Purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F3.

Two low molecular mass endo-1,4-beta-D-xylanases from Fusarium oxysporum were purified to homogeneity by gel-filtration and ion-exchange chromatography. They exhibit molecular masses of 20.8 (xylanase I) and 23.5 (xylanase II) kDa, and isoelectric points of 9.5 and 8.45-8.70, respectively. Both xylanases display remarkable pH (9.0) stability. At 40 to 55 degrees C xylanase II is more thermostable than xylanase I but less active on xylan. In contrast to xylanase I, xylanase II is able to hydrolyze 1-O-4-methylumbelliferyl-beta-D-glucopyranosyl)-beta-D-xylopyranoside (muxg). Neither of these enzymes hydrolyze xylotriose. They bind on crystalline cellulose but not on insoluble xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that both enzymes cleave preferentially the internal glycosidic bonds of xylopentaose and oat spelts xylan. Thus the purified enzymes appeared to be true endo-beta-1,4-xylanases. The amino terminal sequences of xylanases I and II show to homology. Xylanase I shows high similarity with alkaline low molecular mass xylanases of family G/11.

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.

The alkaline xylanase III from Fusarium oxysporum F3 belongs to family F/10.

Xylanase III from Fusarium oxysporum F3 was purified to homogeneity by ion-exchange chromatography and gel filtration. The enzyme has a molecular mass of 38 kDa, an isoelectric point of 9.5, and is maximally active on oat spelt xylan at pH 7 and 45 degrees C with a Km of 0.8 mg/mL. The xylanase displays remarkable stability at pH 9.0. It is not active on xylotriose but hydrolyzes the 4-methylumbelliferyl glycosides of beta-xylobiose and beta-D-glucopyranosyl-(1-->4)-beta-D-xylopyranose, and to a lower extent 4-methylumbelliferyl beta-cellobioside. When acted on xylooligosaccharides and xylan, analysis of reaction mixtures by high-pressure liquid chromatography shows preferred internal glycoside cleavage. Thus the purified enzyme appears to be a true endo-beta-1,4-xylanase. Partial amino acid analysis of xylanase III shows high sequence homology with xylanases of family F/10.

Purification and characterisation of a major xylanase with cellulase and transferase activities from Fusarium oxysporum.

A major xylanase from Fusarium oxysporum was purified to homogeneity by gel filtration, affinity, and ion-exchange chromatographies. It has a molecular mass of 60.2 kDa and pI of 6.6 and was optimally active at pH 7.4 and at 50 degrees C. The enzyme was stable over the pH range 5.8-8.2 at 40 degrees C for 24 h and lost 45% of its original activity at pH 9.0 under the identical conditions. The enzyme rapidly hydrolysed xylans from oat spelts (husks) and birchwood, but the activities on carboxymethylcellulose (CMC), filter paper, and Avicel were very low. Determination of kcat/Km revealed that the enzyme hydrolysed oat spelts and birchwood xylans, 15-30 times more efficiently than CMC. In a 24 h incubation, at pH 7.0 and 9.0, the enzyme hydrolysed oat spelts and birchwood xylans by 75 and 65%, respectively. However, at pH 7.0, the enzyme released almost equal amounts of xylose and xylobiose from both xylans, whereas at pH 9.0, the concentration of xylobiose was twice as muchi as that of xylose and xylotriose. Xylanase attacked preferentially the internal glycosidic bonds of xylo- and 4-methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n]. The enzyme catalysed transglycosylation reaction with xylotriose, xylotetraose, and xylopentaose as donors and 4-methylumbelliferyl beta-D-glucoside (MeUmbGlc) as an acceptor.

Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Thermomyces lanuginosus ATCC 46882.

An endoxylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) from the culture filtrates of T. lanuginosus ATCC 46882 was purified to homogeneity by DEAE-Sepharose and Bio-Gel P-30 column chromatographies. The purified endoxylanase had a specific activity of 888.8 mumol min-1 mg-1 protein and accounted for approximately 30% of the total protein secreted by this fungus. The molecular mass of native (non-denatured) and denatured endoxylanase were 26.3 and 25.7 kD as, respectively. Endoxylanase had a pI of 3.7 and was optimally active between pH 6.0-6.5 and at 75 degrees C. The enzyme showed > 50% of its original activity between pH 5.5-9.0 and at 85 degrees C. The pH and temperature stability studies revealed that this endoxylanase was almost completely stable between pH 5.0-9.0 and up to 60 degrees C for 5 h and at pH 10.0 up to 55 degrees C for 5 h. Thin-layer chromatography (TLC) analysis showed that endoxylanase released mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methyl-D-glucuronoxylan and rhodymenan (a beta-(1-->3)-beta(1-->4)-xylan). Also, the enzyme released an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. The enzyme hydrolysed [1-3H]-xylo-oligosaccharides in an endofashion, but the hydrolysis of [1-3H]-xylotriose appeared to proceed via transglycosylation. since the xylobiose was the predominant product. Endoxylanase was not active on pNPX and pNPC at 40 and 100 mM for up to 6 h, but showed some activity toward pNPX at 100 mM after 20-24 h. The results suggested that the endoxylanase from T. lanuginosus belongs to family 11.

Mode of action of a minor xylanase from Thermoascus aurantiacus on polysaccharides and model substrates.

The mode of action of a minor xylanase on a variety of polysaccharides and model substrates was investigated. The enzyme was excreted by Thermoascus aurantiacus grown in solid state fermentation (SSF). The purified enzyme had a molecular mass of 33,000. Thin layer chromatography analysis showed that the endoxylanase liberated short fragments from polysaccharides. The enzyme hydrolysed aryl-beta-D-cellobioside and the chromogenic (fluorogenic) 4-methylumbelliferyl-beta-glycosides of xylobiose (MeUmbXyl2) and xylotriose (MeUmbXyl3) at the agluconic linkage. The results suggested that the endoxylanase belonged to family 10.

A novel lipolytic activity of Rhodotorula glutinis cells: production, partial characterization and application in the synthesis of esters.

Cell-bound lipase activity (10 pNPL units/g dry cell weight) was released when the yeast Rhodotorula glutinis was cultured in a 7-l stirred tank fermentor using palm-oil as the sole carbon source. The enzyme showed relative specificity towards medium chain organic acids since the apparent K(m) values for pNPB (p-NitroPhenyl-Butyrate) and pNPL (p-NitroPhenyl-Laurate) were equal to 2.7 and 0.7 mM, respectively. In addition, 80% of this activity could be detected on the surface of the cells. The cell-bound nature of the enzyme increased its thermal stability showing half-life times of 200 and 60 min at 50 and 60 degrees C, respectively, as well as good stability in organic solvents. Freeze-dried cell preparations were successfully used to catalyze the synthesis of fatty acid esters of butanol and heptanol in nearly anhydrous organic solvents. A conversion of 60-62% was obtained upon esterification of palmitic or oleic acid with butanol, within 96 h. The enzyme preparation was used in four consecutive batch reactions with only 10% loss of activity.

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.

Enzymatic synthesis of trisaccharides and alkyl beta-D-glucosides by the transglycosylation reaction of beta-glucosidase from Fusarium oxysporum.

Purified beta-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl beta-D-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl beta-D-glucosides were 22-37% and 10-33% of the total sugar, respectively. The enzyme retained 70-80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, beta-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl beta-D-glucosides.

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.

Factors affecting the specificity of beta-glucosidase from Fusarium oxysporum in enzymatic synthesis of alkyl-beta-D-glucosides.

Fusarium oxysporum beta-glucosidase has been used to catalyze the production of alkyl-beta-D-glucosides from various disaccharides, based on the transglucosylation reaction, in the presence of primary, secondary and tertiary alcohols as glucosyl acceptors. Primary alcohols were found to be the best acceptors. The influence of the glucosyl donor concentration, as well as the enzyme specificity towards the cleaved glucosidic bond and the aglucone part of the donor, have also been investigated. The enzyme does not exhibit regiospecificity and seems to be unspecific towards the aglucone part. The specificity of the beta linkage has been confirmed by proton nuclear magnetic resonance (1H NMR) analysis.

