Biotechnological potential of an exo-polygalacturonase of the new strain Penicillium janthinellum VI2R3M: biochemical characterization and clarification of fruit juices.

AIMS: The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M. METHODS AND RESULTS: The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent Km , Vmax and Kcat values for hydrolyzing polygalacturonic acid were 2·56 mg ml-1 , 163·1 U mg-1 and 277 s-1 respectively. The polygalacturonase presented exo activity and was activated by Mg2+ . The juices treated with polygalacturonase presented increases in transmittance with reduction in colour. CONCLUSIONS: The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.

GH11 xylanase from Aspergillus tamarii Kita: Purification by one-step chromatography and xylooligosaccharides hydrolysis monitored in real-time by mass spectrometry.

The present study describes the one-step purification and biochemical characterization of an endo-1,4-ß-xylanase from Aspergillus tamarii Kita. Extracellular xylanase was purified to homogeneity 7.43-fold through CM-cellulose. Enzyme molecular weight and pI were estimated to be 19.5kDa and 8.5, respectively. The highest activity of the xylanase was obtained at 60°C and it was active over a broad pH range (4.0-9.0), with maximal activity at pH 5.5. The enzyme was thermostable at 50°C, retaining more than 70% of its initial activity for 480min. The K0.5 and Vmax values on beechwood xylan were 8.13mg/mL and 1,330.20µmol/min/mg of protein, respectively. The ions Ba2+ and Ni2+, and the compounds ß-mercaptoethanol and DTT enhanced xylanase activity, while the heavy metals (Co2+, Cu2+, Hg+, Pb2+ and Zn2+) strongly inhibited the enzyme, at 5mM. Enzymatic hydrolysis of xylooligosaccharides monitored in real-time by mass spectrometer showed that the shortest xylooligosaccharide more efficiently hydrolyzed by A. tamarii Kita xylanase corresponded to xylopentaose. In agreement, HPLC analyzes did not detect xylopentaose among the hydrolysis products of xylan. Therefore, this novel GH11 endo-xylanase displays a series of physicochemical properties favorable to its application in the food, feed, pharmaceutical and paper industries.

Characterization of trehalase activities from the thermophilic fungus Scytalidium thermophilum.

The thermophilic fungus Scytalidium thermophilum produced large amounts of intracellular and extracellular trehalase activity when grown on starch as the sole carbon source. The specific activity of the purified proteins: 1700 U (mg protein)-1 (extracellular) and 3700 U (mg protein)-1 (intracellular), was many times higher than the values reported for other microbial sources. The apparent molecular mass of the native enzymes was estimated to be 370 kDa (extracellular trehalase) and 398 kDa (intracellular trehalase) by gel-filtration chromatography. Analysis by SDS-PAGE showed unique polypeptide bands of approx. 82 kDa (extracellular trehalase) and 85 kDa (intracellular trehalase), suggesting that the native enzymes were composed of five subunits. The carbohydrate content of extracellular and intracellular trehalases was estimated to be 81% and 51%, respectively. Electrofocusing indicated a pI of 3.7 and 3.4, respectively, for the extracellular and intracellular enzymes. Both trehalases were highly specific for trehalose and were stimulated by calcium and manganese. Calcium and manganese also protected both trehalases from thermoinactivation. Inhibition was observed in the presence of aluminium, mercurium, copper, zinc, EDTA, ADP, and ATP. Apparent Km values, for the extracellular and intracellular trehalases, were 3.58 mM and 2.24 mM, respectively. The optimum of pH for the extracellular and the intracellular trehalase was 6.0, and the optimum of temperature 60 degrees C and 65 degrees C, respectively.