Production and action pattern of inulinase from Aspergillus Niger-245: hydrolysis of inulin from several sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evidence of thermostable amylolytic activity from Rhizopus microsporus var. rhizopodiformis using wheat bran and corncob as alternative carbon source

Rhizopus microsporus var. rhizopodiformis produced high levels of alpha-amylase and glucoamylase under solid state fermentation, with several agricultural residues, such as wheat bran, cassava flour, sugar cane bagasse, rice straw, corncob and crushed corncob as carbon sources. These materials were humidified with distilled water, tap water, or saline solutions-Segato Rizzatti (SR), Khanna or Vogel. The best substrate for amylase production was wheat bran with SR saline solution (1:2 v/v). Amylolytic activity was still improved (14.3%) with a mixture of wheat bran, corncob, starch and SR saline solution (1:1:0.3:4.6 w/w/w/v). The optimized culture conditions were initial pH 5, at 45 degrees C during 6 days and relative humidity around 76%. The crude extract exhibited temperature and pH optima around 65 degrees C and 4-5, respectively. Amylase activity was fully stable for 1 h at temperatures up to 75 degrees C, and at pH values between 2.5 and 7.5.

Xylanase production by Bacillus circulans D1 using maltose as carbon source

Bacillus circulans D1 is a good producer of extracellular thermostable xylanase. Xylanase production in different carbon sources was evaluated and the enzyme synthesis was induced by various carbon sources. It was found that D-maltose is the best inducer of the enzyme synthesis ( 7.05 U/ mg dry biomass at 48 h), while D-glucose and D-arabinose lead to the production of basal levels of xylanase. The crude enzyme solution is free of cellulases, even when the microorganism was cultivated in a medium with D-cellobiose. When oat spelt xylan was supplemented with D-glucose, the repressive effect of this sugar on xylanase production was observed at 24 h, only when used at 5.0 g/ L, leading to a reduction of 60% on the enzyme production. on the other hand, when the xylan medium was supplemented with D- xylose ( 3.0 or 5.0 g/ L), this effect was more evident ( 80 and 90% of reduction on the enzyme production, respectively). Unlike that observed in the xylan medium, glucose repressed xylanase production in the maltose medium, leading to a reduction of 55% on the enzyme production at 24 h of cultivation. Xylose, at 1.0 g/ L, induced xylanase production on the maltose medium. on this medium, the repressive effect of xylose, at 3.0 or 5.0 g/ L, was less expressive when compared to its effect on the xylan medium.

Production and characterization of poly-(3-hydroxybutyrate) from recombinant Escherichia coli grown on cheap renewable carbon substrates

Although the biopolymer poly-(3-hydroxybutyrate), P[3HB], presents physicochemical properties that make it an alternative material to conventional plastics, its biotechnological production is quite expensive. As carbon substrates contribute greatly to P[3HB] production cost, the utilization of a cheaper carbon substrate and less demanding micro-organisms should decrease its cost. In the present study a 23 factorial experimental design was applied, aiming to evaluate the effects of using hydrolysed corn starch (HCS) and soybean oil (SBO) as carbon substrates, and cheese whey (CW) supplementation in the mineral medium (MM) on the responses, cell dried weigh (DCW), percentage P[3HB] and mass P[3HB] by recombinant Escherichia coli strains JM101 and DH10B, containing the P[3HB] synthase genes from Cupriavidus necator (ex-Ralstonia eutropha). The analysis of effects indicated that the substrates and the supplement and their interactions had positive effect on CDW. Statistically generated equations showed that, at the highest concentrations of HCS, SO and CW, theoretically it should be possible to produce about 2 g L(1) DCW, accumulating 50% P[3HB], in both strains. To complement this study, the strain that presented the best results was cultivated in MM added to HCS, SBO and CW ( in best composition observed) and complex medium (CM) to compare the obtained P[3HB] in terms of physicochemical parameters. The obtained results showed that the P[3HB] production in MM (1.29 g L(-1)) was approximately 20% lower than in CM (1.63 g L(-1)); however, this difference can be compensated by the lower cost of the MM achieved by the use of cheap renewable carbon sources. Moreover, using differential scanning calorimetry and thermogravimetry analyses, it was observed that the polymer produced in MM was the one which presented physicochemical properties (Tg and Tf) that were more similar to those found in the literature for P[3HB].

Carbon and nitrogen stock and fluxes in coastal Atlantic Forest of southeast Brazil: potential impacts of climate change on biogeochemical functioning

The Atlantic Forest is one of the most important biomes of Brazil. Originally covering approximately 1.5 million of km(2), today this area has been reduced to 12% of its original size. Climate changes may alter the structure and the functioning of this tropical forest. Here we explore how increases in temperature and changes in precipitation distribution could affect dynamics of carbon and nitrogen in coastal Atlantic Forest of the southeast region of Brazil The main conclusion of this article is that the coastal Atlantic Forest has high stocks of carbon and nitrogen above ground, and especially, below ground. An increase in temperature may transform these forests from important carbon sinks to carbon sources by increasing loss of carbon and nitrogen to the atmosphere. However, this conclusion should be viewed with caution because it is based on limited information. Therefore, more studies are urgently needed to enable us to make more accurate predictions.