Purification and characterization of an extracellular alpha-L-arabinofuranosidase from Fusarium oxysporum.

An alpha-L-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60 degrees C. It hydrolyzed aryl alpha-L-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect between the alpha-L-arabinofuranosidase and an endo-(1-->4)-beta-D-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan.

Mechanism of benzoic acid uptake by Saccharomyces cerevisiae.

A fast uptake of the preservative benzoic acid was observed in Saccharomyces cerevisiae, reaching saturation in about two min and then remaining constant at this level. The strong dependence of benzoic acid uptake on pH was due to the relative distribution of molecular and ionic forms in solution and not to the pH itself. The molecular form was the only one taken up by the cells. The specificity of the uptake mechanism was evidenced by the pattern of irreversible heat inactivation of the uptake system resembling protein denaturation by heat. Furthermore, the effect of temperature on the uptake was similar to that observed in enzymic reactions, whereas the kinetic data of uptake conformed to the Michaelis-Menten curve of saturation with a Km of 1.54 X 10(-2) M and Vmax of 3 X 10(-3) M/10s. The evidence presented in this paper indicates that compounds of protein nature are involved in the uptake of this preservative.

Production and Characterization of Cellulase and beta-Glucosidase from a Mutant of Alternaria alternata.

A mutant of Alternaria alternata excreted enhanced levels of carboxymethylcellulase and particularly beta-glucosidase when grown in cellulose liquid media. Both enzymes were purified two- to four-fold by ammonium sulfate precipitation and gel filtration, and the kinetic data showed K(m) values of 16.64 mg/ml of culture fluid for carboxymethylcellulase and 0.14 mM p-nitrophenyl-beta-d-glucoside and 0.81 mM cellobiose for beta-glucosidase at pH 5. Carboxymethylcellulase and extracellular beta-glucosidase functioned optimally at pH 5 to 6 and 4.5 to 5 and at temperatures of 55 to 60 and 70 to 75 degrees C, respectively. Both temperature optima and thermostability of beta-glucosidase were among the highest ever reported for the same enzyme excreted from cellulase and beta-glucosidase hyperproducing microorganisms.

Production of a Thermostable beta-d-Galactosidase by Alternaria alternata Grown in Whey.

In the course of exploring new microbial sources of extracellular beta-d-galactosidase (EC. 3.2.1.23), Alternaria alternata was found to excrete elevated quantities of a thermostable form of the enzyme when cultivated in whey growth medium. Optimum cultural conditions for maximum enzyme production were a whey lactose concentration of 6%, supplementation of the medium with 0.050 M (NH(4))(2)SO(4), an inoculum size of 10 conidia per ml, and a cultivation time at 28 to 30 degrees C of 5 days. The fungus utilized whey lactose for the production of the enzyme most efficiently, and the observed maximum yield, 280 nanokatals of hydrolyzed o-nitrophenyl-beta-d-galactopyranoside per g of whey lactose, was comparable to maximum yields reported for certain commercial fungi. The optimum pH and temperature of the enzymatic reaction were 4.5 to 5.5 and 60 to 70 degrees C, respectively, and the enzyme lost half of its activity when heated at 65 degrees C for 84 min. These properties make the enzyme particularly suitable for processing acid and less-acid (pH 5 to 6) dairy products and by-products.

Characterization of Extracellular beta-d-Galactosidase from Fusarium moniliforme Grown in Whey.

Extracellular lactase (beta-d-galactosidase, EC 3.2.1.23) was prepared as an ethanol precipitate from a culture of Fusarium moniliforme grown on whey. The enzyme functioned optimally at pH 3.8 to 5.0 and at 50 to 60 degrees C on both o-nitrophenyl-beta-d-galactopyranoside (ONPG) and lactose. The activation energy of the enzymic hydrolysis of ONPG and lactose in the range of 20 to 55 degrees C was 8,500 and 7,200 cal (ca. 3.57 x 10 and 3.02 x 10 J)/mol, respectively. The K(m) values were 4.4 and 12.4 mM for ONPG and lactose, respectively. At optimum pH, the enzyme lost half of its activity when it was heated at 50 degrees C for 6 h; at the same pH, the loss was only 5% when the enzyme was heated at 37 degrees C for 6 h. At optimum conditions, 50% of the lactose in whey was hydrolyzed by 10 U of this enzyme in 50 h.

