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

Abstract

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 Neocallimastixmore » 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.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Univ. of Georgia, Athens, GA (US)
Sponsoring Org.:
USDOE
OSTI Identifier:
20000238
DOE Contract Number:  
FG02-93ER20127
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 65; Journal Issue: 9; Other Information: PBD: Sep 1999; Journal ID: ISSN 0099-2240
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; ESTERASES; FUNGI; DNA-CLONING; CELLULOSE; CHEMICAL COMPOSITION

Citation Formats

Blum, D.L., Li, X.L., Chen, H., and Ljungdahl, L.G. Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2. United States: N. p., 1999. Web.
Blum, D.L., Li, X.L., Chen, H., & Ljungdahl, L.G. Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2. United States.
Blum, D.L., Li, X.L., Chen, H., and Ljungdahl, L.G. Wed . "Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2". United States.
@article{osti_20000238,
title = {Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2},
author = {Blum, D.L. and Li, X.L. and Chen, H. and Ljungdahl, L.G.},
abstractNote = {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.},
doi = {},
journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 9,
volume = 65,
place = {United States},
year = {1999},
month = {9}
}