Xylooligosaccharides Production from Alkali-Pretreated Sugarcane Bagasse Using Xylanases from Thermoascus aurantiacus

Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of L-arabinose, 4-O-methyl-D-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50 degrees C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligosaccharides (DP 2-5) were investigated using substrate concentration in the range 0.5-3.5% (w/v), enzyme load 40-80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 22 factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.

Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 A degrees C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 A degrees C and pH 6.5 for A. terricola, and 65 A degrees C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 A degrees C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t (50) of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4-3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and beta-xylosidase were detected which might act synergistically with xylanase.

Xylanases from Aspergillus niger, Aspergillus niveus and Aspergillus ochraceus produced under solid-state fermentation and their application in cellulose pulp bleaching

This study describes the production of xylanases from Aspergillus niveus, A. niger, and A. ochraceus under solid-state fermentation using agro-industrial residues as substrates. Enzyme production was improved using a mixture of wheat bran and yeast extract or peptone. When a mixture of corncob and wheat bran was used, xylanase production from A. niger and A. ochraceus increased by 18%. All cultures were incubated at 30 A degrees C at 70-80% relative humidity for 96 h. For biobleaching assays, 10 or 35 U of xylanase/g dry cellulose pulp were incubated at pH 5.5 for 1 or 2 h, at 55 A degrees C. The delignification efficiency was 20%, the brightness (percentage of ISO) increased two to three points and the viscosity was maintained confirming the absence of cellulolytic activity. These results indicated that the use of xylanases could help to reduce the amount of chlorine compounds used in cellulose pulp treatment.

An insight into the thermostability of a pair of xylanases: the role of hydrogen bonds

Xylanases are enzymes that are very tolerant to temperature. Their potential use in several biotechnological applications, such as animal food manufacture and pulp bleaching, is due to their intrinsic thermostability. The present report deals with two xylanases, the mesophilic xylanase from Bacillus circulans, BCX, and the thermophilic xylanase from Thermomyces lanuginosus,TLX. These enzymes belong to family 11, and they are the most structurally similar mesophilic-thermophilic pair. Molecular dynamics simulations were employed to investigate the factors responsible for the different thermostabilities exhibited by these structurally similar enzymes. Their active site is their most rigid region, and it is equally rigid at all temperatures. Inter and intramolecular interactions, hydrogen bonds in particular, are the key to the main differences between BCX and TLX. The intramolecular hydrogen bonds and salt bridges are important for maintenance of the backbone rigidity even at high temperature, and the highly solvated surface is a clear optimization in TLX compared with BCX. The main differences between these two enzymes can be found on the fingers domain, which indicates that this domain must be the target for the site-directed mutagenesis responsible for improving the temperature tolerance of this family of enzymes.

Studies on Production of Bacterial Xylanases

Xylanases with hydrolytic activity on xylan, one of the hemicellulosic materials present in plant cell walls, have been identified long back and the applicability of this enzyme is constantly growing. All these applications especially the pulp and paper industries require novel enzymes. There has been lot of documentation on microbial xylanases, however, none meeting all the required characteristics. The characters being sought are: higher production, higher pH and temperature optima, good stabilities under these conditions and finally the low associated cellulase and protease production. The present study analyses various facets of xylanase biotechnology giving emphasis on bacterial xylanases. Fungal xylanases are having problems like low pH values for both enzyme activity and growth. Moreover, the associated production of cellulases at significant levels make fungal xylanases less suitable for application in paper and pulp industries.Bacillus SSP-34 selected from 200 isolates was clearly having xylan catabolizing nature distinct from earlier reports. The stabilities at higher temperatures and pH values along with the optimum conditions for pH and temperature is rendering Bacillus SSP-34 xylanase more suitable than many of the previous reports for application in pulp and paper industries.Bacillus SSP-34 is an alkalophilic thertmotolerant bacteria which under optimal cultural conditions as mentioned earlier, can produce 2.5 times more xylanase than the basal medium.The 0.5% xylan concentration in the medium was found to the best carbon source resulting in 366 IU/ml of xylanase activity. This induction was subjected to catabolite repression by glucose. Xylose was a good inducer for xylanase production. The combination of yeast extract and peptone selected from several nitrogen sources resulted in the highest enzyme production (379+-0.2 IU/ml) at the optimum final concentration of 0.5%. All the cultural and nutritional parameters were compiled and comparative study showed that the modified medium resulted in xylanase activity of 506 IU/ml, 5 folds higher than the basal medium.The novel combination of purification techniques like ultrafiltraton, ammonium sulphate fractionation, DEAE Sepharose anion exchange chromatography, CM Sephadex cation exchange chromatography and Gel permeation chromatography resulted in the purified xylanase having a specific activity of 1723 U/mg protein with 33.3% yield. The enzyme was having a molecular weight of 20-22 kDa. The Km of the purified xylanase was 6.5 mg of oat spelts xylan per ml and Vmax 1233 µ mol/min/mg protein.Bacillus SSP-34 xylanase resulted in the ISO brightness increase from 41.1% to 48.5%. The hydrolytic nature of the xylanase was in the endo-form.Thus the organism Bacillus SSP-34 was having interesting biotechnological and physiological aspects. The SSP-34 xylanase having desired characters seems to be suited for application in paper and pulp industries.

