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 rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino 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 coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K{sub m} of 0.9 mM and a V{sub max} of 785 {micro}mol min{sup {minus}} mg{sup {minus}1}. It had temperature and pH optima of 30 C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum.
- Research Organization:
- Univ. of Georgia, Athens, GA (US)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG02-93ER20127
- OSTI ID:
- 20000238
- Journal Information:
- Applied and Environmental Microbiology, Vol. 65, Issue 9; Other Information: PBD: Sep 1999; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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