Browsing by Author "VICUNA, R"
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- ItemASSESSMENT OF VARIOUS COMMERCIAL ENZYMES IN THE BLEACHING OF RADIATA PINE KRAFT PULPS(1995) VICUNA, R; OYARZUN, E; OSSES, MFour xylanase preparations that are commercially available, namely Cartazyme from Sandoz, Ecopulp from Alko-ICI, Irgazyme from Ciba-Genencor and Pulpzyme HB from Novo Nordisk, were tested in bleaching experiments of kraft pulps from Pinus radiata. The main objective of this study was to optimize a reduction in the consumption of chlorine dioxide in the bleaching sequences C-90/D(10)E(o)DED, C-70/D(30)E(o)DED and D(100)EDED. Enzymatic treatments led to savings of ClO2 between 3.5 and 3.9 kg per air-dried tons (ADT) in the three bleaching sequences, without affecting the target brightness of the pulps. In these assays, some minor although reproducible differences in the performance of the enzymes were observed. In most cases, xylanase treatment partially affected the beatability of the pulps, measured as the number of revolutions in the PFI mill required to reach the same tensile index as the respective controls.
- ItemBENZALDEHYDE LYASE, A NOVEL THIAMINE PPI-REQUIRING ENZYME, FROM PSEUDOMONAS-FLUORESCENS BIOVAR-I(1989) GONZALEZ, B; VICUNA, RPseudomonas fluorescens biovar I can grow on benzoin as the sole carbon and energy source. This ability is due to benzaldehyde lyase, a new type of enzyme that irreversibly cleaves the acyloin linkage of benzoin, producing two molecules of benzaldehyde. Benzaldehyde lyase was purified 70-fold and found to require catalytic amounts of thiamine PPi (TPP) and a divalent cation as cofactors. Optimal activity was obtained with a 1.0 mM concentration of Mn2+, Mg2+, or Ca2+. Gel permeation chromatography indicated a native molecular weight of 80,000, whereas the enzyme migrated in sodium dodecyl sulfate-containing polyacrylamide gels as a single polypeptide with a molecular weight of 53,000. Benzaldehyde lyase is highly specific; of a variety of structurally related compounds tested, only benzoin and benzoin and anisoin (4,4''-dimethoxybenzoin) acted as substrates, their apparent Kms being 9.0 .times. 10-3 and 3.25 .times. 10-2 mM, respectively. A catalytic mechanism for the enzyme is proposed.
- ItemBIOCHEMICAL AND GENETIC-STUDIES OF BACTERIA METABOLIZING LIGNIN-RELATED COMPOUNDS(1988) VICUNA, R; GONZALEZ, B; RUTTIMANN, C; SAPAG, A; SEELENFREUND, D
- ItemCATABOLISM OF 1,2-DIARYLETHANE LIGNIN MODEL COMPOUNDS BY 2 BROWN-ROT FUNGI(1990) ESPEJO, E; AGOSIN, E; VICUNA, RThe catabolism of dimethoxybenzil, anisoin and hydroanisoin in nitrogen-limited stationary cultures of the brown-rot fungi Wolfiporia cocos and Gloephyllum trabeum was analyzed. These three 1,2-diarylethane lignin model compounds, which differ in the degree of oxidation of the alkylic chain, gave rise to p-anisaldehyde in both cultures, suggesting that cleavage between the two aliphatic carbons had occurred. In turn, both strains reduced dimethoxybenzil and anisoin to hydroanisoin, whereas only Wolfiporia cocos was able to oxidize hydroanisoin to anisoin. On the other hand, chemically derived hydroxyl radical, but not superoxide radical, produced p-anisaldehyde plus other unidentified compounds from anisoin and hydroanisoin. Neither radical modified dimethoxybenzil significantly.
- ItemCONDITIONS AFFECTING DNA CLEAVAGE BY TTHI AT A TTHI ENDONUCLEASE-DAM METHYLASE OVERLAPPING SEQUENCE(1981) VENEGAS, A; MOTLES, M; VASQUEZ, C; VICUNA, R
- ItemDEGRADATION OF DIARYLETHANE STRUCTURES BY PSEUDOMONAS-FLUORESCENS BIOVAR-I(1988) GONZALEZ, B; OLAVE, I; CALDERON, I; VICUNA, RPseudomonas fluorescens biovar I was isolated from a pulp mill effluent based on its ability to grow on synthetic media containing 1,2-diarylethane structures as the sole carbon and energy source. Analysis of samples taken from cultures of this strain in benzoin or 4,4''-dimethoxybenzoin (anisoin), showed that cleavage between the two aliphatic carbons takes place prior to ring fission. Intermonomeric cleavage was also obtained with crude extracts. Substrates of this reaction were only those 1,2-diarylethane compounds that supported growth of the bacterium. The purification and partial characterization of an enzyme that catalyzes the NADH-dependent reduction of the carbonyl group of benzoin and anisoin is also reported.
