Structural and Gene Characterization of a New Antifungal Peptide Obtained from Penicillium crustosum FP11 Strain

A novel antifungal peptide, PcAFP (6.48 kDa, pI 8.83 ), was obtained from the culture supernatant of the fungus Penicillium crustosum . The gene encoding the PcAFP peptide was isolated b ased on its homologue in Penicillium chrysogenum , PgAFP. PcAFP is a small, cystine-rich peptide, and th e mature peptide consists of 58 amino acid residues. The i mmature P. crustosum antifungal protein (AFP) showed 95.65% identity to the antifungal prote in of P. chrysogenum , while the mature peptide showed 98.28% identity with PgAFP. Molecular modeling of the tertiary structure of the mature peptide revealed details of the conserved stru cture of the AFPs, such as the ? -barrel motif stabilized by three disulfide bonds and the ? -core motif. Analysis of the extract by 16% tricine SD S- PAGE showed a 6.9 kDa peptide, which was close to the pr edicted molecular mass of the mature peptide of 6.48 kDa. Assays of antimicrobial activity , performed by broth microdilution using the crude extract obtained from the culture medium, showe d activity against Candida albicans . These results demonstrate the conservation of the PcAPF gene and the high level of identity with the PgAFP antifungal protein of P. chrysogenum . Given these structural and biochemical characteristics, PcAFP could be a potential candidate for future investigations that may aid in the development of new antifungal compounds.

Enhance of Cellulase Production and Biomass Degradation by Transformation of the Trichoderma reesei RUT-C30∆ zface1 Strain

Abstract The second-generation bioethanol employs lignocellulosic materials degraded by microbial cellulases in their production. The fungus Trichoderma reesei is one of the main microorganisms producing cellulases, and its genetic modification can lead to the optimization in obtaining hydrolytic enzymes. This work carried out the deletion of the sequence that encodes the zinc finger motif of the transcription factor ACE1 (cellulase expression repressor I) of the fungus T. reesei RUT-C30. The transformation of the RUT-C30 lineage was confirmed by amplification of the 989 bp fragment relative to the selection marker, and by the absence of the zinc finger region amplification in mutants, named T. reesei RUT-C30Δzface1. The production of cellulases by mutants was compared to RUT-C30 and measured with substrates carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel®) and Whatman filter paper (PF). The results demonstrated that RUT-C30Δzface1 has cellulolytic activity increased 3.2-fold in Avicel and 2.1-fold in CMC and PF. The mutants presented 1.4-fold higher sugar released in the hydrolysis of the biomass assays. These results suggest that the partial deletion of ace1 gene is an important strategy in achieving bioethanol production on an industrial scale at a competitive price in the fuel market.

Biotechnological potential of alternative carbon sources for production of pectinases by Rhizopus microsporus var. rhizopodiformis

Fungi collected from Brazilian soil and decomposing plants were screened for pectinase production. R. microsporus var. rhizopodiformis was the best producer and was selected to evaluate the pectic enzyme production under several nutritional and environmental conditions. The pectinase production was studied at 40ºC, under 28 carbon sources-supplemented medium. The inducer effect of several agro-industrial residues such as sugar cane bagasse, wheat flour and corncob on polygalacturonase (PG) activity was 4-, 3- and 2-fold higher than the control (pectin). In glucose-medium, a constitutive pectin lyase (PL) activity was detected. The results demonstrated that R. microsporus produced high levels of PG (57.7 U/mg) and PL (88.6 U/mg) in lemon peel-medium. PG had optimum temperature at 65 ºC and was totally stable at 55 ºC for 90 min. Half-life at 70 ºC was 68 min. These results suggested that the versatility of waste carbon sources utilization by R. microsporus, produce pectic enzymes, which could be useful to reduce production costs and environmental impacts related to the waste disposal.