Purification and characterization of an extracellular xylanase produced by the endophytic fungus, Aspergillus terreus, grown in submerged fermentation

Aspergillus terreus produced high levels of a thermotolerant extracellular xylanase and showed low cellulase activity when cultured at 30°C for 48 h, in liquid medium supplemented with wheat bran as carbon source. Xylanase was purified 45-fold to homogeneity with a recovery yield of 67% by carboxymethyl (CM)-cellulose chromatography. The enzyme, a glycoprotein with 33% of carbohydrate content, appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel with a molecular mass corresponding to 23 kDa. Optimal temperature and pH were 55°C and 4.5, respectively. The enzyme was thermotolerant at 45 and 50°C, with a half-life of 55 and 36 min, respectively. The Km was calculated as 22 mg/ml and Vmax as 625 mg/ml of protein using birchwood xylan as substrate. Metal ions, such as Ag+, Cu+2, Fe+2, Hg+ and Zn+2 strongly inhibited xylanase, whereas K+ and Mn+2 resulted in activation. Xylanase hydrolyzed birchwood xylan and oatspelt xylan, mostly yielding xylooligosaccharides, suggest that it is an endoxylanase (EC. 3.2.1.37).Keywords: Aspergillus terreus, endoxylanase, thermostabilit

Production of cellulase-free xylanase by Aspergillus flavus: Effect of polyols on the thermostability and its application on cellulose pulp biobleaching

The production of xylanase without cellulase is required for prebleaching of pulp in pulp and paper industry. Aspergillus flavus produced high levels of xylanase on agricultural residues with wheat bran and sugarcane bagasse (4.17 U/mg), and wheat bran and corncob (2.97 U/mg). Xylanase was found to be stable at 45°C with 100% of its original activity remaining after 2 h incubation. At 50°C, xylanase was stable for the first twenty minutes, and had half-life of 50 min. The pH stability for the xylanase from A. flavus was most stable in the range of pH 3.0-8.0 retaining more that 100% activity after 1 h. The addition of 5% glycerol, mannitol or xylitol protected the xylanase from thermal inactivation at 50°C. The protective effect by glycerol, xylitol and mannitol resulted in increases of 162, 262.5 and 150% when compared with the control at 120 min, approximately. Increasing the polyols concentration up to 20% (w/v) further improved the thermostability of xylanase after 120 min at 50°C by 300% when compared with the control (no additive). The kappa number reduced 2.56 points, which corresponds to 18.34 kappa efficiency. This xylanase is an attractive enzyme for potential future application in the pulp and paper industries, since industrial application requires a cellulase-free activity, maintenance of high temperature and enzyme stability are desirable.Key words: Aspergillus flavus, polyols, xylanase, biobleaching cellulose pulp

Eksperimentalno ispitivanje modela oksidacije 5-CQA klorogene kiseline pomoću polifenolne oksidaze

A set of experiments to investigate the enzymatic oxidation of 5-o-caffeoylquinic acid with polyphenol oxidases was carried out in the temperature range from 18 to 39 °C. The kinetic data were obtained in a batch isothermal reactor made of quartz and operated at an optimum pH (approx. 7.0) for enzyme activity, using citrate-phosphate buffer. The decay of 5-o-caffeoylquinic acid concentrations was experimentally monitored with a spectrophotometer at 323 nm. A coefficient of molar absorptivity equal to 1.96·103 mM–1m–1 was found for 5-o-caffeoylquinic acid, based on absorbance measurements with standard aqueous solutions. The kinetic experimental results were used to calculate the parameters Km and vmax of the Michaelis-Menten model, which were 0.24 mM and 2.77·10–4 mM/s at 25 °C, respectively. The effects of temperature on the maximum velocity of oxidation of 5-o-caffeoylquinic acid and on the rate of enzyme deactivation were well described using the Arrhenius equation.Da bi se ispitala oksidacija 5-CQA klorogene kiseline pomoću polifenolne oksidaze, proveden je niz pokusa u temperaturnom rasponu od 18 do 39 °C. Kinetički su podaci dobiveni u šaržnom izotermičkom reaktoru izrađenom od kvarca, pri pH-vrijednosti optimalnoj za aktivnost enzima, korigiranoj pomoću citratnog pufera. Smanjivanje koncentracije kiseline praćeno je spektrofotometrijski pri valnoj duljini od 323 nm. Mjerenjem apsorbancije uz pomoć standardnih vodenih otopina ustanovljen je koeficijent molarne apsorpcije 5-CQA od 1,96×103 mM-1m-1. Dobiveni kinetički podaci upotrijebljeni su za izračun parametara Michaelis-Mentenova modela, te je utvrđeno da Km pri temperaturi od 25 °C iznosi 0,24 mM, a vmax 2,77×10-4 mM/s. Učinak temperature na maksimalnu brzinu oksidacije 5-CQA i deaktivaciju enzima dobro je opisan pomoću Arrheniusove jednadžbe

