Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site.
Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.
Keskar
S S
SS
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Srinivasan
M C
MC
Deshpande
V V
VV
D015077:2-Hydroxy-5-nitrobenzyl Bromide; D001665:Binding Sites; D001974:Bromosuccinimide; D003545:Cysteine; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006902:Hydroxymercuribenzoates; D013302:Streptomyces / Q000201:enzymology*; D014364:Tryptophan; D043325:Xylan Endo-1,3-beta-Xylosidase
D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D015077:2-Hydroxy-5-nitrobenzyl Bromide; D014364:Tryptophan; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase; D003545:Cysteine; D001974:Bromosuccinimide