Purification and biochemical properties of multiple xylanases from Aspergillus ochraceus tolerant to Hg2+ ion and a wide range of pH.

Production of multiple xylanases, in which each enzyme has a specific characteristic, can be one strategy to achieve the effective hydrolysis of xylan. Three xylanases (xyl 1, xyl 2, and xyl 3) from Aspergillus ochraceus were purified by chromatography using diethylaminoethyl (DEAE) cellulose, Biogel P-60, and Sephadex G-100 columns. These enzymes are glycoproteins of low molecular weight with an optimum temperature at 60 °C. The glycosylation presented is apparently not related to thermostability, since xyl 3 (20 % carbohydrate) was more thermostable than xyl 2 (67 % carbohydrate). Xyl 3 was able to retain most of its activity in a wide range of pH (3.5-8.0), while xyl 1 and xyl 2 presented optimum pH of 6.0. Xyl 1 and xyl 2 were activated by 5 and 10 mM MnCl2 and CoCl2, while xyl 3 was activated by 1 mM of the same compounds. Interestingly, xyl 2 presented high tolerance toward mercury ion. Xylanases from A. ochraceus hydrolyzed xylans of different origins, such as birchwood, oat spelt, larchwood, and eucalyptus (around 90 % or more), except xyl 2 and xyl 3 that hydrolyzed with lesser efficiency eucalyptus (66.7 %) and oat spelt (44.8 %) xylans.

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

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular ß-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. ß-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. ß-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 ß-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-ß-D-xylopyranoside, exhibiting apparent K(m) and V(max) values of 0.66 mM and 39 U (mg protein)⁻¹ respectively, and to a lesser extent p-nitrophenyl-ß-D-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel ß-D-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by ß-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.

Endo-xylanase GH11 activation by the fungal metabolite eugenitin.

Eugenitin, a chromone derivative and a metabolite of the endophyte Mycoleptodiscus indicus, at 5 mM activated a recombinant GH11 endo-xylanase by 40 %. The in silico prediction of ligand-binding sites on the three-dimensional structure of the endo-xylanase revealed that eugenitin interacts mainly by a hydrogen bond with a serine residue and a stacking interaction of the heterocyclic aromatic ring system with a tryptophan residue. Eugenitin improved the GH11 endo-xylanase activity on different substrates, modified the optimal pH and temperature activities and slightly affected the kinetic parameters of the enzyme.

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii.

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60°C and 4.0, respectively. The enzyme was stable for 1 h at 55°C, showing a t50 of 53 min at 60°C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).

Production of saccharogenic and dextrinogenic amylases by Rhizomucor pusillus A 13.36.

A newly-isolated thermophilic strain of the zygomycete fungus Rhizomucor pusillus 13.36 produced highly active dextrinogenic and saccharogenic enzymes. Cassava pulp was a good alternative substrate for amylase production. Dextrinogenic and saccharogenic amylases exhibited optimum activities at a pH of 4.0-4.5 and 5.0 respectively and at a temperature of 75 degrees C. The enzymes were highly thermostable, with no detectable loss of saccharogenic or dextrinogenic activity after 1 h and 6 h at 60 degrees C, respectively. The saccharogenic activity was inhibited by Ca(2+) while the dextrinogenic was indifferent to this ion. Both activities were inhibited by Fe(2+) and Cu(2+) Hydrolysis of soluble starch by the crude enzyme yielded 66% glucose, 19.5% maltose, 7.7% maltotriose and 6.6% oligosaccharides.