Catalytically Important Amino Acid Residues in Endoxylanases from Aspergillus Awamori

ABSTRACT

Two endoxylanases, Xyl-II (23 kDa) andXyl III (27 kDa), from Aspergillus awamori K-1 were modified with several specific reagents. The catalytic role of the carboxyl groups was estimated through chemical modification studies. Water-soluble carbodiimide (CMC) inactivated the xylanases rapidly and completely in a pseudo-first-order process indicating involvement of carboxyl groups in the catalysis. Treatment of the enzymes with diethyl pyrocarbonate (DEPC) resulted in modification of 1.56 histidine residues in the Xyl III (100% retention of the original enzyme activity in the presence of substrate - 0.4% xylan). Titration of the enzymes with 5.5' -dithiobis-(2-nitrobenzoic acid) (DTNB) did not result in the release of 5-mercapto-2- nitrobenzoic acid, suggesting that no free thiol groups exist in Xyl II and Xyl III. The lack of reactive thiol groups in the intact enzymes was confirmed using the specific cysteine-residue modifiers—iodoacetimide and p-chloromercuribenzoate, neither of which had any noticeable effect on xylanase activities after 60 min of incubation. N-Bromosuccinimide (NBS) showed a strong inhibitory effect on both endoxylanases, suggesting that trypthophan residues are essential for the activity, since the presence of substrate protected the enzymes against inactivation.

Keyword:

• xylanase
• chemical modification
• endoxylanase
• Aspergillus