Production and Characterization of Amylase from Calvatia gigantea.

alpha-Amylase (EC 3.2.1.1) was excreted by Calvatia gigantea in liquid growth media containing different sources of starch. Among the factors affecting enzyme production in shake flasks were the type and the concentration of starch and the nitrogen source supplied. Optimum cultural conditions for maximum enzyme production were: soluble starch concentration, 5%; inoculum size, 3.75 x 10 conidia per ml; 5-day cultivation time at 28 to 30 degrees C. The observed maximum yield of 81.3 U of saccharifying enzyme activity per ml of growth medium was the highest ever reported in the literature for submerged cultures. Partially purified enzyme functioned optimally at pH 4.5 to 5.5 and 53 to 58 degrees C. The activation energy of enzymic hydrolysis of starch in the range of 20 to 40 degrees C was 8,125 cal/mol (ca. 3.41 x 10 J). The apparent K(m) value of the enzyme at 25 degrees C was 7.68 x 10 g/ml. Some of the properties of the enzyme under investigation were similar to those of alpha-amylases excreted from molds producing large amounts of the enzyme.

Growth of Fusarium moniliforme on carob aqueous extract and nutritional evaluation of its biomass.

Fusarium moniliforme was cultured semicontinuously on a carob medium in a 14-liter fermentor (8.5-liter working volume). The growth medium provided 2.4% carob sugar, 0.72% NH4H2PO4, and 0.03% MgSO4-7H2O. The biomass harvest was 8.8 g/liter per day. Ninety percent of the sugars were consumed, and the pH dropped from 5.9 to about 3.7. The crude protein (N X 6.25) of the spray-dried mycelium was 380 g/kg, 300 g/kg for the true protein (Lowry), and 4.8 g/kg for the (Folin-Denis) tannic acid. The mycelium was evaluated nutritionally with the weanling rat as experimental animal. The protein efficiency ratio and net protein utilization values for the unsupplemented mycelium were 1.15 and 0.42, respectively, and for the mycelium supplemented with DL-methionine (5 g/kg) they were 2.31 and 0.72, respectively. No growth depression was observed in the experimental rats, and on dissection of the carcasses the internal organs were found to be normal.

Protein content and amino acid composition of certain fungi evaluated for microbial protein production.

The protein and total amino acid contents of four mycelial fungal strains and one yeast were approximately the same for cultures harvested in the mid-log and early stationary growth phases. It was found that Fusarium oxysporum and Fusarium moniliforme contained approximately 30% more protein and total amino acids than Aspergillus niger. The amino acid composition of mycelial protein compares favorably with that of British Petroleum yeast protein Toprina produced commercially on hydrocarbon substrates. Fusarium spp. may be suitable for commercial production of microbial protein, especially when low-cost agricultural or industrial waste products are readily available as energy sources. Genetic manipulation of these fungi, such as induction of mutant strains through irradiation, may be desirable to obtain a mycelial product of improved yield and/or quality.

Correlating the effect of pretreatment on the enzymatic hydrolysis of straw.

Avicell, Alkali-treated straw cellulose (ATSC), and wheat straw were ball-milled to reduce crystallinity; wheat straw was delignified by hot (120 degrees C) sodium hydroxide solutions of various concentrations. The physically and chemically pretreated cellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Enzymic hydrolysis data were fitted by the hyperbolic correlation of Holtzapple, which involves two kinetic parameters, the maximum conversion (X(max)), and the enzymic hydrolysis time corresponding to 50% of X(max) (t(1/2)). An empirical correlation between X(max) and cellulose crystallinity, lignin content, and degree of delignification has been found under our experimental conditions. Complete cellulose hydrolysis is shown to be possible at less than 60% crystallinity indices or less than 10% lignin content.