Characterisation of a xylanolytic amyloglucosidase of Termitomyces clypeatus.
A xylanolytic amyloglucosidase of Termitomyces clypeatus was characterised with respect to other amyloglucosidases. The enzyme contained high alpha-helix destabilising amino acids but no sulphur amino acid. It contained high threonine and serine, analogous to other raw starch hydrolysing enzymes. Both xylanase and amyloglucosidase activities were gradually lost with the progress of tryptophan oxidation by NBS and total inactivation occurred after oxidation of 4-5 tryptophan residues. In the presence of substrates (either starch or xylan), complete inactivation of either activities was not noticed even after oxidation of 7.7 mol of tryptophan residues. Inactivation by HNBB was not possible in the absence of any denaturant. Only 4.9 mol of tryptophan could be modified in the presence of 5 M urea which resulted in only 42% inhibition of activity. Thus modified enzyme had higher Vm/Km and lower pH optima in comparison to those of native enzyme. It was suggested that tryptophan was present at the substrate binding site and not at the active site. No such change in activity was noticed after modification of tyrosine, lysine or arginine residues. HPGPLC analysis of both dilute and concentrated enzyme solution indicated that the enzyme existed as an equilibrium mixture of protomer-oligomer. Perhaps for this reason molar mass of NAI modified enzyme appeared to be almost half of that modified by NAI in presence of substrate. Arrhenius plot of the enzyme also indicated reversible oligomerisation as a function of temperature.
Ghosh
A K
AK
Applied Biochemistry Department, Indian Institute of Chemical Biology, Calcutta, India.
Naskar
A K
AK
Sengupta
S
S
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D002384:Catalysis; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014990:Xylans / Q000378:metabolism*
D000596:Amino Acids; D014990:Xylans; D005087:Glucan 1,4-alpha-Glucosidase; D001974:Bromosuccinimide