[Show abstract][Hide abstract] ABSTRACT: Brazil is a major producer of agro-industrial residues, such as sugarcane bagasse, which could be used as raw material for microbial production of cellulases as an important strategy for the development of sustainable processes of second generation ethanol production. For this purpose, this work aimed at screening for glycosyl hydrolase activities of fungal strains isolated from the Brazilian Cerrado. Among 13 isolates, a Trichoderma harzianum strain (L04) was identified as a promising candidate for cellulase production when cultured on in natura sugarcane bagasse. Strain L04 revealed a well-balanced cellulolytic complex, presenting fast kinetic production of endoglucanases, exoglucanases and β-glucosidases, achieving 4,022, U.L(-1) (72 h), 1,228 U.L(-1) (120 h) and 1,968 U.L(-1) (48 h) as the highest activities, respectively. About 60% glucose yields were obtained from sugarcane bagasse after 18 hours hydrolysis. This new strain represents a potential candidate for on-site enzyme production using sugarcane bagasse as carbon source.
[Show abstract][Hide abstract] ABSTRACT: Used for millennia to produce beverages and food, Saccharomyces cerevisiae also became a workhorse in the production of biofuels, most notably bioethanol. Yeast strains have acquired distinct characteristics that are the result of evolutionary adaptation to the stresses of industrial ethanol production. JP1 is a dominant industrial S. cerevisiae strain isolated from a sugarcane mill and is becoming increasingly popular for bioethanol production in Brazil. In this work, we carried out the genetic characterization of this strain and developed a set of tools to permit its genetic manipulation. Using flow cytometry, mating type, and sporulation analysis, we verified that JP1 is diploid and homothallic. Vectors with dominant selective markers for G418, hygromycin B, zeocin, and ρ-fluoro-DL-phenylalanine were used to successfully transform JP1 cells. Also, an auxotrophic ura3 mutant strain of JP1 was created by gene disruption using integration cassettes with dominant markers flanked by loxP sites. Marker excision was accomplished by the Cre/loxP system. The resulting auxotrophic strain was successfully transformed with an episomal vector that allowed green fluorescent protein expression.
Journal of Industrial Microbiology 08/2012; 39(11):1673-83. · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial peptides (AMPs) are effective antibiotic agents commonly found in plants, animals, and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. It is vital to understand the molecular details that define the mechanism of action of resistance to AMPs for a rational planning of the next antibiotic generation and also to shed some light on the complex AMP mechanism of action. Here, the antibiotic resistance of Escherichia coli ATCC 8739 to magainin I was evaluated in the cytosolic subproteome. Magainin-resistant strains were selected after 10 subsequent spreads at subinhibitory concentrations of magainin I (37.5 mg · liter⁻¹), and their cytosolic proteomes were further compared to those of magainin-susceptible strains through two-dimensional electrophoresis analysis. As a result, 41 differentially expressed proteins were detected by in silico analysis and further identified by tandem mass spectrometry de novo sequencing. Functional categorization indicated an intense metabolic response mainly in energy and nitrogen uptake, stress response, amino acid conversion, and cell wall thickness. Indeed, data reported here show that resistance to cationic antimicrobial peptides possesses a greater molecular complexity than previously supposed, resulting in cell commitment to several metabolic pathways.
Antimicrobial Agents and Chemotherapy 01/2012; 56(4):1714-24. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since the discovery of the apoptotic pathway in Saccharomyces cerevisiae, several compounds have been shown to cause apoptosis in this organism. While the toxicity of polyunsaturated fatty acids (PUFA) peroxides towards S. cerevisiae has been known for a long time, studies on the effect of nonoxidized PUFA are scarce. The present study deals specifically with linoleic acid (LA) in its nonoxidized form and investigates its toxicity to yeast. Saccharomyces cerevisiae is unable to synthesize PUFA, but can take up and incorporate them into its membranes. Reports from the literature indicate that LA is not toxic to yeast cells. However, we demonstrated that yeast cell growth decreased in cultures treated with 0.1 mM LA for 4 h, and 3-(4,5 dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (a measure of respiratory activity) decreased by 47%. This toxicity was dependent on the number of cells used in the experiment. We show apoptosis induction by LA concomitant with increases in malondialdehyde, glutathione content, activities of catalase and cytochrome c peroxidase, and decreases in two metabolic enzyme activities. While the main purpose of this study was to show that LA causes cell death in yeast, our results indicate some of the molecular mechanisms of the cell toxicity of PUFA.
