Browsing by Author "Lobos, S"
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- ItemCharacterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora(2000) Tello, M; Corsini, G; Larrondo, LF; Salas, L; Lobos, S; Vicuña, RThree new genes (Cs-mnp2A, Cs-mnp2B and Cs-mnp3) coding for manganese-dependent peroxidase (MnP) have been identified in the white-rot basidiomycete Ceriporiopsis subvermispora. The mature proteins contain 366 (MnP2A and MnP2B) and 364 (MnP3) amino acids, which are preceded by leader sequences of 21 and 24 amino acids, respectively. Cs-mnp2A and Cs-mnp2B appear to be alleles, since the corresponding protein sequences differ in only five residues, The upstream region of Cs-mnp2B contains a TATA box, AP-1 and AP-2 sites, as well as sites for transcription regulation by metals (two), cAMP (two) and xenobiotics (one). Some of these elements are also found in the regulatory region of Cs-MnP3, Transcription of Cs-mnp2A and Cs-mnp2B, but not that of Cs-mnp3, is activated by manganese. (C) 2000 Elsevier Science B.V. All rights reserved.
- ItemCloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora(1998) Lobos, S; Larrondo, L; Salas, L; Karahanian, E; Vicuña, RA cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G + C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I-P-X-P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5 kb contained the complete sequence of the Cs-mnp1 gene, including 162 bp and 770 bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT-AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9 kb was amplified using inverse PCR. A putative TATAA element was identified 5' of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified. (C) 1998 Elsevier Science B.V.
- ItemEnzymology and molecular genetics of the ligninolytic system of the basidiomycete Ceriporiopsis subvermispora(2001) Lobos, S; Tello, M; Polanco, R; Larrondo, LF; Manubens, A; Salas, L; Vicuña, RLignin, the most abundant renewable source of aromatic carbon on earth, consists in a highly irregular three-dimensional biopolymer of oxygenated phenylpropanoid units. In natural environments, lignin is only degraded efficiently by some fungi belonging to the group of basidiomycetes. These microorganisms secrete an array of oxidases and peroxidases for this purpose, which may be produced in various combinations. This article summarizes our studies on a particular strain called Ceriporiopsis subvermispora, a fungus which is highly agressive towards lignin when growing on wood.
- ItemIsoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium(2001) Larrondo, LF; Lobos, S; Stewart, P; Cullen, D; Vicuña, RWe expressed cDNAs coding for manganese peroxidases (MnPs! from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha -amylase promoter from. Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar Levels and had molecular masses, both before and after deglycosylation, that were the same as those described For the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pls between 4.83 and 4.06, and one of these pls coincided with the pi described for H4 isolated from P. chrysosporium (pl 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.
- ItemIsolation and characterization of homokaryotic strains from the ligninolytic basidiomycete Ceriporiopsis subvermispora(2001) Tello, M; Seelenfreund, D; Lobos, S; Gaskell, J; Cullen, D; Vicuña, RGenetic analyses of the lignin-degrading fungus Ceriporiopsis subvermispora is complicated by a dikaryotic nuclear condition and the absence of sport: forms. Previous investigations had identified a family of closely related sequences encoding manganese peroxidase (MnP), but the relationship between genes and allelic variants could not be experimentally established. Addressing this issue, homokaryotic derivatives of C. subvermipora strain FP105752 were isolated from regenerated protoplasts. Designated CsA and CsB. their homokaryotic nature was established by polymerase chain reaction amplification and sequence analysis of the allelic variants of three MnP genes. Isoelectrofocusing revealed fewer MnP isoenzymes in filtrates of homokaryon cultures relative to the parental strain. The homokaryotic strains will simplify genetic analyses, particularly the identification of neu genes. (C) 2001 Federation of European Microbiological Societies. published by Elsevier Science B.V. All rights reserved.
- Itemlip-like genes in Phanerochaete sordida, and Ceriporiopsis subvermispora, white rot fungi with no detectable lignin peroxidase activity(1996) Rajakumar, S; Gaskell, J; Cullen, D; Lobos, S; Karahanian, E; Vicuna, RLignin peroxidase-like genes were PCR amplified from Phanerochaete sordida and Ceriporiopsis subvermispora, fungi lacking lignin peroxidase (LiP) activity. Amplification products were highly similar to previously described LiP genes. Using reverse transcription-coupled PCR a LiP-like cDNA clone was amplified from P. sordida RNA. In contrast, no evidence was obtained for transcription of C. subvermispora LiP genes.
- ItemStructure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora(1998) Karahanian, E; Corsini, G; Lobos, S; Vicuña, RA gene encoding laccase has been isolated from a genomic library of the white-rot basidiomycete Ceriporiopsis subvermispora constructed in Lambda GEM-ii. This gene (Cs-lcs1) contains an open reading frame of 2215 bp, encoding a mature protein of 499 amino acids with a 21-residue signal peptide. The protein sequence exhibits between 63 and 68% identity with laccases from other basidiomycetes and shares with all of them 10 conserved histidines and one cysteine involved in the coordination of copper atoms at the active site of the enzyme. The gene possesses ii introns, with splicing junctions and internal lariat formation sites adhering to the GT-AG and CTRAY rules, respectively. The upstream region of Cs-lcsl contains a TATA box, two CAAT sites, five putative metal response elements and a ACE1 element. In agreement with the presence of the latter element, transcription of Cs-lcsl is activated by copper and silver, as shown by Northern blot and reverse transcription followed by DNA amplification analyses. Based on Southern blot analysis, Cs-lcsl appears to be the only gene encoding laccase in C. subvermispora. (C) 1998 Elsevier Science B.V. All rights reserved.