Biomedical subjects

R Vicuña

Publications and source records attributed to R Vicuña.

At least 19 recordsLinked to original sources

PubMed2004

Molecular approach for analysis of model fungal genes encoding ligninolytic peroxidases in wood-decaying soil systems.

AIMS: Test the use of nondegenerated consensus polymerase chain reaction (PCR) primers targeting lip and mnp sequences to detect ligninolytic fungi in wood-decaying soil systems, avoiding the need for enrichment or isolation on traditional fungal media culture. METHODS AND RESULTS: The PCR primers were tested with total DNA isolated from incubations of wood-soil systems inoculated or not with the white-rot fungi Phanerochaete chrysosporium, or a white-rot sample obtained from a Nothofagus forest. The PCR products for lip and mnp sequences were only obtained in soil with P. chrysosporium-colonized wood chips. In these soil samples, reverse transcription-PCR analysis of lip and mnp PCR products indicated expression of LipA, LipB, LipJ and MnP isoenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first assessment of the use of consensus PCR primers for direct detection of ligninolytic peroxidase genes in wood-decaying soil systems.

Basidiomycota↗

PubMed2001

Isolation and characterization of homokaryotic strains from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

Genetic analyses of the lignin-degrading fungus Ceriporiopsis subvermispora is complicated by a dikaryotic nuclear condition and the absence of spore 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 new genes.

Amino Acid Sequence↗

PubMed2001

Isoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium.

We 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 pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 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.

Aspergillus nidulans↗

PubMed2000

Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

Three 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.

Amino Acid Sequence↗

PubMed2000

Ligninolysis. A very peculiar microbial process.

Lignin, 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 basidioamycetes that secrete an array of enzymes for this purpose. Recent advances in our understanding of the mechanism of lignin breakdown have revealed several features that make this process highly unique from a biochemical standpoint. This article summarizes some of them.

Biopolymers↗

PubMed1999

Oxalate oxidase from Ceriporiopsis subvermispora: biochemical and cytochemical studies.

The enzyme oxalate oxidase was identified in mycelial extracts of the basidiomycete Ceriporiopsis subvermispora and thereafter purified to homogeneity. The purification procedure included only three steps: Q-Sepharose chromatography, precipitation at pH 3.0, and phosphocellulose chromatography. The enzyme is a 400-kDa homohexamer, as determined by gel permeation in Sephadex G-200 and SDS-polyacrylamide gel electrophoresis. Isoelectrofocusing revealed a pI of 4.2. Optimal activity was obtained at pH 3.5 and at 45 degrees C. The purified enzyme has Km and kcat values of 0.1 mM and 88 s-1, respectively. It is highly specific for oxalate, although it is inhibited at concentrations of this substrate above 2.5 mM. Hystochemistry studies conducted over mycelium slices showed reactions products in both endocellular and periplasmic associated elements. A possible connection between the intracellular metabolism of oxalate and the extracellular ligninolytic activity of the fungus is proposed.

Enzyme Activation↗

PubMed1998

Kinetics of Mn3+-oxalate formation and decay in reactions catalyzed by manganese peroxidase of Ceriporiopsis subvermispora.

The kinetics of Mn3+-oxalate formation and decay were investigated in reactions catalyzed by manganese peroxidase (MnP) from the basiomycete Ceriporiopsis subvermispora in the absence of externally added hydrogen peroxide. A characteristic lag observed in the formation of this complex was shortened by glyoxylate or catalytic amounts of Mn3+ or hydrogen peroxide. MnP titers had a minor effect on this lag and did not influence the decay rate of the complex. In contrast, Mn2+ and oxalate drastically affected maximal concentrations of the Mn3+-oxalate complex formed, the decay of which was accelerated at high Mn2+ levels. The highest concentration of complex was obtained at pH 4.0, whereas an inverse relationship was found between the pH of the reaction and the decay rate of the complex with MnP present. In the absence of MnP, the best fit for the decay kinetics of the complex gave an order of 3/2 at concentrations in the range of 30-100 microM, with a kobs = 0.012 min-1 M-0.5 at pH 4.0. The rate constant increases at lower pH values and decreases at high oxalate concentrations. The physiological relevance of these findings is discussed.

