroxidases of Marasmius scorodonius degrade β-carotene
Applied Microbiology and Biotechnology, 2008
Two extracellular enzymes (MsP1 and MsP2) capable of efficient β-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of ~150 kDa and ~120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1604 to 1923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of ~23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).
Applied Microbiology and Biotechnology, 2009
Tobacco aroma compounds were generated via lutein cleavage by the combined action of a yeast and a bacterium identified as Trichosporon asahii and Paenibacillus amylolyticus, respectively. In this study, an inverse relationship between glucose concentration and the generation of three compounds, present in the tobacco aroma profile, was observed in mixed cultures. In order to identify the organism sensitive to the sugar effect, both were grown separately. The presence of glucose suppressed β-ionone production by T. asahii grown with lutein. However, the biotransformation of the ionone into its reduced derivatives (7,8-dihydro-β-ionone and 7,8-dihydro-β-ionol) by P. amylolyticus was not affected by the sugar . This pointed to the cleavage of lutein, a step within the process necessary for the synthesis of β-ionone, as the target of the glucose effect. In vitro studies with crude extracts and concentrated cell-free medium derived from T. asahii cultures showed that the carotenoid breakdown activity was located extracellularly and only detected in supernatants from yeast cells grown in the absence of the sugar. Rather than an inhibition or a mechanism affecting the enzyme secretion, the glucose effect on lutein degradation comprised another regulatory level. Further experiments showed that the enzyme responsible for lutein breakdown and susceptible to the sugar effect exhibited a high degree of identity to fungal peroxidases, studied as well, for their involvement in carotenoid cleavage.
Homology among multiple extracellular peroxidases from Phanerochaete chrysosporium
Journal of Biological Chemistry, 1987
The extracellular peroxidases of Phanerochaete chrysosporium were separated into 21 proteins by analytical isoelectric focusing. Fifteen of these enzymes oxidized veratryl alcohol (lignin peroxidases) in the presence of HzOz. Six enzymes were Mn(I1)-dependent peroxidases. The Mn(I1)-dependent enzymes appeared and reached their maximal activity earlier than the lignin peroxidases in the cultures. Peptide mapping, amino acid analysis, and reaction against specific antibodies showed that all the Mn(I1)-dependent peroxidases were probably products of one gene. A great degree of homology was also present among the various lignin peroxidases. Biological lignin degradation has gained much attention since the original discovery of an enzyme from culture filtrates of Phanerochaete chrysosporium able to partially depolymerize lignin and oxidize several lignin-related aromatic compounds (1-3). During the last 3 years, this enzyme has been called a lignin degrading enzyme (l), lignin degrading H202dependent oxygenase (3), diary1 propane oxygenase (2), and ligninase (4). Recently this enzyme was shown to be a peroxidase (5,6) which oxidizes different aromatic substrates by a one-electron transfer mechanism. The existence of multiple molecular forms of this lignin peroxidase has been reported (7-9). The lignin peroxidase can oxidize molecules that are not normally its substrates through electron transfer mediators like veratryl alcohol and dimethoxybenzene (10-12). In culture filtrates of P. chrysosporium Kuwahara et al. (13) also found another type of peroxidase that is Mn(I1)-dependent. This enzyme has been purified and characterized (14,15). It oxidizes Mn(I1) to Mn(III), which is able to oxidize various substrates. This enzyme has also been separated into multiple molecular forms (7, 15). The question of homology among the multiple forms of P. chrysosporium peroxidases and the function of the hemoproteins separated thus far (7, 8, 15) has not been satisfactorily answered. In this paper we show that, in the culture fluid of P. chrysosporium, more than 20 extracellular hemoproteins can be found which all have peroxidative activity. Furthermore, on the basis of reaction specificity, immunological properties, peptide mapping, and amino acid composition, these peroxidases can be divided into two basic groups. MATERIALS AND METHODS Production of Peroxidases-P. chrysosporium (ATCC 24725) was grown in nitrogen-or carbon-limited cultures. Medium composition
Purification and Characterization of a Versatile Peroxidase from Edible Mushroom Pleurotus eryngii
Chinese Journal of Chemical Engineering, 2010
A versatile peroxidase (VP-Peco60-7) was generated and purified from the liquid culture of Pleurotus eryngii. The purification procedure included ammonium sulfate precipitation, ion exchange chromatography, and gel chromatography. The molecular weight and isoelectric point (pI) of VP-Peco60-7 were determined to be approximately 40 kDa and 4.1, respectively. By N-terminal sequence determination and peptide mapping analysis, VP-Peco60-7 was found to be similar to the versatile peroxidase isoenzyme VPL1, which was previously isolated from liquid cultures of the same species. However, the molecular weight and pI of VP-Peco60-7 were different from those of versatile peroxidases of liquid cultures, implying that the VP-Peco60-7 in this study is of a novel type. With 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate, the maximal enzyme activity was obtained at 50 C and pH 3.0. The catalysis of ABTS by VP-Peco60-7 was expressed by the Michaelis-Menten equation. At 50 C and pH 3.0, the maximum velocity (V max) was 188.68 U•mg 1 and the michaelis constant (K m) was 203.09 mol•L 1 .
