Basil J. Macris

Production of cellulolytic and xylanolytic enzymes by Fusarium oxysporum grown on corn stover in solid state fermentation

Corn stover is an abundant, potential fermentation substrate. Production of cellulolytic and xylanolytic enzymes by the mesophilic fungus Fusarium oxysporum under solid state culture (SSC) on corn ...

Production of xylanases, mannanases, and pectinases by the thermophilic fungus Thermomyces lanuginosus

A group of 17 strains of the thermophilic fungus Thermomyces lanuginosus was examined for the production of xylanases, β-mannanases, arabinanases, and pectinases. All strains were found to be xylanolytic, and several were proven to be outstanding producers of microbial xylanase on glucuronoxylan and corn cobs. The strains hyperproducing xylanase secreted low amounts of xylan-debranching enzymes and did not produce β-mannan and arabinan-degrading enzyme systems. Only the strains showing lower xylanase production exhibited a higher degree of xylan utilization and also the ability to produce a mannanolytic enzyme system. One of the mannanolytic strains was found to be capable of producing arabinan-degrading enzymes. This strain also showed the best production of pectinolytic enzymes during growth on citrus pectin or sugar beet pulp. Some of the strains have good potential for use as sources of important industrial enzymes of high thermal stability.

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.

An alternative approach to the bioconversion of sweet sorghum carbohydrates to ethanol

The ethanol fermentation of juice and press cake, resulting from the squeezing of sweet sorghum stalks at high pressure, was investigated. The juice was fermented by Saccharomyces cerevisiae and yielded 4.8 g ethanol per 100 g of fresh stalks. The press cake was fermented directly to ethanol by a mixed culture of Fusarium oxysporum and Saccharomyces cerevisiae and yielded 5.1 g ethanol per 100 g of fresh stalks. An overall ethanol concentration and yield of 5.6% (w/v) and 9.9 g of ethanol per 100 g of fresh stalks respectively was obtained. Based on soluble carbohydrates, the ethanol yield from press cake was doubled while the overall theoretical yield was enhanced by 20.7% due to the bioconversion of a significant portion of cell wall polysaccharides to ethanol. The process was found promising for further investigation.

Optimizing production of extracellular lipase from Rhodotorula glutinis

Production of extracellular lipase byRhodotorula glutinis was substantially enhanced when the type and concentration of carbon and nitrogen source, the initial pH of culture medium and the growth temperature were consecutively optimized. Lipase activity as high as 30.4 U/ml of culture medium was obtained at optimum conditions, comparing favourably with most of the activities reported for other lipase hyperproducing microorganisms. The enzyme was optimally active at pH 7.5 and 35°C and had, at optimum pH, half-lives of 45 and 11.8 min at 45 and 55°C respectively. The high activity and kinetic characteristics of the enzyme make this process worthy of further investigation.

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.

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.

A new glucose oxidase from Aspergillus niger: characterization and regulation studies of enzyme and gene

A new glucose oxidase from Aspergillus niger was isolated and characterized. The enzyme showed different kinetic and stability characteristics when compared to a commercially available batch of A. niger glucose oxidase. The gene encoding the new glucose oxidase was isolated and DNA sequence analysis of the coding region showed 80% identity to the sequence of a glucose oxidase gene previously published. However, the similarity of the non-coding sequences up- and downstream of the open reading frame was much less, showing only 66% and 50% identity respectively. Despite the low degree of similarity between the promotor region of the new gene and the previously published one, the new glucose oxidase was likewise induced by calcium carbonate. In addition, we showed that this induction occurred on the transcriptional level. Observations concerning the effect of gluconolactone and the levels of glucose-6-phosphate isomerase upon calcium carbonate induction suggested that the enhancement of glucose oxidase biosynthesis by calcium carbonate was accompanied by a metabolic shift from glycolysis to the pentose phosphate pathway.

Purification and characterization of a Fusarium oxysporum feruloyl esterase (FoFAE-I) catalysing transesterification of phenolic acid esters

An extracellular feruloyl esterase (FoFAE-I) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by ion-exchange, hydrophobic interaction and gel filtration chromatograp ...

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.