B.J. Macris

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.

Purification and characterization of a feruloyl esterase from Fusarium oxysporum catalyzing esterification of phenolic acids in ternary water–organic solvent mixtures

An extracellular feruloyl esterase (FAE-II) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by SP-Sepharose, t-butyl-HIC and Sephacryl S-200 column chromatography. The protein corresponded to molecular mass and pI values of 27 kDa and 9.9, respectively. The enzyme was optimally active at pH 7 and 45 degrees C. The purified esterase was fully stable at pH 7.0-9.0 and temperature up to 45 degrees C after 1 h incubation. Determination of k(cat)/K(m) revealed that the enzyme hydrolysed methyl sinapinate 6, 21 and 40 times more efficiently than methyl ferulate, methyl coumarate and methyl caffeate, respectively. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 but inactive to the C-2 positions of arabinofuranose such as 4-nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside. In the presence of Sporotrichum thermophile xylanase, there was a significant release of ferulic acid from destarched wheat bran by FAE-II, indicating a synergistic interaction between FAE-II and S. thermophile xylanase. FAE-II by itself could release only little ferulic acid from destarched wheat bran. The potential of FAE-II for the synthesis of various phenolic acid esters was tested using as a reaction system a surfactantless microemulsion formed in ternary mixture consisting of n-hexane, 1-propanol and water.

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.

Bioethanol from sweet sorghum: Simultaneous saccharification and fermentation of carbohydrates by a mixed microbial culture

Sweet sorghum carbohydrates were simultaneously saccharified and fermented to ethanol by a mixed culture of Fusarium oxysporum and Saccharomyces cerevisiae in a bioreactor. Fusarium oxysporum was grown aerobically for the production of the enzymes necessary for the saccharification of sorghum cellulose and hemicellulose. Saccharomyces cerevisiae, together with F. oxysporum, converted the soluble sugars to ethanol. Three batches of sorghum were used, harvested at different periods of the year. The optimum yield of bioconversion and ethanol concentration was 5·2–8·4 g ethanol/100 g of fresh sorghum and 3·5–4·9% (w/v), respectively, depending on the composition of sorghum stalks. In all experiments, the ethanol yield exceeded the theoretical, based on soluble sugars, by 20·0–32·1% due to bioconversion of polysaccharides to ethanol.

Factors regulating production of glucose oxidase by Aspergillus niger

Certain factors affecting production of extracellular and cell-bound glucose oxidase by Aspergillus niger were investigated. The intention was to maximize total glucose oxidase activity of academic and potential commercial application by the selection of the appropriate strain and consecutive optimization of growth media and conditions. It was possible to identify combinations resulting in the utilization of molasses as the best carbon source and enhancing enzyme activity approximately 40-fold. Glucose oxidase activities as high as 5.7 U ml−1 were produced, comparing favorably with those reported for other enzyme-producing microorganisms. These activity levels were obtained with molasses, indicating an economically attractive process for enzyme production. In addition, our work identified CaCO3 as a particularly strong inducer of glucose oxidase activity.

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.

Synergy between enzymes involved in the degradation of insoluble wheat flour arabinoxylan

Two microbial endo-β-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 were examined for their ability to release ferulic acid (FA) from water-unextractable arabinoxylan (WU-AX) in the presence of a feruloyl esterase (FoFAE-II) from Fusarium oxysporum. WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 (XYLI) and a Sporotrichum thermophile family 11 (XYLA) endoxylanases. At 10 g/l arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates for FA release were 0.18 and 0.44 nM for the xylanases from T. aurantiacus and S. thermophile, respectively. Determination of Vmax/KE revealed that the family 10 enzyme performed 4.3 times more efficiently than the family 11 enzyme in liberation of FA when a feruloyl esterase is present. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The results showed that the degradation of the xylan backbone was influenced strongest by the action of xylanases while the presence of the esterase mainly resulted in the release of ferulic acid from the produced short chain feruloylated xylo-oligosaccharides by the action of xylanases.

Purification and mode of action of a low molecular mass endo-1,4-β-d-glucanase from Fusarium oxysporum

Abstract A low molecular mass (23.2 kDa) endo-1,4-β- d -glucanase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme was optimally active at pH 6.0 and at 50 ° C. It had a pI value of 8.6 and was stable at 55 ° C for 1 h. It hydrolyzed carboxymethylcellulose, cello-oligosaccharides (Glcn) and 4-methylumbelliferylcello-oligosaccharides but did not hydrolyze cellobiose, p-nitrophenyl β-o-glucoside, p-nitrophenyl β- d -xyloside, Avicel, filter paper and xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that this enzyme cleaved preferentially the internal glycoside bonds of higher cello-oligosaccharides. The enzyme also catalyzed the formation of transfer products in the presence of cellotriose, cellotetraose and 4-methylumbelliferylglucoside (MeUmbGlc).

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 ...

Effect of alkali delignification on wheat straw saccharification by Fusarium oxysporum cellulases

Abstract The effect of alkaline delignification of wheat straw on the chemical composition and the subsequent enzymic hydrolysis of the pretreated straw are reported. Both hot (120°°C) and cold (20–36°°C) delignification were investigated, using either aqueous or organic alkaline solutions. The treated lignocellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Both delignification and saccharification yield showed linear relationships with the level of alkali used. Under the chosen experimental conditions 70–100% hydrolysis was achieved either by hot or cold delignification. Delignification to at least 50% appeared crucial for total polysaccharide conversion.