RESUMO
Four cutinase genes are encoded in the genome of the saprophytic fungus Aspergillus nidulans, but only two of them have proven to codify for active cutinases. However, their overall roles in cutin degradation are unknown, and there is scarce information on the regulatory effectors of their expression. In this work, the expression of the cutinase genes was assayed by multiplex qRT-PCR in cultures grown in media containing both inducer and repressor carbon sources. The genes ancut1 and ancut2 were induced by cutin and its monomers, while ancut3 was constitutively expressed. Besides, cutin induced ancut4 only under oxidative stress conditions. An in silico analysis of the upstream regulatory sequences suggested binding regions for the lipid metabolism transcription factors (TF) FarA for ancut1 and ancut2 while FarB for ancut3. For ancut4, the analysis suggested binding to NapA (the stress response TF). These binding possibilities were experimentally tested by transcriptional analysis using the A. nidulans mutants ANΔfarA, ANΔfarB, and ANΔnapA. Regarding cutin degradation, spectroscopic and chromatographic methods showed similar products from ANCUT1 and ANCUT3. In addition, ANCUT1 produced 9,10-dihydroxy hexadecanoic acid, suggesting an endo-cleavage action of this enzyme. Regarding ANCUT2 and ANCUT4, they produced omega fatty acids. Our results confirmed the cutinolytic activity of the four cutinases, allowed identification of their specific roles in the cutinolytic system and highlighted their differences in the regulatory mechanisms and affinity towards natural substrates. This information is expected to impact the cutinase production processes and broaden their current biotechnological applications.
Assuntos
Aspergillus nidulans/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lipídeos de Membrana/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Hidrolases de Éster Carboxílico/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Biochemical characterization of purified ANCUT2 cutinase from Aspergillus nidulans is described. The identified amino acid sequence differs from that predicted in Aspergillus genomic databases in amino acids not relevant for catalysis. The enzyme is thermo-alkaline, showing its maximum activity at pH 9 and 60 °C, and it retains more than 60% of its initial activity after incubation for 1 h at 60 °C for pH values between 6 and 10. ANCUT2 is more active towards long-chain esters and it hydrolyzes cutin; however, it also hydrolyzes short-chain esters. Cutinase is inhibited by metal ions, PMSF, SDS, and EDTA (10 mM). It retains 50% of its activity in most of the solvents tested, although it is more stable in hydrophobic solvents. According to its found biochemical properties, preliminary assays demonstrate its ability to synthesize methyl esters from sesame oil and the most likely application of this enzyme remains in detergent formulations.