Role of invariant water molecules and water-mediated ionic interactions in D-xylose isomerase from Streptomyces rubiginosus

The enzyme, D-xylose isomerase (D-xylose keto-isomerase; EC 5.3.1.5) is a soluble enzyme that catalyzes the conversion of the aldo-sugar D-xylose to the keto-sugar D-xylulose. A total of 27 subunits of D-xylose isomerase from Streptomyces rubiginosus were analyzed in order to identify the invariant water molecules and their water-mediated ionic interactions. A total of 70 water molecules were found to be invariant. The structural and/or functional roles of these water molecules have been discussed. These invariant water molecules and their ionic interactions may be involved in maintaining the structural stability of the enzyme D-xylose isomerase. Fifty-eight of the 70 invariant water molecules (83%) have at least one interaction with the main chain polar atom.

Xylan-Degrading Enzymes from the Thermophilic Fungus Humicola Zanuginosa (Griffon and Maublanc) Bunce: Action Pattern of Xylanase and beta-Glucosidase on Xylans, Xylooligomers and Arabinoxylooligomers

The mode of action of xylanase and beta-glucosidase purified from the culture filtrate of Humicola lanuginosa (Griffon and Maublanc) Bunce on the xylan extracted from sugarcane bagasse and on two commercially available larchwood and oat spelt xylans, on xylooligomers and on arabinoxylooligomers was studied. While larchwood and oat spelt xylans were hydrolyzed to the same extent in 24 h, sugarcane bagasse xylan was hydrolyzed to a lesser extent in the same period. It was found that the rate of hydrolysis of xylooligomers by xylanase increased with increase in chain length, while beta-glucosidase acted rather slowly on all the oligomers tested. Xylanase exhibited predominant ''endo'' action on xylooligomers attacking the xylan chain at random while beta-glucosidase had ''exo'' action, releasing one xylose residue at a time. On arabinoxylooligomers, however, xylanase exhibited ''exo'' action. Thus, it appears that the presence of the arabinose substituent has, in some way, rendered the terminal xylose-xylose linkage more susceptible to xylanase action. It was also observed that even after extensive hydrolysis with both the enzymes, substantial amounts of the parent arabinoxylooligomer remained unhydrolyzed together with the accumulation of arabinoxylobiose. It can therefore be concluded that the presence of the arabinose substituent in the xylan chain results in linkages that offer resistance to both xylanase and beta-glucosidase action.

Isolation and Culture of a Thermophilic Fungus, Melanocarpus albomyces, and Factors Influencing the Production and Activity of Xylanase

Summary: An uncommon thermophilic fungus, Melanocarpus albomyces, was isolated from soil and compost by incubating samples in a glucose/sorbose/asparagine liquid medium, followed by enrichment culture in medium containing sugarcane bagasse as carbon source. The culture filtrate protein of the fungus grown in the presence of bagasse or xylose hydrolysed xylan and some other polysaccharides but cellulose was not hydrolysed. High extracellular xylanase (EC 3.2.1.8) activity was produced by cultures grown on xylose or hemicellulosic materials. The enzyme was induced in glucose-grown washed mycelia in response to addition of xylose or xylan but not by alkyl or aryl β-D-xylosides. Cultures produced higher enzyme yields in shaken flasks than in a fermenter. Gel-filtration chromatography of culture filtrate protein showed the presence of two isoenzymes of xylanase, whose relative proportions varied with the carbon source used for growth. The extent of hydrolysis of heteroxylans or the hemicellulosic fraction of bagasse by culture filtrate protein preparations was greater when the cultures had been grown on bagasse rather than xylose as the inducing substrate. The activity of xylanase preparations was increased when an exogenous β-glucosidase was added.

Rice husk filtrate as a nutrient medium for the growth of Desulfotomaculum nigrificans: characterisation and sulfate reduction studies

The filtrate obtained by interacting a known amount of rice husk with deionised, Milli-Q water was assessed as a carbon source and nutrient medium for the growth of Desulfotomaculum nigrificans, a typical sulfate-reducing bacterium. The filtrate contained essential growth constituents such as magnesium, potassium, phosphorous apart from calcium, sodium, chloride and sulfate ions. Based on the 1H and 13C NMR characterization studies, the organic composition of the components dissolved from the rice husk, was found to be: (i) 66% lignocellulosic material, (ii) 24% xylose + arabinose and (iii) 10% galactose. The growth studies indicated a 15-fold increase in the bacterial cell number in about 20 days. Nearly 81% and 66% reduction in sulfate concentration could be achieved in about 28 days, from the solutions containing initial sulfate concentrations of 550 mg/l and 1200 mg/l respectively. In both the cases studied, the iron concentration could be reduced by over 85%.