FEMS Yeast Research 04/2011; 11(5):408-17. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH(4) (+) and inhibited by Cu(+2) and Hg(+2). Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to K(m) values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1(w), points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels.
[Show abstract][Hide abstract] ABSTRACT: Urate oxidase (EC 126.96.36.199) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of approximately 60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37 degrees C, respectively, and retained 90% of its activity after 72 hours of incubation at -20 degrees C and 4 degrees C.
BioMed Research International 01/2010; 2010:674908. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The National Alcohol Program--PróAlcool, created by the government of Brazil in 1975 resulted less dependency on fossil fuels. The addition of 25% ethanol to gasoline reduced the import of 550 million barrels oil and also reduced the emission CO(2) by 110 million tons. Today, 44% of the Brazilian energy matrix is renewable and 13.5% is derived from sugarcane. Brazil has a land area of 851 million hectares, of which 54% are preserved, including the Amazon forest (350 million hectares). From the land available for agriculture (340 million hectares), only 0.9% is occupied by sugarcane as energy crop, showing a great expansion potential. Studies have shown that in the coming years, ethanol yield per hectare of sugarcane, which presently is 6000 L/ha, could reach 10,000 L/ha, if 50% of the produced bagasse would be converted to ethanol. This article describes the efforts of different Brazilian institutions and research groups on second generation bioethanol production, especially from sugarcane bagasse.
[Show abstract][Hide abstract] ABSTRACT: The yeast Cryptococcus flavus secretes a glycosylated alpha-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 microg mL(-1) affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 microg mL(-1) TM. SDS-PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K(M) values of native and non-glycosylated alpha-amylases were 0.052 and 0.098 mg mL(-1), and V(max) values were 0.038 and 0.047 mg min(-1), respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg(2+) and Cu(2+) suggesting that N-glycosylation is involved in the folding of Amy1.
Carbohydrate research 07/2009; 344(13):1682-6. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to characterize the xylanolytic activity of Cryptococcus flavus isolate I-11. This microorganism was isolated from the Brazilian Cerrado, and enzyme plate assays showed that it also produces amylase and CMCase activity. The xylanolytic production of C. flavus isolate I-11 was improved by using a suitable combination of the carbon and nitrogen sources, reaching 130 U/mL. A zymogram assay was performed showing three xylanase activity bands. The cDNA of one xylanase gene, CfXYN1, was obtained and preliminary expression analysis was performed on RNA samples collected after yeast growth on different carbon sources. This indicated that the CfXYN1 gene is transcribed in the presence of xylose, sugar cane bagasse and carboxymethyl cellulose, but not in the presence of glucose, as carbon source. The cDNA of CfXYN1 was cloned and expressed in Saccharomyces cerevisiae. The recombinant enzyme was partially characterized and showed an optimum at a pH of 3.0 and temperature of 50 °C. The recombinant enzyme retained 70% of its initial activity after pre-incubation for 30 min at the optimum pH and temperature. Computational analysis predicted a molecular weight of 21.2 kDa, and an isoelectric point of 7.02. The Cfxyn1p has 209 amino acids, including a signal peptide consisting of 16 amino acids.
Journal of Molecular Catalysis B: Enzymatic. 01/2009;
[Show abstract][Hide abstract] ABSTRACT: Kexin-like proteins are proteinases belonging to the subtilase family which are involved in the processing of pro-proteins to their active forms. In fungi, kexin-like proteins are involved in several important cellular processes, including mating and dimorphism. Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis undergoes a thermo-regulated dimorphic transition which is essential for the establishment of the infection. Although the molecular mechanisms which rule this process are still unknown, several genes identified in P. brasiliensis have been implicated in dimorphism, including kex2, a kexin-like protein. In this work we have used the baker's yeast Saccharomyces cerevisiae as a host to perform heterologous expression analysis of the P. brasiliensis kex2 gene. Our data shows that kex2 can complement the functions of a S. cerevisiae kex2 mutant strain and could therefore be considered its functional homologue.
Medical Mycology 07/2008; 46(4):385-8. · 1.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yeasts can metabolize xylose by the action of two key enzymes: xylose reductase and xylitol dehydrogenase. In this work, we present data concerning the cloning of the XYL2 gene encoding xylitol dehydrogenase from the yeast Candida tropicalis. The gene is present as a single copy in the genome and is controlled at the transcriptional level by the presence of the inducer xylose. XYL2 was functionally tested by heterologous expression in Saccharomyces cerevisiae to develop a yeast strain capable of producing ethanol from xylose. Structural analysis of C. tropicalis xylitol dehydrogenase, Xyl2, suggests that it is a member of the medium-chain dehydrogenase (MDR) family. This is supported by the presence of the amino acid signature [GHE]xx[G]xxxxx[G]xx[V] in its primary sequence and a typical alcohol dehydrogenase Rossmann fold pattern composed by NAD(+) and zinc ion binding domains.
