title: Purification and characterization of an acidophilic xylanase from Aureobasidium pullulans var. melanigenum and sequence analysis of the encoding gene.
abstract: An extracellular endo-1,4-beta-xylanase was purified from the culture supernatant of Aureobasidium pullulans var. melanigenum (ATCC 20524) grown on oat-spelt xylan. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with an apparent M(r) of 24 kDa and had an isoelectric point of 6.7. Xylanase activity was optimal at pH 2.0 and 50 degrees C. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. Southern blot analysis indicated that the xylanase gene (xynI) was present as 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 deduced amino acid sequence showed 94% identity with that of a previously reported 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-joining tree showed that the Aureobasidium enzymes are closely related to several other family-11 xylanases from black aspergilli and Penicillium purpurogenum.
authors.affiliation: Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan. k.ohta@cc.miyazaki-u.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Ohta
authors.forename: S
authors.initials: S
authors.lastname: Moriyama
authors.forename: H
authors.initials: H
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Shige
authors.forename: H
authors.initials: H
authors.lastname: Akimoto