Isolation, analysis, and expression of two genes from Thermoanaerobacterium sp. strain JW/SL YS 485: A {beta}-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity

Journal Article · Mon Sep 01 04:00:00 EDT 1997 · Journal of Bacteriology

OSTI ID:625668

Lorenz, W W; Wiegel, J ^[1]

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1. Univ. of Georgia, Athens, GA (United States)

The genes encoding acetyl xylan esterase 1 (axe1) and a {beta}-xylosidase (xylB) have been cloned and sequenced from Thermoanaerobacterium sp. strain JW/SL YS485. axe1 is located 22 nucleotides 3{prime} of the xylB sequence. The identity of axe1 was confirmed by comparison of the deduced amino acid sequence to peptide sequence analysis data from purified acetyl xylan esterase 1. The xylB gene was identified by expression cloning and by sequence homology to known {beta}-xylosidases. Plasmids which independently expressed either acetyl xylan esterase 1 (pAct1BK) or {beta}-xylosidase (pXylo-1.1) were constructed in Escherichia coli. Plasmid pXylAct-1 contained both genes joined at a unique EcoRI site and expressed both activities. Substrate specificity, pH, and temperature optima were determined for partially purified recombinant acetyl xylan esterase 1 and for crude recombinant {beta}-xylosidase. Similarity searches showed that the axe1 and xylB genes were homologs of the ORF-1 and xynB genes, respectively, isolated from Thermoanaerobacterium saccharolyticum. Although the deduced sequence of the axe1 product had no significant amino acid sequence similarity to any reported acetyl xylan esterase sequence, it did not have strong similarity to cephalosporin C deacetylase from Bacillus subtilis. Recombinant acetyl xylan esterase 1 was found to have thermostable deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid.

• OSTI ID: 625668
• Journal Information: Journal of Bacteriology, Journal Name: Journal of Bacteriology Journal Issue: 17 Vol. 179; ISSN JOBAAY; ISSN 0021-9193
• Country of Publication: United States
• Language: English

Related Subjects

55 BIOLOGY AND MEDICINE
BASIC STUDIES
AMINO ACID SEQUENCE
BACILLUS SUBTILIS
CLONING
ESCHERICHIA COLI
ESTERASES
PH VALUE
PLASMIDS