RESUMO
KEY MESSAGE: The gene coding for F5H from Eucalyptus globulus was cloned and used to transform an f5h -mutant of Arabidopsis thaliana , which was complemented, thus verifying the identity of the cloned gene. Coniferaldehyde 5-hydroxylase (F5H; EC 1.14.13) is a cytochrome P450-dependent monooxygenase that catalyzes the 5-hydroxylation step required for the production of syringyl units in lignin biosynthesis. The Eucalyptus globulus enzyme was characterized in vitro, and results showed that the preferred substrates were coniferaldehyde and coniferyl alcohol. Complementation experiments demonstrated that both cDNA and genomic constructs derived from F5H from E. globulus under the control of the cinnamate 4-hydroxylase promoter from Arabidopsis thaliana, or a partial F5H promoter from E. globulus, can rescue the inability of the A. thaliana fah1-2 mutant to accumulate sinapate esters and syringyl lignin. E. globulus is a species widely used to obtain products that require lignin removal, and the results suggest that EglF5H is a good candidate for engineering efforts aimed at increasing the lignin syringyl unit content, either for kraft pulping or biofuel production.
Assuntos
Acroleína/análogos & derivados , Arabidopsis/enzimologia , Eucalyptus/enzimologia , Lignina/metabolismo , Oxigenases de Função Mista/genética , Acroleína/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sistema Enzimático do Citocromo P-450/genética , Eucalyptus/genética , Expressão Gênica , Cinética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Mutação , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1-6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.
Assuntos
Biotecnologia/métodos , Coriolaceae/metabolismo , Recuperação e Remediação Ambiental/métodos , Ganoderma/metabolismo , Corantes/metabolismo , Coriolaceae/enzimologia , Proteínas Fúngicas/metabolismo , Ganoderma/enzimologia , Lacase/metabolismo , Lignina/metabolismo , Peroxidases/metabolismoRESUMO
Ganoderma australe is a basidiomycete responsible for a natural process of selective and extensive lignin degradation. Fatty acids, thiobarbituric acid reactive substances (TBARS), Fe3+-reduction and enzymatic activities were monitored in cultures of G. australe growing on Drimys winteri wood chips. Linoleic acid was de novo synthesized, and steadily increased during 12 weeks of cultivation. Part of the unsaturated fatty acids underwent peroxidation as TBARS accumulated with biodegradation time. TBARS accumulation was proportional to the wood weight and component losses. Manganese-dependent peroxidase and lignin peroxidase were not detected in the culture extracts, whereas laccase-induced oxidation of syringaldazine peaked after 2 weeks (104+/-9 micromol oxidized min(-1) kg(-1) of dry wood), subsequently decreasing. On the other hand, nonenzymatic Fe3+-reducing activity increased as a function of cultivation time and could be involved in the initiation of lipid peroxidation.