Characterisation of a recombinant β-xylosidase (xylA) from Aspergillus oryzae expressed in Pichia pastoris

β-xylosidases catalyse the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. In this study we report the heterologous expression of Aspergillus oryzae β-xylosidase (XylA) in Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The recombinant enzyme was optimally active at 55°C and pH 4.5 with Km and Vmax values of 1.0 mM and 250 μmol min−1 mg−1 respectively against 4-nitrophenyl β-xylopyranoside. Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax). The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X2, X3 and X4). Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length

Bioprocess and biotecnology: effect of xylanase from Aspergillus niger and Aspergillus flavus on pulp biobleaching and enzyme production using agroindustrial residues as substract

This study compares two xylanases produced by filamentous fungi such as A. niger and A. flavus using agroindustrial residues as substract and evaluated the effect of these enzymes on cellulose pulp biobleaching process. Wheat bran was the best carbon source for xylanase production by A. niger and A. flavus. The production of xylanase was 18 and 21% higher on wheat bran when we compare the xylanase production with xylan. At 50°C, the xylanase of A. niger retained over 85% activity with 2 h of incubation, and A. flavus had a half-life of more than 75 minutes. At 55°C, the xylanase produced by A. niger showed more stable than from A. flavus showing a half-life of more than 45 minutes. The xylanase activity of A. niger and A. flavus were somehow protected in the presence of glycerol 5% when compared to the control (without additives). On the biobleaching assay it was observed that the xylanase from A. flavus was more effective in comparison to A. niger. The kappa efficiency corresponded to 36.32 and 25.93, respectively. That is important to emphasize that the cellulase activity was either analyzed and significant levels were not detected, which explain why the viscosity was not significantly modified.This work was supported by grants from Conselho de Desenvolvimento Científico e Tecnológico (CNPq). This work was part of Master Dissertation of NCAG (Laboratório de Bioquímica / CCBS, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brasil). The authors gratefully for the support from the USP/Ribeirão Preto – SP, Brazil.This work was supported by grants from Conselho de Desenvolvimento Científico e Tecnológico (CNPq). This work was part of Master Dissertation of NCAG (Laboratório de Bioquímica / CCBS, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brasil). The authors gratefully for the support from the USP/Ribeirão Preto – SP, Brazil

A novel xylan degrading β-D-xylosidase: purification and biochemical characterization

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular b-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. b-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 C and 3.0–5.5, respectively.b-xylosidase was stable in acidic pH (3.0–6.0) and 70 C for 1 h. The enzyme was activated by 5 mM MnCl2 (28 %)and MgCl2 (20 %) salts. The b-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-b- D-xylopyranoside, exhibiting apparent Km and Vmax values of 0.66 mM and 39 U (mg protein)-1 respectively, and to a lesser extent p-nitrophenyl-b-D-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources,suggesting a novel b-D-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by b-xylosidase. TLC confirmed the capacity.This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and the Conselho de Desenvolvimento Científico e Tecnológico (CNPq). J. A. J. and M. L. T. M. P are Research Fellows of CNPq. M. M. was a recipient of a FAPESP fellowship and this work is part of her Doctoral Thesis. It is also part of the project SISBIOTA CNPq: 563260/2010-6 and FAPESP: 2010/52322-3