- ItemDEOXYRIBONUCLEIC-ACID POLYMERASE FROM THE MARINE PSEUDOMONAS SP BAL-31(1980) VICUNA, R; VALDES, F; MEDINA, A; YUDELEVICH, AA DNA-dependent DNA polymerase (DNA nucleotidyltransferase) was purified 3000-fold from the marine Pseudomonas sp. BAL-31. The MW of the native enzyme was estimated by glycerol gradient sedimentation to be 110,000. The enzyme migrated in sodium dodecyl sulfate-acrylamide gels as a single polypeptide with a MW of 105,000. An absolute requirement for divalent cation was satisfied by Mg2+ or Mn2+ at concentrations of 1 mM. Monovalent cations at concentrations higher than 50 mM showed an inhibitory effect. The polymerase activity was resistant to N-ethylmaleimide and showed a wide pH optimum.
- ItemDNA-POLYMERASES FROM THE EXTREMELY THERMOPHILIC BACTERIUM THERMUS-THERMOPHILUS HB-8(1985) RUTTIMANN, C; COTORAS, M; ZALDIVAR, J; VICUNA, R
- ItemEFFECT OF STREPTOMYCES-VIRIDOSPORUS T7A ON KRAFT LIGNIN(1990) SEELENFREUND, D; VICUNA, RThe ability of the lignino-cellulolytic actinomycete Streptomyces virdosprous T7A to attack purified fractions of kraft lignin was examined. In the presence of 0.3% yeast extract, high-molecular weight kraft lignin (MW > 3000, ether-insoluble fraction) does not affect growth of this microorganism significantly, whereas low-molecular weight kraft lignin (MW < 3000, ether-soluble fraction) inhibits its development. Accordingly, average molecular weight of the ether-insoluble fraction after bacterial growth remained unaltered, as measured by Sephadex G-50 gel permeation chromatography. Slight modifications were detected by high performance liquid chromatography in the ether-soluble fraction after incubation with the microorganism. S. viridosporus T7A partially decolorized Remazol Brilliant Blu R during growth on wheat lignocellulose. However, decolorization of either fraction of kraft lignin was not observed. These results suggest that the filamentous bacterium S. viridosporous T7A is not suitable for pulp mill effluent treatment.
- ItemELECTRON-MICROSCOPIC MAPPING OF THERMUS-THERMOPHILUS RNA-POLYMERASE BINDING-SITES ON PLASMID PBR322(1985) GONZALEZ, B; DAVAGNINO, J; VICUNA, RThe binding of RNA polymerase from the extreme thermophile T. thermophilus HB8 to plasmid pBR322 was measured by EM. DNA-protein complexes were prepared at 35 and 60.degree. C. At both temperatures the enzyme binds strongly to sites which coincide with promoters P1, P2, P3 and P4 present in pBR322. At 60.degree. C, an additional binding site appears, which is located between P3 and P4. There is a high degree of correlation between RNA polymerase binding sites and the location of A-T rich regions on pBR322 DNA.
- ItemELECTRON-MICROSCOPY MAPPING OF ESCHERICHIA-COLI RNA POLYMERASE-BINDING SITES ON PLASMIDS FROM THERMOPHILIC BACTERIA(1984) GONZALEZ, B; VASQUEZ, C; BULL, P; VICUNA, RThe binding sites of E. coli RNA polymerase to plasmid DNA from extremely thermophilic bacteria were mapped by EM. Templates used in these studies included plasmids pTF62 (from Thermus flavus AT62) and pTT8 (from T. thermophilus HB8) and hybrid molecules constructed by ligation of these plasmids to pBR322. Although the affinity of the enzyme for heterologous DNA was .apprx. 1/3 of that for pBR322, it was possible to localize preferred binding sites on pTF62 and pTT8. Six binding sites were identified in pTT8, mapping close to 7, 28, 47, 61, 65 and 81 map units (1 unit = 1% of the length of the DNA). Seven such regions located at 3, 27, 48, 60, 67, 81 and 86 map units were found in pTF62. RNA polymerase binding sites found in pBR322 coincided with promoters identified previously by EM analysis of transcriptional complexes prepared in vitro. Evidently, E. coli RNA polymerase binds preferentially to specific sequences in plasmids from thermophilic bacteria, suggesting possible promoter locations in these plasmids.
- ItemISOENZYMES OF MANGANESE-DEPENDENT PEROXIDASE AND LACTASE PRODUCED BY THE LIGNIN-DEGRADING BASIDIOMYCETE CERIPORIOPSIS-SUBVERMISPORA(1994) LOBOS, S; LARRAIN, J; SALAS, L; CULLEN, D; VICUNA, RThe 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.