Purification and characterization of two xylanases from alkalophilic and thermophilic Bacillus licheniformis 77-2

The alkalophilic bacteria Bacillus licheniformis 77-2 produces significant quantities of thermostable cellulase-free xylanases. The crude xylanase was purified to apparent homogeneity by gel filtration (G-75) and ionic exchange chromatography (carboxymethyl sephadex, Q sepharose, and Mono Q), resulting in the isolation of two xylanases. The molecular masses of the enzymes were estimated to be 17 kDa (X-I) and 40 kDa (X-II), as determined by SDS-PAGE. The K(m) and V(max) values were 1.8 mg/mL and 7.05 U/mg protein (X-I), and 1.05 mg/mL and 9.1 U/mg protein (X-II). The xylanases demonstrated optimum activity at pH 7.0 and 8.0-10.0 for xylanase X-I and X-II, respectively, and, retained more than 75% of hydrolytic activity up to pH 11.0. The purified enzymes were most active at 70 and 75 degrees C for X-I and X-II, respectively, and, retained more than 90% of hydrolytic activity after 1 h of heating at 50 degrees C and 60 degrees C for X-I and X-II, respectively. The predominant products of xylan hydrolysates indicated that these enzymes were endoxylanases.

Purification and Characterization of Two Extracellular Xylanases from Penicillium sclerotiorum: A Novel Acidophilic Xylanase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Xylanases from fungi: properties and industrial applications

Xylan is the principal type of hemicellulose. It is a linear polymer of beta-D-xylopyranosyl units linked by (1-4) glycosidic bonds. In nature, the polysaccharide backbone may be added to 4-O-methyl-alpha-D-glucuronopyranosyl units, acetyl groups, alpha-L-arabinofuranosyl, etc., in variable proportions. An enzymatic complex is responsible for the hydrolysis of xylan, but the main enzymes involved are endo-1,4-beta-xylanase and beta-xylosidase. These enzymes are produced by fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans, insect, seeds, etc., but the principal commercial source is filamentous fungi. Recently, there has been much industrial interest in xylan and its hydrolytic enzymatic complex, as a supplement in animal feed, for the manufacture of bread, food and drinks, textiles, bleaching of cellulose pulp, ethanol and xylitol production. This review describes some properties of xylan and its metabolism, as well as the biochemical properties of xylanases and their commercial applications.

Purification and characterization of xylanases from Aspergillus giganteus

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50°C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50°C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu 2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.

Purification and biochemical characterization of two xylanases produced by Aspergillus caespitosus and their potential for kraft pulp bleaching

Two extracellular xylanases produced by the thermotolerant fungus Aspergillus caespitosus grown in sugar cane bagasse were purified and characterized. Estimated molecular masses were 26.3 and 27 kDa (xyl I); 7.7 and 17.7 kDa (xyl II) for gel filtration and SDS-PAGE, respectively. Optimal temperature for both xylanases was 50-55°C. Optimal pH was 6.5-7.0 for xyl I, and 5.5-6.5 for xyl II. The thermostability (T half) at 55°C was 27.3 min (xyl I) and >90 min (xyl II). Xylanase activity was inhibited by several ions. β-mercaptoethanol activated 59 and 102% xyl I and xyl II activities, respectively. These enzymes preferentially hydrolyzed birchwood xylan, and the K m and V max values were 2.5 mg/ml and 1679 U/mg protein (xyl I), and 3.9 mg/ml and 113 U/mg protein (xyl II). The action of both xylanases mainly that of xyl II, on kraft pulp reduced kappa number and increased pulp viscosity. © 2004 Elsevier Ltd. All rights reserved.

Molecular and Kinetic Characterization of Two Extracellular Xylanases Isolated from Leucoagaricus gongylophorus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Thermophilic fungi as new sources for production of cellulases and xylanases with potential use in sugarcane bagasse saccharification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Atividade de extratos de Arrabidaea chica (Humb. & Bonpl.) Verlot obtidos por processos biotecnológicos sobre a proliferação de fibroblastos e células tumorais humanas

Arrabidaea chica (H&B) Verlot is a plant popularly known as Pariri and this species is a known source of anthocyanins, flavonoids and tannins. This report describes an approach involving enzymatic treatment prior to extraction procedures to enhance A chica crude extract anticancer activity. Anticancer activity in human cancer cell lines in vitro using a 48 h SRB cell viability assay was performed to determine growth inhibition and cytotoxic properties. The final extraction yield without enzyme treatment was higher (24.28%) compared to the enzyme-treated material (19.03%), with an enhanced aglycones anthocyanin ratio as determined by HPLC- DAD and LC-MS with direct infusion.

Biochemical properties of a beta-mannanase and a beta-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan, Its activity was optimal at pH 4.5. The K(m) value with galactoglucomannan as substrate was 0.50 mg ml (1). One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 degrees C and pH 8.0. Its K(m) value with birchwood xylan as substrate was 1.65 mg ml (1). Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn(+2), Fe(+3), and Cu(+2) were strong inhibitors for the mannanase. (C) 2008 Elsevier Ltd. All rights reserved.