Extracellular collagenase isolated from Streptomyces antibioticus UFPEDA 3421: purification and biochemical characterization.

Collagenases are proteases able to degrade native and denatured collagen, with broad applications such as leather, food, and pharmaceutical industries. The aim of this research was to purify and characterize a collagenase from Streptomyces antibioticus. In the present work, the coffee ground substrate provided conditions to obtaining high collagenase activity (377.5 U/mL) using anion-exchange DEAE-Sephadex G50 chromatographic protocol. SDS-PAGE revealed the metallo-collagenase with a single band of 41.28 kDa and was able to hydrolyzed type I and type V collagen producing bioactive peptides that delayed the coagulation time. The enzyme activity showed stability across a range of pH (6.0-11) and temperature (30-55 °C) with optima at pH 7.0 and 60 °C, respectively. Activators include Mg+2, Ca+2, Na+, K+, while full inhibition was given by other tested metalloproteinase inhibitors. Kinetic parameters (Km of 27.14 mg/mol, Vmax of 714.29 mg/mol/min, Kcat of 79.9 s-1 and Kcat/Km of 2.95 mL/mg/s) and thermodynamic parameters (Ea of 65.224 kJ/mol, ΔH of 62.75 kJ/mol, ΔS of 1.96 J/mol, ΔG of 62.16 kJ/mol, ΔGE-S of 8.18 kJ/mol and ΔGE-T of -2.64 kJ/mol) were also defined. Coffee grounds showed to be an interesting source to obtaining a collagenase able to produce bioactive peptides with anticoagulant activity.

A novel ß-fructofuranosidase produced by Penicillium citreonigrum URM 4459: purification and biochemical features.

Fructooligosaccharides (FOS) are prebiotics of interest to the food industry. These compounds can be produced through the transfructosylation reaction by the enzyme fructofuranosidase. This enzyme is widely produced by fungi in a medium rich in sugar. Therefore, in this work, the main objectives were production, purification, biochemical characterization of a novel fructofuranosidase enzyme by Penicillium citreonigrum URM 4459 and synthesize and evaluate the antibacterial potential of fructooligosaccharides. With respect to sucrose hydrolysis, the optimal pH was 5.5, the apparent Km for purified FFase was 3.8 mM, the molecular mass was 43.0 kDa, estimated by gel filtration on Superdex increase G75 controlled by AKTA Avant 25 and confirmed by 10% SDS-PAGE under denaturing condition. Also, the isoelectric point was 4.9. The fractions obtained with enzymatic activities, both stable at acidic pH and high temperatures, as well as being able to produce FOS. Regarding antibacterial activity, the FOS produced in this study showed better results than commercial FOS and other carbon sources. Thus, this work presents relevant data for the use of P. citreonigum to produce fructofuranosidase and consequently FOS and can be used in the food and pharmaceutical industry.

Evaluation of the influence of temperature on the protein-tannic acid complex.

Precipitation of blood products from plasma fractionation has played a fundamental role in the industrial purification of important therapeutic products. Only a few studies have been reported by using tannins as proteins precipitant agent from whole plasma while, several conditions have been analyzed. Here, we decided to verify the effect of the temperature on the precipitation process of plasma proteins using tannic acid (TA). Plasma proteins were precipitated with tannic acid by using different temperature incubations. Subsequently, the protein-TA complex was analyzed by SDS-PAGE and quantified. In addition, the protein activity of the complex was measured after heating, as well as the structural changes of the complexes were accompanied by thermogravimetric analysis, differential scanning calorimetry and circular dichroism. In all conditions tested, tannic acid was able to precipitate without selectively separating the proteins in the mixture by using different temperatures during the precipitation process. Furthermore, the protein concentration from the plasma precipitate was not affected by different temperatures and the plasma precipitate was able to dissolve fibrin clots in vitro.