Enzimas ligninolíticas en Fomes sclerodermeus

• Autores: Papinutti, Víctor Leandro
• Año de publicación: 2003
• Idioma: español castellano
• Tipo de recurso: tesis doctoral
• Colaborador/a o director/a de tesis: Forchiassin, Flavia
• Institución: Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales

White rot fungi are the only organisms capable to mineralize the lignin to CO2. These fungiposses at least three enzymes impicated in the process: lignin peroxidase, manganese dependentperoxidase and laccase. The screening of 13 strains of white-rot fungi showed that F.sclerodermeus was the more efficient ligninase producer fungus and the decolorization of thepolymeric dye poly R was fast. Lip activity was not detected in any of the media tested. Optimal media conditions for laccase and MnP production in synthetic liquid media were found. The Doehlert uniform factorial experimental design was applied to study the effect of MnSO4, CuSO4 and asparagine. Among the aromatic compounds evaluated in GA medium, vanillin andfenilic acid were the more efficient laccase and MnP inducers. On the other hand, insemidefined medium (YPG) aromatic compounds had not effect on any activity. Under thisconditions only copper and Mn increased laccase and MnP activities, respectively. Solid state fermentation on wheat or soy bran, high laccase and MnP were produced. Neitheraromatic compounds, MnSO4 nor CuSO4 increased none of the activities. The effect on known ligninolytic inducers was studied on a crystalline cellulose (CC) basedmedium. Cellulolytic activities decreased 3-fold when CuSO4 or MnSO4 were added to themedium. MnP activity was only detected in the media containing MnSO4 as inducer, while thelaccase activity was constitutiver expressed under all the conditions tested, and it was increasedto a major extent when copper was added to the medium. Wood blocks of poplar incubated during six months with F. sclerodermeus registered dryweight losses of 51%. Differential hydrolysis of the wood blocks revealed that the remainingcontent of cellulose and lignin were 58 y 56%, respectively. On the other hand, thehydrosolubles increased 3-fold, due to the degradation products. Observation of the woodshowed the degradation of parenchyma rays and thinning of the cell walls. Growing on sawdustmedium, enzyme activities were produced only with poplar, whereas on cedar laccase and MnPwere inhibited. Laccase and MnP produced in YPG medium by F. sclerodcrmeus were purified toelectrophoretic homogeneity and characterized biochemically. Two isoenzymes of laccase (Lac I y Lac II) and two MnP (MnP I y MnP II) were found. Isoforms of both systems showed slightdifferences in their isoelectric points. Laccase activity produced on wheat bran based medium revealed three bands in PAGE withdifferential patterns of inactivation. Among the compounds tested, the CuSO4 1,25 mM was thebest stabilizer. The combination of both CuSO4 and glycerol 0,2% further increased the stabilityof the enzyme. Laccase activity produced in the optimized conditions was used in assays of decoloration anddetoxification of the fungicide malachite green. P. chrysosporium was used as biotest, this Fungus showed a high sensitivity to the fungicide, while degradation by F. sclerodermeuslaccase rendered a not toxic compound. The presence of I-HBT accelerated the detoxification. The detoxification response of other white-rot fungi was similar to that observed for P.chrysosporium.
Fil: Papinutti, Víctor Leandro. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.