Effect of compressed fluids treatment on ß-galactosidase activity and stability.

This study aimed at assessing the influence of different pressurized fluids treatment on the enzymatic activity and stability of a lyophilized ß-galactosidase. The effects of system pressure, exposure time and depressurization rate, using propane, n-butane, carbon dioxide and liquefied petroleum gas on the enzymatic activity were evaluated. The ß-galactosidase activity changed significantly depending on the experimental conditions investigated, allowing the selection of the proper compressed fluid for advantageous application of this biocatalyst in enzymatic reactions. The residual activity ranged from 32.1 to 93.8 % after treatment. The storage stability of the enzyme after high-pressure pre-treatment was also monitored, and results showed that the biocatalyst activity presents strong dependence of the fluid used in the pretreatment. The activity gradually decreases over the time for the enzyme treated with LGP and propane, while the enzyme treated with n-butane maintained 96 % of its initial activity until 120 days. For CO(2), there was a reduction of around 40 % in the initial activity 90 days of storage. The enzyme treated with n-butane also showed a better thermostability in terms of enzymatic half-life.

Production of multifunctional lipases by Penicillium verrucosum and Penicillium brevicompactum under solid state fermentation of babassu cake and castor meal.

The main objective of this work was to optimize lipase production, in terms of hydrolytic and esterification activities, by Penicillium brevicompactum and Penicillium verrucosum in solid state fermentation using agroindustrial residues as raw material. Maxima hydrolytic activities of 48.6 and 87.7 U/g were achieved when P. brevicompactum was cultured in babassu cake and castor meal, respectively. Higher esterification activities (around 244 U/g) were achieved when P. brevicompactum was used as microorganism and babassu cake as raw material. Different experimental conditions led to these promising values, clearly showing that no correlation can be attributed between hydrolytic and esterification activities. In spite of the several applications of lipases which are capable of catalyze synthesis reactions, only few works in this subject are presented in the literature, especially when low cost raw materials are used.

Interference of salts used on aqueous two-phase systems on the quantification of total proteins.

In this study the interference of potassium phosphate, sodium citrate, sodium chloride and sodium nitrate salts on protein quantification by Bradford's method was assessed. Potassium phosphate and sodium citrate salts are commonly used in aqueous two-phase systems for enzyme purification. Results showed that the presence of potassium phosphate and sodium citrate salts increase the absorbance of the samples, when compared with the samples without any salt. The increase in absorptivity of the solution induces errors on protein quantification, which are propagated to the calculations of specific enzyme activity and consequently on purification factor. The presence of sodium chloride and sodium nitrate practically did not affect the absorbance of inulinase, probably the metals present in the enzyme extract did not interact with the added salts.

Synthesis of fructooligosaccharides from Aspergillus niger commercial inulinase immobilized in montmorillonite pretreated in pressurized propane and LPG.

Commercial inulinase from Aspergillus niger was immobilized in montmorillonite and then treated in pressurized propane and liquefied petroleum gas (LPG). Firstly, the effects of system pressure, exposure time, and depressurization rate, using propane and LPG, on enzymatic activity were evaluated through central composite design 2³. Residual activities of 145.1 and 148.5% were observed for LPG (30 bar, 6 h, and depressurization rate of 20 bar min⁻¹) and propane (270 bar, 1 h, and depressurization rate of 100 bar min⁻¹), respectively. The catalysts treated at these conditions in both fluids were then used for the production of fructooligosaccharides (FOS) using sucrose and inulin as substrates in aqueous and organic systems. The main objective of this step was to evaluate the yield and productivity in FOS, using alternatives for enhancing enzyme activity by means of pressurized fluids and also using low-cost supports for enzyme immobilization, aiming at obtaining a stable biocatalyst to be used for synthesis reactions. Yields of 18% were achieved using sucrose as substrate in aqueous medium, showing the potential of this procedure, hence suggesting a further optimization step to increase the process yield.

Enzymatic synthesis of fructooligosaccharides by inulinases from Aspergillus niger and Kluyveromyces marxianus NRRL Y-7571 in aqueous-organic medium.

This work is focused on the synthesis of the fructooligosaccharides (FOS) from sucrose and inulin, using free, immobilized and pre-treated immobilized inulinase from Kluyveromyces marxianus NRRL Y 7571 and Aspergillus niger in an aqueous-organic system. Initially, the influence of pre-treatment using four different gases, propane, n-butane, CO(2) and liquefied petroleum gas (LPG), was investigated towards FOS production and best results were found when both enzymes were previously treated with LPG. The best reaction yields were obtained when the immobilized enzymes were treated with LPG. Considering FOS synthesis using the enzyme from A. niger, yields of 26.62% of GF2 (kestose), 30.62% of GF3 (nystose) and 8.47% of GF4 (fructosyl nystose) were achieved using sucrose as substrate. Using inulinases from K. marxianus NRRL Y 7571, 11.89% of GF2 and 20.83% of GF3 were obtained, using inulin as substrate. However, promising results were achieved using the free form of inulinase from A. niger (77.19% of GF2; 14.03% of GF3 and 0.07% of GF4) using inulin as substrate.

Concentration, partial characterization, and immobilization of lipase extract from P. brevicompactum by solid-state fermentation of babassu cake and castor bean cake.

One relevant limitation hindering the industrial application of microbial lipases has been attributed to their production cost, which is determined by the production yield, enzyme stability among other. The objective of this work was to evaluate the concentration and immobilization of lipase extracts from Penicillium brevicompactum obtained by solid-state fermentation of babassu cake and castor bean cake. The precipitation with ammonium sulfate 60% of saturation of crude extract obtained with babassu cake as raw material showed an enhancement in hydrolytic and esterification activities from 31.82 to 227.57 U/g and from 170.92 to 207.40 U/g, respectively. Concentrated lipase extracts showed preference to medium-chain triglycerides and fatty acids. It is shown that the enzyme activity is maintained during storage at low temperatures (4 and -10°C) for up to 30 days. Higher esterification activities were achieved when the lipase extract was immobilized in sodium alginate and activated coal.

Partial characterization of inulinases obtained by submerged and solid-state fermentation using agroindustrial residues as substrates: a comparative study.

Inulinase belongs to an important class of enzymes as it can be used to produce high-fructose syrups by enzymatic hydrolysis of inulin and fructooligosaccharides, which has been used as functional food. This work aimed to carry out a partial characterization of the crude enzymatic extract of two different inulinases, obtained by solid-state fermentation (SSF) and submerged fermentation (SmF), using agroindustrial residues as substrates. The crude enzymatic extract obtained by SmF showed an optimal pH and temperature for hydrolytic activity of 4.5 and 55 degrees Celsius, respectively; and that obtained by SSF conducted to optimal pH and temperature of 5.0 and 55 degrees Celsius, respectively. Both enzymes presented high thermostability, with a D value of 230.4 h and 123.1 h for SmF and SSF, respectively. The inulinase produced by SmF showed highest stability at pH 4.4, while inulinase obtained by SSF was more stable at pH 4.8. The results showed that inulinase obtained by SmF is less susceptible to pH effect and the inulinase obtained by SSF is more resistant to higher temperatures.