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The present study compared the production and the catalytic properties of amylolytic enzymes obtained from the fungi Lichtheimia ramosa (mesophilic) and Thermoascus aurantiacus (thermophilic). The highest amylase production in both fungi was observed in wheat bran supplemented with nutrient solution (pH 4.0) after 96 hours of cultivation, reaching 417.2 U/g of dry substrate (or 41.72 U/mL) and 144.5 U/g of dry substrate (or 14.45 U/mL) for L. ramosa and T. aurantiacus, respectively. The enzymes showed higher catalytic activity at pH 6.0 at 60°C. The amylases produced by L. ramosa and T. aurantiacus were stable between pH 3.5–10.5 and pH 4.5–9.5, respectively. The amylase of L. ramosa was stable at 55°C after 1 hour of incubation, whereas that of T. aurantiacus maintained 60% of its original activity under the same conditions. Both enzymes were active in the presence of ethanol. The enzymes hydrolyzed starch from different sources, with the best results obtained with corn starch. The enzymatic complex produced by L. ramosa showed dextrinizing and saccharifying potential. The enzymatic extract produced by the fungus T. aurantiacus presented only saccharifying potential, releasing glucose monomers as the main hydrolysis product.

A B S T R A C TThe use of microbial enzyme cocktails for the conversion of polysaccharides of plant origin into fermentable sugars is a global trend. The objectives of this study were to optimize the production of cellulases and hemicellulases by the fungus Lichtheimia ramosa, evaluate the catalytic properties of the produced enzymes, and apply these biocatalysts in the saccharification of sugarcane bagasse. The production of carboxymethylcellulase (CMCase), β-glucosidase, xylanase, and β-xylosidase by L. ramosa were 168.1 ± 2.2, 270.4 ± 8.9, 34 ± 0.8, and 199.2 ± 2.6 U/g of dry substrate, respectively. The optimum pH for the activity of the enzymes ranged between 4.5 and 5.5, and the optimum temperature varied between 55°C and 65°C. The enzymes were stable within a wide range of pH, and approximately 95% of their original activity was preserved when incubated for 1 h at 55°C. The half-lives (t 1/2 ) of CMCase, β-glucosidase, xylanase, and β-xylosidase were 68, 59, 52, and 54 min, respectively, when incubated at 60°C. The enzymes were stable in solutions containing ethanol (10%), and the kinetic parameters (K m and V max ) demonstrated that β-glucosidase from L. ramosa was competitively inhibited by glucose. The enzyme extract produced by L. ramosa was used for the saccharification of sugarcane bagasse pretreated with glycerol and the highest yield of glucose (10.66%) was obtained at 24 h of hydrolysis. The characteristics of the enzymes, combined with the efficiency in hydrolyzing sugarcane bagasse, allow for the application of this enzyme extract to processes of cellulose saccharification for the production of second-generation ethanol.

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Capsular polysaccharide, extracted from microorganism cultivations, is the principal antigen for elaboration of vaccine against the disease caused by Neisseria meningitidis serogroup C. The final protein content allowed in this vaccine is 1%. In order to find a relationship between nitrogen consumption and cell growth, including polysaccharide production, and cell nitrogen content, cultivations were carried out in an 80 liters bioreactor (total capacity), under the following conditions: Frantz medium; temperature of 35ºC; air flow of 5 L/min (0.125 vvm); agitation frequency of 120 rpm and vessel pressure of 6 psi (kLa = 0.07 min-1). Concentrations of biomass, total polysaccharide, cellular nitrogen, residual organic and inorganic nitrogen in the medium were measured during cultivation. From five cultivations carried out under the same conditions, a mean cell nitrogen percentage of 12.6% (w/w) in respect to the dry biomass was found. The inorganic nitrogen in the medium did not change significantly along the cultivation time, whereas the organic nitrogen consumption was linearly related to cell growth, with constant yield factors (average of 8.44). Polysaccharide production kinetics followed the cell growth kinetics until the beginning of the stationary growth phase. A supplemental polysaccharide production was observed until the end of cultivation, but without cell nitrogen absorption. Thus, the results indicate that polysaccharide is produced in two phases, being the first one biomass formation followed by non-associated to growth.

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Profiling microorganisms with potential for amylase production in low cost culture media has been widely recognized due to its broad applicability. The aim of this study was to select yeast strains with potential to produce amylolytic enzymes by solid state fermentation. Fifty-four (54) strains were assessed and three exhibited ability to produce amylases: Candida parapsilosis with 14.68 U/mL (146.8 U/g substrate); Rhodotorula mucilaginosa with 25.0 U/mL (250 U/g substrate), and Candida glabrata with 25.39 U/mL (253.9 U/g substrate), in solid state fermentation, for 120 h at 28°C, using wheat bran with 70% moisture. The enzymes exhibited maximum activity at a pH of 7.0 and at 60°C. Amylases demonstrated satisfactory structural stability, maintaining their catalytic activity after 1 h at 50°C. All enzymes were ethanol tolerant and retained more than 70% of their original activities in 15% ethanol solution. Corn starch was efficiently hydrolyzed by enzymes and the extracts produced by C. parapsilosis and C. glabrata exhibited dextrinizing activity, while those produced by R. mucilaginosa exhibited saccharifying activity.

Invertases are used for several purposes; one among these is the production of fructooligosaccharides. The aim of this study was to biochemically characterize invertase from industrial Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa isolated from Cerrado soil. The optimum pH and temperature were 4.0 and 70 °C for Rhodotorula mucilaginosa invertase and 4.5 and 50 °C for Saccharomyces cerevisiae invertase. The pH and thermal stability from 3.0 to 10.5 and 75 °C for R. mucilaginosa invertase, respectively. The pH and thermal stability for S. cerevisiae CAT-1 invertase from 3.0 to 7.0, and 50 °C, respectively. Both enzymes showed good catalytic activity with 10% of ethanol in reaction mixture. The hydrolysis by invertases occurs predominantly when sucrose concentrations are ≤5%. On the other hand, the increase in the concentration of sucrose to levels above 10% results in the highest transferase activity, reaching about 13.3 g/L of nystose by S. cerevisiae invertase and 12.6 g/L by R. mucilaginosa invertase. The results demonstrate the high structural stability of the enzyme produced by R. mucilaginosa, which is an extremely interesting feature that would enable the application of this enzyme in industrial processes.