Browsing by Author "SALAS, L"

Now showing 1 - 4 of 4
  • No Thumbnail Available
    Item
    ISOENZYMES OF MANGANESE-DEPENDENT PEROXIDASE AND LACTASE PRODUCED BY THE LIGNIN-DEGRADING BASIDIOMYCETE CERIPORIOPSIS-SUBVERMISPORA
    (1994) LOBOS, S; LARRAIN, J; SALAS, L; CULLEN, D; VICUNA, R
    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 revels 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. Immunoblotting studies showed that the molecular mass of MnP isoenzymes from liquid cultures was about 52.5 kDa, whereas the molecular masses of MnPs extracted from wood varied from 52.5 kDa to 62.5 kDa upon ageing of the cultures. The amino terminal sequences of seven MnP isoenzymes were determined. The consensus sequences of MnPs from liquid and solid cultures were clearly distinct, although both showed homology to MnPs from related white-rot fungi.
  • No Thumbnail Available
    Item
    LIGNINOLYTIC ENZYMES OF THE WHITE ROT BASIDIOMYCETES PHLEBIA-BREVISPORA AND CERIPORIOPSIS-SUBVERMISPORA
    (1992) RUTTIMANN, C; SCHWEMBER, E; SALAS, L; CULLEN, D; VICUNA, R
  • No Thumbnail Available
    Item
    MICROBIAL AND BIOCHEMICAL-CHARACTERIZATION OF A BACTERIAL CONSORTIUM ISOLATED FROM DECAYING WOOD BY GROWTH ON A BETA-O-4 LIGNIN-RELATED DIMERIC COMPOUND
    (1992) CESPEDES, R; SALAS, L; CALDERON, I; GONZALES, B; VICUNA, R
    As an approach to evaluate the contribution of bacteria to lignin degradation in wood, we have chosen to study these microorganisms in the natural wood decay ecosystem known as Palo Podrido. Initially, the characterization of bacteria able to metabolize lignin-related compounds present in samples of Palo Podrido was undertaken. For their isolation, minimal salt media containing lignin dimers of either the arylglycerol-beta-aryl ether (beta-O-4) or 1,2-diarylpropane (beta-1) types as the only source of carbon and energy were inoculated with various wood samples exhibiting different degrees of decay. The beta-1 dimers used failed to support bacterial growth. However, three bacterial consortia able to consume quantitatively the beta-O-4 model 1-[3,4-dimethoxyphenyl]-2-[2-methoxyphenoxy]-3-hydroxypropanone (compound I) were isolated. One of these was further characterized. It is composed of eight strains belonging to the families of Streptomycetaceae, Dermatophilaceae and Actinoplanaceae. HPLC and GC-MS analyses revealed that the consortium utilizes two pathways to degrade beta-O-4 dimers, both involving direct cleavage of the ether linkage. The formation of a novel C6-C3 degradation intermediate is described. Some metabolic properties of each strain, as well as those of the intact consortium, are also reported.
  • No Thumbnail Available
    Item
    PROPERTIES OF LACCASE ISOENZYMES PRODUCED BY THE BASIDIOMYCETE CERIPORIOPSIS-SUBVERMISPORA
    (1995) SALAS, C; LOBOS, S; LARRAIN, J; SALAS, L; CULLEN, D; VICUNA, R
    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 Ran(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 nn, 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.