E. Kalogeris

Studies on the solid-state production of thermostable endoxylanases from Thermoascus aurantiacus: Characterization of two isozymes

Production of xylanases by the thermophilic fungus Thermoascus aurantiacus under solid state culture (SSC) was enhanced by optimization of the type of carbon and nitrogen source, inoculum type, moisture level and particle size of the carbon source. Under these conditions, yields as high as 6193 U g−1 of carbon source were obtained. Chromogenic (fluorogenic) 4-methylumbelliferyl-β-glycosides of xylose (MUX) and xylobiose (MUX2) were used to characterize xylanase multienzyme components, after separation by isoelectric focusing. The zymogram indicated one major and two minor xylanases and one β-xylosidase. The major (xylanase I) and one of the minor (xylanase II) xylanases were separated and characterized. Both xylanases exhibited remarkable thermostability.

Performance of an intermittent agitation rotating drum type bioreactor for solid-state fermentation of wheat straw

A laboratory bioreactor, designed for solid-state fermentation of thermophilic microorganisms, was operated for production of cellulases and hemicellulases by the thermophilic fungus Thermoascus aurantiacus. The suitability of the apparatus for the effective control of important operating variables affecting growth of microbes in solid-state cultivation was determined. Application of the optimum conditions found for the moisture content of the medium, growth temperature and airflow rate produced enzyme yields of 1709 U endoglucanase, 4 U cellobiohydrolase, 79 U beta-glucosidase, 5.5 U FPA, 4490 U xylanase and 45 U beta-xylosidase per g of dry wheat straw. The correlation between microorganism growth and production of enzymes was efficiently described by the Le Duy kinetic model.

Antimicrobial activity of acidic xylo-oligosaccharides produced by family 10 and 11 endoxylanases

Acidic oligosaccharides were obtained from birchwood xylan by treatment with a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. The main difference between the products liberated by xylanases of family 10 and 11 concerned the length of the products containing 4-O-methyl-D-glucuronic acid. The xylanase from T. aurantiacus liberate from glucuronoxylan an aldotetrauronic acid as the shortest acidic fragment in contrast with the enzyme from S. thermophile, which liberated an aldopentauronic acid. Acidic xylooligosaccharides were separated from the hydrolysate by anion-exchange and size-exclusion chromatography (SEC) and the primary structure was determined by 13C NMR spectroscopy. The acidic xylo-oligosaccharides were tested against three Gram-positive and three Gram-negative aerobically grown bacteria, as well as against Helicobacter pylori. Aldopentauronic acid was proved more active against the Gram-positive bacteria and against H. pylori.

Hydrothermal processing and enzymatic hydrolysis of sorghum bagasse for fermentable carbohydrates production

Untreated and hydrothermally treated sorghum bagasse (SB) was hydrolyzed to simple sugars by the synergistic action of cellulases and hemicellulases produced by the fungi Fusarium oxysporum and Neurospora crassa. Synergism between the two lignocellulolytic systems was maximized with the application of higher fraction of N. crassa enzymes. Hydrothermolysis of SB was studied at a wide range of treatment times and temperatures. At intense pretreatment conditions (210 degrees C for 20 min; logR(0)=4.54), the residual hemicellulose percentage was 17.45%, while formation of inhibitory products, 5-hydromethyl-furfural (HMF), furfural, acetic and formic acid, (0.21, 0.51, 3.36 and 1.80 g/l, respectively) remained in acceptable levels. Maximum conversion of cellulose and total polysaccharides of the untreated SB were 23.18% and 18.79%, respectively. Combining hydrothermal treatment and enzymatic hydrolysis of released oligosaccharides and insoluble solids resulted in improvement of cellulose (approximately 15% increase) and total polysaccharides (two fold) hydrolysis compared to that of untreated SB.

Design of a solid-state bioreactor for thermophilic microorganisms

A laboratory horizontal bioreactor was designed for cellulase and hemicellulase production by the thermophilic fungus Thermoascus aurantiacus grown in solid-state fermentation. The bioreactor was operated at 49°C using wheat straw as carbon source, at 75% moisture content. High levels of cellulolytic and hemicellulolytic activities were obtained. The overall performance of the bioreactor was promising for further investigation.

Bioconversion of ferulic acid into vanillic acid by the thermophilic fungus Sporotrichum thermophile

Sporotrichum thermophile is capable of promoting the formation of vanillic acid during ferulic acid degradation. Ferulic acid metabolism by S. thermophile apparently occurred via the propenoic chai ...