Catalase↗

PubMed1998

Structure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

A gene encoding laccase has been isolated from a genomic library of the white-rot basidiomycete Ceriporiopsis subvermispora constructed in Lambda GEM-11. 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 11 introns, with splicing junctions and internal lariat formation sites adhering to the GT-AG and CTRAY rules, respectively. The upstream region of Cs-lcs1 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-lcs1 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-lcs1 appears to be the only gene encoding laccase in C. subvermispora.

Amino Acid Sequence↗

PubMed1998

Cloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora.

A 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 2.5kb contained the complete sequence of the Cs-mnp1 gene, including 162bp and 770bp 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 1.9kb 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.

Amino Acid Sequence↗

PubMed1998

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide.

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; CO(2) evolution was monitored after addition of exogenous [C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO(2) from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.

Journal Article↗

PubMed1995

Oxidation reactions catalyzed by manganese peroxidase isoenzymes from Ceriporiopsis subvermispora.

A total of 11 manganese peroxidase isoenzymes (MnP1-MnP11) with isoelectric points (pIs) in the range of 4.58-3.20 were isolated from liquid- and solid-state cultures of the basidiomycete Ceriporiopsis subvermispora. In the presence of hydrogen peroxide, these isoenzymes showed different requirements for Mn(II) in the oxidation of vanillylacetone, o-dianisidine, p-anisidine and ABTS, whereas oxidation of guaiacol by any isoenzyme did not take place when this metal was omitted. Km values for o-dianisidine and p-anisidine in the absence of Mn(II) are in the range of 0.5-1.0 mM and 4.5-42.0 mM, respectively. Oxalate and citrate, but not tartrate, accelerate the oxidation of o-dianisidine, both in the presence and in the absence of Mn(II). MnPs from this fungus are able to oxidize kojic acid without externally added hydrogen peroxide, indicating that they can also act as oxidases. In this reaction, however, the requirement for Mn(II) is absolute.

Basidiomycota↗

PubMed1995

Properties of laccase isoenzymes produced by the basidiomycete Ceriporiopsis subvermispora.

Laccase is one of the ligninolytic enzymes found in liquid cultures of the fungus Ceriporiopsis subvermispora in defined medium. As an approach to a clarification of the role of laccases during the attack on lignin by the fungus, the enzyme has been characterized further. The levels of this phenol oxidase increase 2-fold in the presence of p-anisidine and are severely affected when addition of either Mn(II) or Cu(II) ions to the medium is omitted. Isoelectrofocusing allowed the resolution of two laccase isoenzymes, with pIs of 3.65 and 3.59. In rich medium, laccase activity is 10-fold higher than in salt medium, and it is not affected by the external addition of p-anisidine or Mn(II). Four isoenzymes were detected in these cultures, with pIs between 3.76 and 3.60. In a wheat bran medium, four isoenzymes with pIs in the range 3.63-3.46, plus a fifth isoenzyme of high pI (4.82), were also identified. The absorption spectrum of a pool containing the four isoenzymes from rich medium shows a maximum at 600 nm, typical of laccase possessing a type I copper atom. The molecular mass of the isoenzyme with pI 3.60 is 79 kDa, as determined by SDS/PAGE. Upon treatment with endoglycosidase F, the molecular mass of this isoform decreases to 63 kDa, indicating a high degree of glycosylation. Substrate specificity studies conducted with the four isoenzymes from rich medium and a combination of isoenzymes from salt medium showed marked differences among them. The amino-terminal sequences (24 residues) of three isoenzymes isolated from rich medium were determined. Two of them are identical, whereas the third one differs from these in three amino acid residues. The consensus sequence reveals clear homology with laccases from other microorganisms.

Amino Acid Sequence↗

PubMed1994

Cloning and sequencing of the gene encoding benzaldehyde lyase from Pseudomonas fluorescens biovar I.

The gene (bzl) encoding benzaldehyde lyase (BL) from Pseudomonas fluorescens biovar I has been cloned and characterized. The nucleotide sequence contains an open reading frame encoding a protein of 563 amino acids. The deduced BL protein shares little homology with proteins contained in the NBRF-PIR data bank. However, the higher homologies (up to about 28%) were obtained with enzymes that also utilize thiamine pyrophosphate as a cofactor.

Aldehyde-Lyases↗

PubMed1994

Isoenzymes of manganese-dependent peroxidase and laccase produced by the lignin-degrading basidiomycete Ceriporiopsis subvermispora.

