Edivaldo Ximenes Ferreira Filho

Purification and characterization of a new xylanase from Acrophialophora nainiana.

A new xylanase activity (XynII) was isolated from liquid state cultures of Acrophialophora nainiana containing birchwood xylan as carbon source. XynII was purified to apparent homogeneity by gel filtration and ion exchange chromatographies. The enzyme was optimally active at 55 degrees C and pH 7.0. XynII had molecular mass of 22630+/-3.0 and 22165 Da, as determined by mass spectrometry and SDS-PAGE, respectively. The purified enzyme was able to act only on xylan as substrate. The apparent K(m) values on soluble and insoluble birchwood xylans were 40.9 and 16.1 mg ml(-1), respectively. The enzyme showed good thermal stability with half lives of 44 h at 55 degrees C and ca. 1 h at 60 degrees C The N-terminal sequence of XynII showed homology with a xylanase grouped in family G/11. The enzyme did not show amino acid composition similarity with xylanases from some fungi and Bacillus amyloliquefaciens.

A biotechnology perspective of fungal proteases.

Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

Fungal Enzymes for Bio-Products from Sustainable and Waste Biomass

Lignocellulose, the most abundant renewable carbon source on earth, is the logical candidate to replace fossil carbon as the major biofuel raw material. Nevertheless, the technologies needed to convert lignocellulose into soluble products that can then be utilized by the chemical or fuel industries face several challenges. Enzymatic hydrolysis is of major importance, and we review the progress made in fungal enzyme technology over the past few years with major emphasis on (i) the enzymes needed for the conversion of polysaccharides (cellulose and hemicellulose) into soluble products, (ii) the potential uses of lignin degradation products, and (iii) current progress and bottlenecks for the use of the soluble lignocellulose derivatives in emerging biorefineries.

Purification and characterization of a novel cellulase-free xylanase from Acrophialophora nainiana.

A beta-xylanase (XynIII) of Acrophialophora nainiana was purified to homogeneity from the culture supernatant by ultrafiltration and a combination of ion exchange and gel filtration chromatographic methods. It was optimally active at 55 degrees C and pH 6.5. XynIII had molecular masses of 27.5 and 54 kDa, as estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The purified enzyme hydrolyzed preferentially xylan as the substrate. The half-lives of XynIII at 50 and 60 degrees C were 96 and 1 h, respectively. It was activated by L-tryptophan, dithiothreitol, 5,5-dithio-bis(2-nitrobenzoic acid, L-cysteine and beta-mercaptoethanol and strongly inhibited by N-bromosuccinimide. The presence of carbohydrate was detected in the pure XynIII.

The Production of Hemicellulases by Aerobic Fungi on Medium Containing Residues of Banana Plant as Substrate

Trichoderma harzianum strains T4 and T6, Acrophialophora nainiana, and Humicola grisea var. thermoidea were screened for their ability to produce carbohydrate‐degrading enzyme activities in a medium containing banana plant residue as the carbon source. The best balance of enzyme activities was obtained from cultures of H. grisea var. thermoidea. Xylanase activity from crude extract of A. nainiana had a maximum activity at pH 5.5−7.0 and a temperature range of 50−55 °C. It was stable up to 55 °C at pH 7.0 for at least 2 h. The fungi were also able to produce xylanase and pectinase activities when grown on extractives as substrate.

Purification and Characterization of a Low-Molecular-Weight Xylanase Produced by Acrophialophora nainiana

Abstract. A low-molecular-weight xylanase activity (XynI) was isolated from the fungus Acrophialophora nainiana after growth in a solid medium containing wheat bran. XynI was purified to apparent homogeneity by ultrafiltration and gel filtration chromatography. The purified enzyme had a molecular weight value of approx. 17 kDa, as determined by SDS-PAGE. This enzyme was most active at 50°C and pH 6.0. At 50°C the half-life was 150 min. The apparent Km value for birchwood xylan was much lower than the Km value for oat spelt xylan. XynI was activated by L-cysteine, DTE, β-mercaptoethanol, and L-tryptophan. XynI did not show significant sequence homology with other xylanases. The analysis of hydrolysis products of xylans and wood pulps showed that XynI was able to release xylooligomers ranging from X2 to X3 and X2 to X6, respectively. The enzyme was not active against acetylated xylan. A small amount of xylose was released from deacetylated, birchwood, and oat spelt xylans. The results obtained with enzymatic treatment of Kraft pulp indicated a reduction in the amount of chlorine compounds required for the process and enhanced brightness gain.

