Purification and characterization of a low molecular weight endo-xylanase from mushroom Termitomyces clypeatus.
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.
Soren
Dhananjay
D
Drug Development/Diagnostics & Biotechnology Division, Indian Institute of Chemical Biology, 4, Raja S C Mullick Road, Kolkata 700032, India.
Jana
Mohanlal
M
Sengupta
Subhabrata
S
Ghosh
Anil K
AK
D000595:Amino Acid Sequence; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D055434:Termitomyces / Q000201:enzymology*
D043364:Endo-1,4-beta Xylanases