Obtaining filamentous fungi and lipases from sewage treatment plant residue for fat degradation in anaerobic reactors.

A residue from the primary treatment of a Wastewater Treatment Plant (WWTP) was used to isolate filamentous fungi with lipase production potential. Two of the 27 isolated fungi presented high hydrolysis index and were selected for lipase production by solid-state fermentation (SSF). The fermentations were conducted at 30 °C for 48 h, with moist air circulation, using 20% (w/w) of the residue mixture with a basal medium (agroindustrial residue-babassu cake), obtaining a solid enzymatic preparation (SEP) with lipase activity of 19 U/g with the fungus identified as Aspergillus terreus. Scum, collected in an anaerobic reactor operating in a WWTP, was hydrolyzed with SEP and subjected to anaerobic biodegradability tests at 30 °C. Different dilutions of crude (Control) or hydrolyzed scum in raw sewage were evaluated. The dilution of 5% (v/v) of hydrolyzed scum in raw sewage proved the most adequate, as it resulted in higher methane yield compared to the raw sewage (196 and 133 mL CH4/g CODadded, respectively), without increasing the chemical oxygen demand (COD) of the treated sewage (138 and 134 mg/L). The enzymatic hydrolysis of the scum, followed by dilution in the influent sewage, is technically feasible and increases methane production in anaerobic reactors.

Improving the thermostability and optimal temperature of a lipase from the hyperthermophilic archaeon Pyrococcus furiosus by covalent immobilization.

A recombinant thermostable lipase (Pf2001Δ60) from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL) was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL) and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment) (glyoxyl-DTT PFUL) and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment) (glyoxyl PFUL). The enzyme's properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity), whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity). Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity (E = 22) and enantiomeric excess (ee (%) = 91).

Impact of extraction parameters on the recovery of lipolytic activity from fermented babassu cake.

Enzyme extraction from solid matrix is as important step in solid-state fermentation to obtain soluble enzymes for further immobilization and application in biocatalysis. A method for the recovery of a pool of lipases from Penicillium simplicissimum produced by solid-state fermentation was developed. For lipase recovery different extraction solution was used and phosphate buffer containing Tween 80 and NaCl showed the best results, yielding lipase activity of 85.7 U/g and 65.7 U/g, respectively. The parameters with great impacts on enzyme extraction detected by the Plackett-Burman analysis were studied by Central Composite Rotatable experimental designs where a quadratic model was built showing maximum predicted lipase activity (160 U/g) at 25°C, Tween 80 0.5% (w/v), pH 8.0 and extraction solution 7 mL/g, maintaining constant buffer molarity of 0.1 M and 200 rpm. After the optimization process a 2.5 fold increase in lipase activity in the crude extract was obtained, comparing the intial value (64 U/g) with the experimental design (160 U/g), thus improving the overall productivity of the process.

Adding value to a toxic residue from the biodiesel industry: production of two distinct pool of lipases from Penicillium simplicissimum in castor bean waste.

In countries with a strong agricultural base, such as Brazil, the generation of solid residues is very high. In some cases, these wastes present no utility due to their toxic and allergenic compounds, and so are an environmental concern. The castor bean (Ricinus communis) is a promising candidate for biodiesel production. From the biodiesel production process developed in the Petrobras Research Center using castor bean seeds, a toxic and alkaline waste is produced. The use of agroindustrial wastes in solid-state fermentation (SSF) is a very interesting alternative for obtaining enzymes at low cost. Therefore, in this work, castor bean waste was used, without any treatment, as a culture medium for fungal growth and lipase production. The fungus Penicillium simplicissimum was able to grow and produce an enzyme in this waste. In order to maximize the enzyme production, two sequential designs-Plackett-Burman (variable screening) followed by central composite rotatable design (CCRD)-were carried out, attaining a considerable increase in lipase production, reaching an activity of 155.0 U/g after 96 h of fermentation. The use of experimental design strategy was efficient, leading to an increase of 340% in the lipase production. Zymography showed the presence of different lipases in the crude extract. The partial characterization of such extract showed the occurrence of two lipase pools with distinct characteristics of pH and temperature of action: one group with optimal action at pH 6.5 and 45°C and another one at pH 9.0 and 25°C. These results demonstrate how to add value to a toxic and worthless residue through the production of lipases with distinct characteristics. This pool of enzymes, produced through a low cost methodology, can be applied in different areas of biotechnology.

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

Separation and immobilization of lipase from Penicillium simplicissimum by selective adsorption on hydrophobic supports.

Lipases are an enzyme class of a great importance as biocatalysts applied to organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures, organic solvents, and high stereoselectivity presence. The present work's aim was to immobilize the lipases pool produced by Penicillium simplissicimum, a filamentous fungi strain isolated from Brazilian babassu cake residue. P. simplissicimum lipases were separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, and octyl-agarose) at low ionic strength. After immobilization, it was observed that these fractions' hyperactivation is in the range of 131% to 1133%. This phenomenon probably occurs due to enzyme open form stabilization when immobilized onto hydrophobic supports. Those fractions showed different thermal stability, specificity, and enantioselectivity towards some substrates. Enantiomeric ratio for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid ranged from 1 to 7.9 for different immobilized P. simplissicimum lipase fractions. Asymmetry factor for diethyl 2-phenylmalonate hydrolysis ranged from 11.8 to 16.4 according to the immobilized P. simplissicimum lipase fractions. Those results showed that sequential adsorption methodology was an efficient strategy to obtain new biocatalysts with different enantioselectivity degrees, thermostability, and specificity prepared with a crude extract produced by a simple and low-cost technology.

Prostaglandin A1 inhibits the replication of Sindbis virus in monkey kidney and mosquito cells

The present study reports the effect of prostaglandin A1 (PGA1) on the replication of Sindbis virus in monkey kidney and mosquito cells. In PGA1 treated cells we observed a severe reduction of virus yield. In both cells lines the highest nontoxic concentration of PGA1 (10 ìg/mL) decreased virus replication, dose dependently, by more than 90 por cento. SDS-PAGE analysis of [35S] methionine labeled proteins showed that viral proteins (E1/E2 and C) were normally synthesized in PGA1 treated Vero cells, and induction of stress proteins (HSP70 and HSP90 ) was detected in uninfected and infected cells. In Vero cells the inhibition of virus replication was accompanied by a decrease in [3H] glucosamine incorporation into the virus glycoproteins.

Lipase production by solid-state fermentation: cultivation conditions and operation of tray and packed-bed bioreactors.

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30 degrees C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28 degrees C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.