Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp strain PC-2

A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA ) was cloned from the anaerobic lumen fungus Orpinomyces sp. strain PC-2. T he gene had an open reading frame of 939 bp encoding a polypeptide of 313 a mino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia c oli. Two active forms of the esterase were purified from recombinant E. col i cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released aceta te from several acetylated substrates, including acetylated xylan. The acti vity toward acetylated xylan was tripled in the presence of recombinant xyl anase A from the same fungus. Using p-nitrophenyl, acetate as a substrate, the enzyme had a K-m of 0.9 mM and a V-max of 785 mu mol min(-1) mg(-1). It had temperature and pH optima of 30 degrees C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocalli mastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic r epeated peptide domain (NCRPD) found at the carboxy terminus of the Neocall imastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cel lulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum. The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter bein g a component of a cellulase-hemicellulase complex. Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.