Purification and Characterization of a Low Molecular Weight Endo-xylanase from Mushroom <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis> |
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Authors: | Dhananjay Soren Mohanlal Jana Subhabrata Sengupta Anil K Ghosh |
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Institution: | (1) Drug Development/Diagnostics & Biotechnology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700032, India;(2) Department of Biotechnology, Heritage Institute of Technology, Chowbaga Road, Anandapur, East Kolkata Township, Kolkata, 700107, India; |
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Abstract: | 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°C and pH 5.0, being
most stable at pH 6.5. The Km 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 (HgCl2), 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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