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Identification of Amino Acid Residues Involved in Xylanase Activity from Bacillus alcalophilus AX2000 by Chemical Modifiers  

Park Young-Seo (Division of Biotechnology, Kyungwon University)
Publication Information
Microbiology and Biotechnology Letters / v.34, no.2, 2006 , pp. 121-128 More about this Journal
Abstract
The purified xylanase from Bacillus alcalophilus AX2000 was modified with various chemical modifiers to determine amino acid residues in the active site of the enzyme. Treatment of the enzyme with group-specific reagents such as carbodiimide or N-bromosuccinimide resulted in complete loss of enzyme activity. These results suggested that these reagents reacted with glutamic acid or aspartic acid and tryptophan residues located at or near the active site. In each case, inactivation was performed by pseudo first-order kinetics. Inhibition of enzyme activity by carbodiimide and N-bromosuccinimide showed non-competitive and competitive inhibition type, respectively. Addition of xylan to the enzyme solution containing N-bromosuccinimide prevented the inactivation, indicating the presence of tryptophan at the substrate binding site. Analysis of kinetics for inactivation showed that the loss of enzyme activity was due to modification of two glutamic acid or aspartic acid residues and single tryptophan residue.
Keywords
Bacillus alcalophilus; xylanase; chemical modification; active site;
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