Production and characterization of hemicellulase activities from Trichoderma harzianum strain T4

Xylan and mannan are the major constituent groups of hemicellulose in the cell wall of higher plants. The mesophilic fungus Trichoderma harzianum strain T4 produces extracellular xylanase and mannanase activities when grown in the presence of oat (Avena sativa)‐spelt xylan and wheat bran as the carbon sources respectively. After the growth procedure, the crude extracts were submitted to ultrafiltration in an Amicon system fitted with a 10 kDa‐cut‐off membrane. Mannanase activity was only detected in the concentrated sample, whereas xylanase was also found in the permeate after ultrafiltration. Xylanase from the concentrated sample showed highest activity at 40 °C and pH 5.0. Mannanase activity was optimal at 65 °C and pH 2.6. Xylanase was stable in the temperature range 40–70 °C, presenting full stability for at least 48 h. Xylanase retained 100% of its original activity after incubation for 48 h at 70 °C. Xylanase was also stable at pH 5.0 and 6.0 for 48 h. However, mannanase activity was markedly less stable. The enzyme lost 50% of its activity at 55 °C after 45 min, whereas at 60 °C its half‐life was 20 min. The Michaelis–Menten constant Km and Vmax for mannanase and xylanase activities were also calculated. Xylanase had more affinity for soluble xylan, with Km and Vmax values of 1.61 mg/ml and 10.03 units/ml respectively. The Km and Vmax values for crude mannanase were 6.0 mg/ml and 20.1 units/ml respectively. Xylanase and mannanase were activated by dithiothreitol, l‐cysteine and l‐tryptophan. Xylanase was partially purified by gel‐filtration (Sephadex G‐50) and hydrophobic‐interaction (Phenyl‐Sepharose) chromatographies. The partially purified enzyme was stable over the pH range 5–7 and temperature range of 40–60 °C. It was more active on soluble oat‐spelt xylan and was activated by dithiothreitol, l‐cysteine and l‐tryptophan.

A new xylanase from a Trichoderma harzianum strain

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45°C and was stable at 50°C and pH 5.0 up to 4 h xylanase. XYL2 had a low Km with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp.

Biomass-Derived Inhibitors of Holocellulases

Enzymes constitute a major monetary cost in the bioconversion of holocellulose to ethanol. Identifying enzyme inhibitors and moderating their effects is one approach that may help to overcome this issue. Most inhibitors that reduce the hydrolysis activity of holocellulases are released as the holocellulosic biomass is broken down in the pretreatment and hydrolysis steps. Recent reports in the literature have shown that the major inhibitors or deactivators of cellulases are phenols and xylooligosaccharides. The bioconversion of hemicelluloses by hemicellulases also has important practical applications in various agro-industrial processes in addition to the conversion of hemicellulosic biomass to fuels and chemicals. Hemicellulases, such as β-xylosidases, may also help alleviate the inhibitory effect of xylooligosaccharides to cellulases. However, compared to cellulases, less is known about the inhibition or deactivation of hemicellulases and pectinases, especially for inhibitors that are generated during pre-treatment and the hydrolysis of lignocellulosic substrates. Considering the importance of such enzymes for the complete degradation of lignocellulosic substrates, this review provides a broad view of the effect of inhibitors of holocellulases (cellulases, hemicellulases, and pectinases).

Purification and characterization of a low molecular weight xylanase from solid-state cultures of Aspergillus fumigatus Fresenius

Uma enzima xilanolitica (xilanase II) foi purificada a partir de culturas de estado solido de Aspergillus fumigatus Fresenius. O peso molecular de xilanase II foi estimado em 19 e 8,5 kDa por SDS-PAGE e FPLC, respectivamente. A enzima purificada apresentou maior atividade a 55°C e pH 5,5, alem de hidrolisar especificamente xilana. Os valores aparentes de Km e Vmax de xilanas soluveis e insoluveis, isoladas de cereal e madeira, mostrou que xilanase IIa foi mais ativa em xilana soluvel de madeira. Estudos sobre produtos de hidrolise de xilanas e xilooligomeros por xilanase II em HPLC revelou que a enzima liberou uma variedade de xilooligomeros (xilobiose-xilohexose) e uma pequena quantidade de xilose a partir de xilooligomeros, apresentando atividade de transferase.