Catalytic properties of the endoxylanase I from Thermoascus aurantiacus

Endo-β-1,4-xylanase I previously purified from Thermoascus aurantiacus solid state culture was further characterized. The enzyme had a molecular weight of 33 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 31 kDa by gel filtration. Thin layer chromatography (TLC) analysis showed that endoxylanase liberates aldotetrauronic acid MeGlcAα-1,2-Xylβ-1,4-Xylβ-1,4-Xyl as the shortest acidic fragment from glucuronoxylan and an isomeric xylotriose (Xyl3) of the structure Xylβ1-3Xylβ1-4Xyl from rhodymenan. The enzyme performed ideally on O-acetyl-4-O-methylglucuronoxylan, liberating large amounts of short acetylated and non-acetylated fragments. Also, the enzyme was capable to hydrolyse arabinoxylan to arabinose (Arab), xylose (Xyl) and xylobiose (Xyl2). The enzyme degraded pNPX (4-nitrophenyl β-d-xylopyranoside) by a complex reaction pathway that involved both hydrolysis and glycosyl transfer reactions. The enzyme tolerates the replacement of β-xylopyranosyl units in several artificial substrates by β-glucopyranosyl, α-l-arabinopyranosyl and α-l-arabinofuranosyl units and was active on pNPC (4-nitrophenyl β-d-cellobioside), pNP-Arap (4-nitrophenyl α-l-arabinopyranoside) and pNPAraf (4-nitrophenyl α-l-arabinofuranoside). The enzyme also hydrolysed the 4-methylumbelliferyl glycosides of β-d-xylobiose and β-d-xylotriose at the agluconic linkage. The results suggested that the xylanase I from T. aurantiacus has catalytic properties similar to those belonging to family 10.

Effects of purified endo-β-1,4-xylanases of family 10 and 11 and acetyl xylan esterases on eucalypt sulfite dissolving pulp

Sulfite dissolving pulp from Eucalyptus grandis contained approximately 3.8% O-acetyl-4-O-methylglucuronoxylan with a molar ratio of xylose:4-O-methylglucuronic acid:acetyl group close to 13.6:1:6.2. The effects produced by purified endo-xylanases from two different glycosyl hydrolase families (family 10 and 11) as well as acetyl xylan esterases were examined and assessed on pulp in relation to their bleaching abilities. The purified endo-xylanases hydrolyzed only a limited portion (less than 30%) of the acetylglucuronoxylan present in the pulp. The enzymes of family 10 produced acetylated xylobiose and xylotriose whereas acetylated xylobiose was not observed among the products released from the pulp by the family 11 xylanases. The esterases however were not capable of deacetylating the acetylated aldouronic acids generated by the xylanases. Regardless of the different mode of action of the endo-xylanases on dissolving pulp, their effect on pulp bleaching was not related to the amount and nature of sugars generated or the glycosyl hydrolase family. No additional brightness gain was obtained when endo-xylanases were used in conjunction with acetyl xylan esterases, suggesting that the latter do not play an important role in biobleaching of eucalypt sulfite dissolving pulps.

Biochemical Characterization of the Multi-enzyme System Produced by Penicillium decumbens Grown on Rutin

Abstract Penicillium decumbens produced a set of enzymes, including a monoxygenase and two glycosidases, which degrade rutin, a nontoxic flavonoid glycoside, to water-soluble products. The monoxygenase (quercetinase) cleaves the heterocyclic ring in quercetin, the aglycone part of rutin. The glycosidases (α-l-rhamnosidase and β-glucosidase) hydrolyze the bonds between quercetin and rutinose, and between glucose and rhamnose, the constituent monosaccharides of rutinose. Simultaneous production of the three enzymes was optimized following the examination of a number of culture conditions. Maximum enzyme activities were observed when the fungus was grown at 30°C with an initial pH of 7.0, using 8.0 g/L rutin and 9.0 g/L di-ammonium hydrogen phosphate as carbon and nitrogen sources, respectively. The enzymes were purified to electrophoretic homogeneity by a series of consecutive chromatographic steps including anion and cation exchange as well as gel filtration. The purified quercetinase revealed an apparent tetrameric structure, with a reduced molecular mass of 45 kDa. α-l-Rhamnosidase showed an apparent molecular mass of 58 kDa and the purified β-glucosidase was a tetramer exhibiting a reduced molecular mass of 120 kDa.

Production of β-Fructofuranosidase from Sporotrichum thermophile and Its Application in the Synthesis of Fructooligosaccharides

Production of β-fructofuranosidase from the thermophilic fungus Sporotrichum thermophile was studied. The effect of nitrogen source, as well as the type and concentration of carbon source on enzyme production was examined. The results from flask experiments were used for the production of the enzyme in 7-l bioreactors. β-Fructofuranosidase from Sporotrichum thermophile showed both transfructosylating and hydrolytic activities. It was optimally active at 60°C, while the optimal pHs for hydrolysis and transfructosylation were 4.0 and 6.0, respectively. Synthesis of fructooligosaccharides was maximized at 20% (w/v) initial sucrose concentration. The major sugar produced by the transfructosylating activity of the enzyme was 6-kestose.