- ItemLIGNINOLYTIC ENZYMES OF THE WHITE ROT BASIDIOMYCETES PHLEBIA-BREVISPORA AND CERIPORIOPSIS-SUBVERMISPORA(1992) RUTTIMANN, C; SCHWEMBER, E; SALAS, L; CULLEN, D; VICUNA, R
- ItemMETABOLISM OF LIGNIN MODEL COMPOUNDS OF THE ARYLGLYCEROL-BETA-ARYL ETHER TYPE BY PSEUDOMONAS-ACIDOVORANS D3(1987) VICUNA, R; GONZALEZ, B; MOZUCH, MD; KIRK, TKA natural bacterial isolate that we have classified as Pseudomonas acidovorans grows on the lignin model compounds 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1''), as well as on the corresponding 1-oxo compounds (2 and 2'') as sole sources of carbon and energy. Metabolic intermediates present in cultures growing on compound 1 included compound 2,2-methoxyphenol (guaiacol [compound 3]), .beta.-hydroxypropioveratrone (compound 4), acetoveratrone (compound 5), and veratric acid (compound 6). Also identified were compounds 1'', 2'', .beta.-hydroxypropiovanillone (compound 4''), and acetovanillone (compound 5''), indicating that 4-O demethylation also occurs. The phenolic intermediates were the same as those found in cultures growing on compound 1''. Compounds 2 and 2'' were in part also reduced to compounds 1 and 1'', respectively. Compound 3 was shown to be derived from the 2-methoxyphenoxy moiety. A suggested degradation scheme is as follows: compound 1 .fwdarw. 2 .fwdarw. (3 + 4) .fwdarw. 5 .fwdarw. 6 (and similarly for 1''). In this scheme, the key reaction is cleavage of the ether linkage between C-2 (C.beta.) of the phenylpropane moiety and the 2-methoxyphenoxy moiety in compounds 2 and 2'' (i.e., .beta.-aryl ether cleavage). On the basis of compounds identified, viz., 3 and 4 (4''), cleavage appears formally to be reductive. Because this is unlikely, the initial cleavage products probably were not detected. The implications of these results for the enzyme(s) responsible are discussed.
- ItemMETABOLISM OF LOW-MOLECULAR-WEIGHT LIGNIN-RELATED COMPOUNDS BY STREPTOMYCES-VIRIDOSPORUS T7A(1987) RUTTIMANN, C; SEELENFREUND, D; VICUNA, RThe ability of the ligninolytic actinomycete Streptomyces viridosporus T7A to degrade selected lignin model compounds, both in the presence and in the absence of lignocellulose, was examined. Compounds studied included benzyl alcohols and aldehydes, plus dimers possessing intermonomeric linkages, which are characteristic of the lignin macromolecule. Oxidation of veratryl alcohol to the corresponding acid was significant only under ligninolytic growth conditions, i.e., in medium containing lignocellulose, while other benzyl alcohols and aldehydes were readily oxidized in its absence. S. viridosporus T7A reduces carbonylic groups of 1,2-diarylethane, but not of 1,2-diarylpropane structures, under both ligninolytic and non-ligninolytic culture conditions. Cleavage of 1,2-diarylpropane (.beta.-1), arylglycerol-.beta.-arylether (.beta.-0-4) and biphenyl structures by this strain could not be detected under either metabolic conditions.
- ItemMICROBIAL 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, RAs 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.
- ItemMONITORING BACTERIAL CONSUMPTION OF LOW-MOLECULAR-WEIGHT LIGNIN DERIVATIVES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY(1986) GOYCOOLEA, M; SEELENFREUND, D; RUTTIMANN, C; GONZALEZ, B; VICUNA, RThe lignolytic capacity of some natural bacterial isolates was examined. Strains were selected from samples of decaying wood by growth in a minimal medium containing aromatic compounds with a structural relationship to lignin as the sole carbon sources. These included derivatives of benzoic and phenylpropanoic acids, as well as a mixture of low molecular weight compounds obtained by fractionation of kraft lignin. Reversed-phase high-performance liquid chromatography analyses before and after cell growth in the latter revealed a degradation pattern of the different compounds present in the culture which was characteristic for each of the strains studied.
- ItemOXIDATION REACTIONS CATALYZED BY MANGANESE PEROXIDASE ISOENZYMES FROM CERIPORIOPSIS-SUBVERMISPORA(ELSEVIER SCIENCE BV, 1995) URZUA, U; LARRONDO, LF; LOBOS, S; LARRAIN, J; VICUNA, RA 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, K-m 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.
- ItemPHYSICAL CHARACTERIZATION OF A PLASMID (PTT1) ISOLATED FROM THERMUS THERMOPHILUS(1981) EBERHARD, MD; VASQUEZ, C; VALENZUELA, P; VICUNA, R; YUDELEVICH, A
- ItemPLASMID CURING IN THERMUS-THERMOPHILUS AND THERMUS-FLAVUS(1983) VASQUEZ, C; VILLANUEVA, J; VICUNA, R