Purification and characterization of an acidophilic xylanase from Aureobasidium pullulans var. melanigenum and sequence analysis of the encoding gene

Citation
K. Ohta et al., Purification and characterization of an acidophilic xylanase from Aureobasidium pullulans var. melanigenum and sequence analysis of the encoding gene, J BIOSCI BI, 92(3), 2001, pp. 262-270
Citations number
38
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biotecnology & Applied Microbiology",Microbiology
Journal title
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
ISSN journal
1389-1723 → ACNP
Volume
92
Issue
3
Year of publication
2001
Pages
262 - 270
Database
ISI
SICI code
1389-1723(200109)92:3<262:PACOAA>2.0.ZU;2-W
Abstract
An extracellular endo-1,4-beta -xylanase was purified from the culture supe rnatant of Aurcobasidium pullulans var. melanigenum (ATCC 20524) grown on o at-spelt xylan. The purified enzyme showed a single band on SDS-polyacrylam ide gel electrophoresis with an apparent M-r of 24 kDa and had an isoelectr ic point of 6.7. Xylanase activity was optimal at pH 2.0 and 50 degreesC. T he genomic DNA and cDNAs encoding this protein were cloned and sequenced. S outhern blot analysis indicated that the xylanase gene (xynI) was present a s a single copy in the genome. An open reading frame, consisting of 663 bp, encoded a presumed prepropeptide of 34 amino acids and a mature protein of 187 amino acid. The DNA region encoding the prepeptide was interrupted by a 59-bp intron. A single transcription start point was observed at position -46 (A) from the start codon. The 5'-noncoding region had a putative TATA box at -91 (TATATAA) and two possible CCAAT boxes at -247 (CAAT) and -283 ( CCAAT). A cloned xynI cDNA was expressed in Saccharomyces cerevisiae. The d educed amino acid sequence showed 94% identity with that of a previously re ported equivalent gene (xynA) encoding a xylanase with an optimal pH of 4.8 from a color variant strain, NRRL Y-2311-1, of A. pullulans. A neighbor-jo ining tree showed that the Aureobasidium enzymes are closely related to sev eral other family-11 xylanases from black aspergilli and Penicillium purpur ogenum.