The white-rot basidiomycete Ceriporiopsis subvermispora produces two families of ligninolytic enzymes, namely manganese-dependent peroxidases (MnPs) and laccases, when growing in liquid cultures of defined composition. In medium containing 11 p.p.m. of Mn(II), up to seven isoenzymes of MnP and four isoenzymes of laccase were resolved by isoelectrofocusing (IEF), with pI values in the range 4.10-4.60 and 3.45-3.65, respectively. Occasionally, a fifth laccase isoform of pI 4.70 was also detected. In cultures with 25 and 40 p.p.m. of Mn(II), mainly the MnPs with higher pI values are produced. The isoenzyme pattern of MnP is not altered throughout the growth period of the fungus. MnP and laccase are also produced by C. subvermispora when growing on wood chips of Pinus radiata. Highest levels of both enzymes were obtained during the first week of incubation. A second peak of MnP activity was observed during the fourth week, whereas very low levels of laccase were extracted from the chips after the second week of growth. IEF analysis showed that the pI values of these laccases are similar to those of laccases produced in liquid cultures, being in the range 3.45-3.65. In contrast, four isoforms of MnP were resolved during the first week of incubation on wood chips, with pI values of 4.40, 4.17, 4.04 and 3.53. This profile underwent a transition during the second week of growth, at the end of which isoforms of MnP with pI values of 3.53, 3.40, 3.30 and 3.20 were resolved by IEF.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Biotechnology in Chile (overview).

This article provides a brief overview of biotechnological activities in Chile. The role played by the scientific community is emphasized and aspects of technology transfer are commented.

Biotechnology↗

PubMed1993

Possible initial steps in the catabolism of 1,2-diphenylethanone (deoxybenzoin) by Pseudomonas fluorescens DB-5.

A natural bacterial strain, identified as Pseudomonas fluorescens DB-5, was isolated in enrichment cultures containing 1,2-diphenylethanone as the only source of carbon and energy. On the basis of characteristic features observed in the mass spectra of degradation intermediates, it is proposed that metabolism of 1,2-diphenylethanone is initiated by two hydroxylations on the benzyl ring. Phenol, presumably arising from the benzoyl ring, was transiently detected as a catabolic intermediate.

Benzoin↗

PubMed1993

Extracellular Enzyme Production and Synthetic Lignin Mineralization by Ceriporiopsis subvermispora.

The ability of the white rot fungus Ceriporiopsis subvermispora to mineralize C-synthetic lignin was studied under different culture conditions, and the levels of two extracellular enzymes were monitored. The highest mineralization rates (28% after 28 days) were obtained in cultures containing a growth-limiting amount of nitrogen source (1.0 mM ammonium tartrate); under this condition, the levels of manganese peroxidase (MnP) and laccase present in the culture supernatant solutions were very low compared with cultures containing 10 mM of the nitrogen source. In contrast, cultures containing a limiting concentration of the carbon source (0.1% glucose) showed low levels of both enzymes and also very low mineralization rates compared with cultures containing 1% glucose. Cultures containing 11 ppm of Mn(II) showed a higher rate of mineralization than those containing 0.3 or 40 ppm of this cation. Levels of MnP and laccase were higher when 40 ppm of Mn(II) was used. Mineralization rates were slightly higher in cultures flushed daily with oxygen, whereas laccase levels were lower and MnP levels were approximately the same as in cultures maintained under an air atmosphere. The presence of 0.4 mM veratryl alcohol reduced both mineralization rates and MnP levels, without affecting laccase levels. Lignin peroxidase activity was not detected under any condition. Addition of purified lignin peroxidase to the cultures in the presence or absence of veratryl alcohol did not enhance mineralization rates significantly.

Journal Article↗

PubMed1991

Limited bacterial mineralization of fungal degradation intermediates from synthetic lignin.

The ability of selected bacterial strains and consortia to mineralize degradation intermediates produced by Phanerochaete chrysosporium from 14C-labeled synthetic lignins was studied. Three different molecular weight fractions of the intermediates were subjected to the action of the bacteria, which had been grown on a lignin-related dimeric compound. Two consortia isolated from wood being decayed naturally by a Ganoderma species of white rot fungus (the palo podrido system) mineralized 10 to 11% of the fraction with a molecular weight of approximately 500 but less than 4% of the higher- and lower-molecular-weight fractions. The consortia mineralized 5 to 9% of the original lignins. The ability of two pseudomonads isolated earlier from lignin-rich environments to mineralize the original lignins or fungus degradation products was much lower.

Bacteria↗