Molecular characterisation of a versatile peroxidase from a strain
J Biotechnol, 2005
The cloning and sequencing of the rbpa gene coding for a versatile peroxidase from a novel Bjerkandera strain is hereby reported. The 1777 bp isolated fragment contained a 1698 bp peroxidase-encoding gene, interrupted by 11 introns. The 367 amino acid-deduced sequence includes a 27 amino acid-signal peptide. The molecular model, built via homology modelling with crystal structures of four fungal peroxidases, highlighted the amino acid residues putatively involved in manganese binding and aromatic substrate oxidation. The potential heme pocket residues (R44, F47, H48, E79, N85, H177, F194 and D239) include both distal and proximal histidines (H48 and H177). RBP possesses potential calcium-binding residues (D49, G67, D69, S71, S178, D195, T197, I200 and D202) and eight cysteine residues (C3, C15, C16, C35, C121, C250, C286, C316). In addition, RBP includes residues involved in substrate oxidation: three acidic residues (E37, E41 and D183)-putatively involved in manganese binding and H83 and W172-potentially involved in oxidation of aromatic substrates. Characterisation of nucleotide and amino acid sequences include RBP in versatile peroxidase group sharing catalytic properties of both LiP and MnP. In addition, the RBP enzyme appears to be closely related with the ligninolytic peroxidases from the Trametes versicolor strain.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1996
Two manganese peroxidase isozymes, MnP1 and MnP2, were purified from the extracellular medium of ligninolytic cultures of Dichomitus squalens. The proteins were purified to homogeneity using DEAE-Sepharose chromatography and Mono Q fast protein liquid chromatography. MnP1 and MnP2 have molecular masses of 48 000 and 48 900 Da, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both isozymes are glycoproteins and each contains one iron protoporphyrin IX as a prosthetic group. The pl values of MnP1 and MnP2 are 4.15 and 3.90, respectively. N-Terminal amino-acid analysis suggests that these proteins are encoded by distinct genes. The Soret bands of the native ferric enzymes (408 nm and 406 nm, respectively) are shifted to 434 nm in the reduced enzymes and to 422 Jam in the reduced-CO complexes. EPR g-values of the native enzymes are essentially identical to those for other MnPs and lignin peroxidases, and they confirm the high-spin state of the iron. The addition of 1 equivalent of H202 to either of the native ferric isozymes yields spectra which are characteristic of compound I. Successive additions of 1 equivalent of ferrocyanide and 1 equivalent of H202 to the native enzymes yield spectra which are characteristic of compound II. Both MnP isozymes oxidize Mn 2+ to Mn 3 + in the presence of organic acid chelators. The MnP isozymes are produced by D. squalens only when the cells are grown in the presence of Mn.
Applied and Environmental Microbiology, 2018
Trametes versicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dyedecolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of T. versicolor BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase TvDyP1 and the versatile peroxidase TvVP2. Both peroxidases were heterologously produced in Escherichia coli, biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and TvDyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while TvVP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and TvDyP1 was globally found to be more stable than TvVP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of TvVP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, TvDyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and TvVP2 was shown to accept a wider range of reducing substrates than TvDyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with TvVP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and TvVP2 presented a greater ability to oxidize and decolorize the dye substrates than TvDyP1.
FEMS Microbiology Letters, 2000
We report cloning and sequencing of gene ps1 encoding a versatile peroxidase combining catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP) isolated from lignocellulose cultures of the white-rot fungus Pleurotus eryngii. The gene contains 15 putative introns, and the deduced amino acid sequence consists of a 339-residue mature protein with a 31-residue signal peptide. Several putative response elements were identified in the promoter region. Amino acid residues involved in oxidation of Mn 2 and aromatic substrates by direct electron transfer to heme and long-range electron transfer from superficial residues as predicted by analogy with Phanerochaete chrysosporium MnP and LiP, respectively. A dendrogram is presented illustrating sequence relationships between 29 fungal peroxidases.
The synthesis of the acidic apo-carotenoid neurosporaxanthin by the fungus Fusarium fujikuroi depends on four enzyme activities: phytoene synthase and carotene cyclase, encoded by the bifunctional gene carRA, a carotene desaturase, encoded by carB, and a postulated cleaving enzyme converting torulene (C40) into neurosporaxanthin (C35). Based on sequence homology to carotenoid oxygenases, we identified the novel fungal enzyme CarT. Sequencing of the carT allele in a torulene-accumulating mutant of F. fujikuroi revealed a mutation affecting a highly conserved amino acid, and introduction of a heterologous carT gene in this mutant restored the ability to produce neurosporaxanthin, pointing to CarT as the enzyme responsible for torulene cleavage. Expression of carT in lycopene-accumulating E. coli cells resulted in the formation of minor amounts of apocarotenoids, but no enzymatic activity was observed in b-carotene-accumulating cells, indicating a preference for acyclic or monocyclic carotenes. The purified CarT enzyme efficiently cleaved torulene in vitro to produce b-apo-4Ј-carotenal, the aldehyde corresponding to the acidic neurosporaxanthin, and was also active on other monocyclic synthetic substrates. In agreement with its role in carotenoid biosynthesis, the carT transcript levels are induced by light and upregulated in carotenoid-overproducing mutants, as already found for other car genes.