Carbohydrate binding specificity of the B-cell maturation mitogen from Artocarpus integrifolia seeds

Artocarpin, a mannose-specific lectin, is a homotetrameric protein (M(r) 65,000) devoid of covalently attached carbohydrates and consists of four isolectins with pI in the range 5-6.5. Investigations of its carbohydrate binding specificity reveal that among monosaccharides, mannose is preferred over glucose. Among mannooligosaccharides, mannotriose (Man alpha 1-3[Man alpha 1-6]Man) and mannopentaose are the strongest ligands followed by Man alpha 1-3Man. Extension of these ligands by GlcNAc at the reducing ends of mannooligosaccharides tested remarkably improves their inhibitory potencies, while substitution of both the alpha 1-3 and alpha 1-6 mannosyl residues of mannotriose and the core pentasaccharide of N-linked glycans (Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc) by GlcNAc or N-acetyllactosamine in beta 1-2 linkage diminishes their inhibitory potencies. Sialylated oligosaccharides are non-inhibitory. Moreover, the substitution of either alpha 1-3 or alpha 1-6 linked mannosyl residues of M5Gn or both by mannose in alpha 1-2 linkage leads to a considerable reduction of their inhibitory power. Addition of a xylose residue in beta 1-2 linkage to the core pentasaccharide improves the inhibitory activity. Considering the fact that artocarpin has the strongest affinity for the xylose containing hepasaccharide from horseradish peroxidase, which differs significantly from all the mannose/glucose-specific lectins, it should prove a useful tool for the isolation and characterization of glycoproteins displaying such structure.

Purification and characterization of an alpha-D-glucuronidase from a thermophilic fungus, Thermoascus aurantiacus

An alpha-D-glucuronidase was purified from the culture filtrates of Thermoascus aurantiacus. A simple colorimetric method for its assay is reported. The enzyme is a single polypeptide chain with a molecular weight of 118,000. It acts optimally at pH 4.5. It shows maximum activity at 65 degrees C. The t 1/2 at 70 degrees C was 40 min. It specifically cleaved the alpha-(1----2) linkage between 4-O-methyl-alpha-D-glucuronic acid and the xylose residue in xylan and several glucurono-xylooligosaccharides.

Purification and characterization of endo-1,4-β-xylanase from Paecilomyces varioti Bainier

An endo-xylanase (1,4-β-d-xylanxylanohydrolase EC 3.2.1.8) was isolated from the culture filtrate of Paecilomyces varioti Bainier. The enzyme was purified 3.2 fold with a 60% yield by gel filtration and ion exchange chromatography. The purified enzyme had a molecular weight of 25,000 with a sedimentation coefficient of 2.2 S. The isoelectric point of the enzyme was 3.9. The enzyme was obtained in crystalline form. The optimum pH range was 5.5–7.0 and the temperature, 65°C. The Michaelis constant was 2.5 mg larchwood xylan/ml. The enzyme was found to degrade xylan by an endo mechanism producing arabinose, xylobiose, xylo- and arabinosylxylo-oligosaccharides, during the initial stages of hydrolysis. On prolonged incubation, xylotriose, arabinosylxylotriose and xylobiose were the major products with traces of xylotetraose, xylose and arabinose.

Crystallization and Preliminary X-ray Diffraction Analysis of Crystals of Thermoascus aurantiacus Xylanase

Crystals suitable for high resolution X-ray diffraction analysis have been grown of the 29,774-Da protein, xylanase (1,-4-beta-xylan xylanohydrolase EC 3.2.1.8) from the thermophilic fungus Thermoascus aurantiacus. This protein, an endoxylanase demonstrates the hydrolysis of β-(1-4)-Image -xylose linkage in xylans and crystallizes as monoclinic pinacoids in the presence of ammonium sulphate buffered at pH 6·5, and also with neutral polyethylene glycol 6000. The crystals belong to space group P 21 and have cell dimensions, a = 41·2 Å, b = 67·76 Å, c = 51·8 Å; β = 113·2°.