A trophic study of the sympatric Amazonian freshwater turtles Podocnemis unifilis and Podocnemis expansa (Testudines, Podocnemidae) using carbon and nitrogen stable isotope analyses

The Yellow-spotted River Turtle (Podocnemis unifilis Troschel, 1848) and the South American River Turtle (Podocnemis expansa (Schweigger, 1812)) are two turtles species that are widely distributed and have ecological, economic, and cultural importance in the Amazon basin. Although sympatric regarding most of their distribution, few studies have addressed the coexistence of these two species. To examine this, we analyzed the trophic level and the primary carbon source from the diets of both species in Baixo Araguaia, Tocantins, Brazil, using stable isotope analyses of carbon (delta C-13) and nitrogen (delta N-15). We also verified possible intraspecific variations (related to sex and body mass) in the trophic levels and primary carbon sources of their diets. Podocnemis unifilis had higher values of delta N-15 than P. expansa, averaging 7.59 parts per thousand and 5.06 parts per thousand, respectively, a difference which may indicate a possible trophic change owing to exploiting different food resources. No differences were found between the two species in relation to delta C-13 (mean values of -26.2 parts per thousand and -26.1 parts per thousand, respectively). The similarity between delta C-13 values suggests that the sources of their basal feeding are the same, consisting mainly of C-3 plants. There was no intraspecific variation in the values of delta C-13 and delta N-15.

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide andO-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. UsingLemna minor, we analyzed the effects of omission of CO2 from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5′-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO2 led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO2 on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO2, APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO2 also recovered both enzyme activities, with OAS again influenced only APR.35SO4 2− feeding showed that treatment in air without CO2 severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of 35S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of 35S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.

Alternative carbon metabolism: Virulence and regulation in Candida albicans

The interaction between C. albicans and innate immune cells is a key determinant to disease progression. Transcriptional profiling showed that C. albicans responds to macrophage phagocytosis by inducing pathways required for alternative carbon metabolism (beta-oxidation, the glyoxylate cycle, and gluconeogenesis), suggesting these pathways are important for virulence of C. albicans. ^ We have shown that deleting key genes (FOX2, FBP1) in these pathways results in virulence defects in an in vivo mouse model for systemic infection. Like icl1Δ/Δ mutants, fbp1Δ/Δ mutants are severely attenuated and fox2Δ/Δ mutants are mildly but significantly attenuated, indicating that carbon starvation is a relevant stress in vivo. ^ However, fox2Δ/Δ mutants also had unexpected phenotypes on certain carbon sources, unlike the case in Saccharomyces cerevisiae, suggesting these pathways are regulated differently in C. albicans. To test this, we identified the C. albicans regulators of these pathways based on those from S. cerevisiae and Aspergillus nidulans. ^ C. albicans has a partly conserved framework, but lacks two regulators (Oaf1p, Pip2p) controlling peroxisome biogenesis and beta-oxidation genes in yeast. Instead, C. albicans has a homolog, CTF1, of the A. nidulans fatty acid catabolism regulators FarA and FarB. We have shown that CTF1 is needed for growth on oleate (like FarA and FarB), expression of beta-oxidation and glyoxylate cycle genes, and full virulence. No function for CTF1 has previously been identified in C. albicans. Our data demonstrate a role for alternative carbon metabolism in the virulence of C. albicans and suggest that the regulation of these pathways is a mixture of the filamentous fungi and budding yeast systems. ^

REGULATION OF ALTERNATIVE CARBON METABOLISM IN CANDIDA ALBICANS

Candida albicans is the most important fungal pathogen of humans. Transcript profiling studies show that upon phagocytosis by macrophages, C. albicans undergoes a massive metabolic reorganization activating genes involved in alternative carbon metabolism, including the glyoxylate cycle, β-oxidation and gluconeogenesis. Mutations in key enzymes such as ICL1 (glyoxylate cycle) and FOX2 (fatty acid β-oxidation) revealed that alternative carbon metabolic pathways are required for full virulence in C. albicans. These studies indicate C. albicans uses non-preferred carbon sources allowing its adaptation to microenvironments were nutrients are scarce. It has become apparent that the regulatory networks required for regulation of alternative carbon metabolism in C. albicans are considerably different from the Saccharomyces cerevisiae paradigm and appear more analogous to the Aspergillus nidulans systems. Well-characterized transcription factors in S. cerevisiae have no apparent phenotype or are missing in C. albicans. CTF1 was found to be a single functional homolog of the A. nidulans FarA/FarB proteins, which are transcription factors required for fatty acid utilization. Both FOX2 and ICL1 were found to be part of a large CTF1 regulon. To increase our understanding of how CTF1 regulates its target genes, including whether regulation is direct or indirect, the FOX2 and ICL1 promoter regions were analyzed using a combination of bioinformatics and promoter deletion analysis. To begin characterizing the FOX2 and ICL1 promoters, 5’ rapid amplification of cDNA ends (5’RACE) was used to identify two transcriptional initiation sites in FOX2 and one in ICL1. GFP reporter assays show FOX2 and ICL1 are rapidly expressed in the presence of alternative carbon sources. Both FOX2 and ICL1 harbor the CCTCGG sequence known to be bound by the Far proteins, hence rendering the motif as a putative CTF1 DNA binding element. In this study, the CCTCGG sequence was found to be essential for FOX2 regulation. However, this motif does not appear to be equally important for the regulation of ICL1. This study supports the notion that although C. albicans has diverged from the paradigms of model fungi, C. albicans has made specific adaptations to its transcription-based regulatory network that may contribute to its metabolic flexibility.