Applied Microbiology and Biotechnology 01/2007; 73(3):631-9. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yeast cells of the human pathogenic fungus Paracoccidioides brasiliensis strain Pb01 were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved by electroporation, with intact or linearized plasmid DNA. The fungus was transformed using 200 mM manitol, 5 or 7 kV/cm field strength, 25 microF capacitance, 400 omega resistance, 5 microg plasmid DNA and 10(7) yeast cells in 400 microl, and selected in BHI medium overlaid with 30 microg/ml hygromycin B (hygB). Mitotic stability was assessed by growing transformants on non-selective BHI medium, followed by plating on hygromycin B (30 microg/ml). Transformants were analyzed by PCR and Southern blotting, confirming the hph gene integration into the transformants genome. A low level of stability of the integrated hph sequence in the transformant genomes was observed, probably because of the multinuclearity of P. brasiliensis yeast cells.
Medical Mycology 01/2006; 43(8):719-23. · 1.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the cloning of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris by a PCR approach. The coding sequence of the PGK1 gene comprises 1251 bp with the potential to encode a polypeptide of 416 amino acid residues, which shows high identity to homologous proteins from other yeasts. The promoter region of this gene (P(PGK1)) contains regulatory cis-elements found in other PGK1 genes, such as TATA box, CT-rich block and a heat shock element. In the 3' downstream region we identified a tripartite element 5'-TAG-TAGT-TTT-3', which is supposed to be important for transcription termination. As in other yeasts, the PGK1 gene from P. pastoris is present as a single-copy gene. Northern blot analysis revealed that the gene is transcribed as a 1.5 kb mRNA; when cells are grown on glucose the levels of this mRNA are increased two-fold in comparison to cells grown on glycerol. The transcriptional regulation of this gene by the carbon source was further confirmed when the alpha-amylase gene from Bacillus subtilis was placed under the control of P(PGK1): higher levels of expression were obtained when cells were grown on glucose as compared to glycerol and methanol. Preliminary results related to the strength of P(PGK1) show that it represents a potential alternative to constitutive heterologous expression in P. pastoris.
[Show abstract][Hide abstract] ABSTRACT: DNA replication, together with repair mechanisms and cell cycle control, are the most important cellular processes necessary to maintain correct transfer of genetic information to the progeny. These processes are well conserved throughout the Eukarya, and the genes that are involved provide essential information for understanding the life cycle of an organism. We used computational tools for data mining of genes involved in these processes in the pathogenic fungus Paracoccidiodes brasiliensis. Data derived from transcriptome analysis revealed that the cell cycle of this fungus, as well as DNA replication and repair, and the recombination machineries, are highly similar to those of the yeast Saccharomyces cerevisiae. Among orthologs detected in both species, there are genes related to cytoskeleton structure and assembly, chromosome segregation, and cell cycle control genes. We identified at least one representative gene from each step of the initiation of DNA replication. Major players in the process of DNA damage and repair were also identified.
[Show abstract][Hide abstract] ABSTRACT: A β-glucosidase gene (bgl4) from Humicola grisea var thermoidea was successfully expressed in Saccharomyces cerevisiae. The recombinant protein (BGL4
) was initially detected associated with yeast cells and later in the culture medium. BGL4
showed optimal pH and temperature of 6.0 and 40 °C, respectively, and an apparent molecular mass of 57kDa. The enzyme showed
activity against cellobiose and synthetic substrates, and was inhibited more than 80% by Fe2+, Cu2+, Zn2+, and Al3+. Using p-nitrophenyl-β-d-glucopyranoside (pNPG) as substrate, BGL4
presented a V
max of 6.72μmolmin−1 mg total protein−1 and a K
m of 0.16mM under optimal conditions. Most important, BGL4
is resistant to inhibition by glucose and the calculated K
i value for this sugar is 70mM. This feature prompts BLG4
as an ideal enzyme to be used in the saccharification process of lignocellulosic materials for ethanol production.
Humicola grisea var. thermoidea
-Glucose tolerance-Biomass conversion
Applied Biochemistry and Biotechnology 160(7):2036-2044. · 1.89 Impact Factor