In the estimation of glycosidase activity by dinitrosalicylic acid (DNS) reagent, the stoichiometry of DNS reduction was reported to increase proportionately with the increase in the number of glycosidic linkages present in oligosaccharides liberated by the enzyme. The relationship between increases in DNS reduction and increases in the number of glycosidic bonds was found to be represented by a part of a rectangular hyperbola. The increase was optimum with disaccharide and insignificant when the degree of polymerization (DP) was > or =10. The difference did not arise as a result of the DNSA discriminating between mono- and oligosaccharide oxidation. The relationship stemmed from the acidity of the hydroxyl group adjacent to the reducing group, which repressed DNS reduction. The acidity is likely to decrease with an increase in oligosaccharide chain length. It is suggested that DNS reduction is actually optimum and uniform for all oligosaccharides of DP > or = 10 and that it is minimum for monosaccharide. Thus the introduction of rectification factors in the estimation of glycosidase activities by the DNS method appears to be justified.

An amyloglucosidase was purified to homogeneity from the culture filtrate of Termitomyces clypeatus, using the following steps: ammonium sulfate fractionation, DEAE‐Sephadex chromatography, and HP‐GPLC on an Ulstropac TSK‐G3000 SWG column. The enzyme was a glycoprotein with a minimum molecular weight of 56 000. It had appreciable activity on glycogen and amylopectin, moderate activity on maltose, and little activity on panose. The enzyme, unlike fungal amyloglucosidase (Aspergillus niger), could liberate glucose from xylans. The enzyme had Km = 1.81 mg/mL and Vm = 82.1 μmol/min/mg for starch hydrolysis and Km = 4.36 mg/mL and Vm = 57.7 μmol/min/mg for the hydrolysis of larch wood xylan. Among the different inhibitors, NBS and CDTA were the most potent. Previously the enzyme was shown [Khowala, S.; et al. Appl. Microbiol. Technol. 1992, 37, 287–292] to have synergistic activity on xylan hydrolysis similar to other xylanolytic enzymes: α‐arabinofuranosidase or α‐glucuronidase. Since the amyloglucosidase was not active on cellulose, arabinogalactan, or β‐glucans, which may be present as contaminants in xylan, the probable liberation of glucose directly from xylan by the enzyme was indicated.

A low molecular weight endo-xylanase (EC 3.2.1.8) was purified from an edible mushroom Termitomyces clypeatus grown in submerged medium with oat spelt xylan. Xylanase was purified to apparent homogeneity by ammonium sulfate fractionation and gel filtration chromatography. Its molecular weight was determined by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 12 kDa. The enzyme was found to be most active at 50 degrees C and pH 5.0, being most stable at pH 6.5. The K(m) for oat spelt xylan was determined to be 10.4 mg/ml. The specificities of the enzyme was observed to be highly specific towards oat spelt xylan and was inhibited by mercuric chloride (HgCl(2)), N-bromosuccinimide, and trans-1,2-diaminocyclohexane-N',N',N',N'-tetraacetic acid strongly. The inhibitory action of N-bromosuccinimide on enzyme confirmed the presence of one tryptophan residue in its substrate-binding site. Amino acid analysis for xylanase showed the presence of high amount of hydrophobic serine, glycine, threonine, and alanine residues. The N-terminal sequencing study for the previously purified and characterized 56 kDa xylanolytic amyloglucosidase reveal the presence of 33.30% identity with glucoamylase chain A from Aspergillus awamori. The N-terminal sequence analysis of the present 12 kDa enzyme showed highest similarity (72.22% identity) towards xylanase from Neurospora crassa.

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