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

An extracellular endo-1,4-β-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 Mr of 24 kDa and had an isoelectric point of 6.7. Xylanase activity was optimal at pH 2.0 and 50°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.