Purification and properties of a family-10 xylanase from Aureobasidium pullulans ATCC 20524 and characterization of the encoding gene.
An extracellular endo-1,4-beta-xylanase was purified from the culture supernatant of the ascomycete Aureobasidium pullulans ATCC 20524 grown on xylan. The purified enzyme was homogeneous as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing, which showed an apparent M (r) of 39 kDa and a pI of 8.9, respectively. Xylanase activity was optimal at pH 6.0 and 70 degrees C. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. The xylanase gene (xynII) encoded a 26 amino acid signal peptide and a 335 amino acid mature protein. DNA regions encoding the signal sequence and the mature protein were interrupted by introns of 56 and 73 bp, respectively. The xynII 5'-noncoding region had two consensus binding sites (5'-GCCARG-3') for the transcription factor PacC mediating pH regulation. Quantitative real-time polymerase chain reaction analysis revealed that the transcription levels at pH 6.0 and 8.0 were 8-fold and 22-fold higher than that at pH 2.7, respectively. A cloned xynII cDNA was expressed and secreted in the yeast Pichia pastoris. Sequence alignment and phylogenetic analysis suggested that the XynII belongs to glycosyl hydrolase family 10 and that it is evolutionarily distant from two clusters formed by other family-10 xylanases.
Tanaka
Hidenori
H
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Japan.
Muguruma
Michio
M
Ohta
Kazuyoshi
K
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA
D003470:Culture Media; D043364:Endo-1,4-beta Xylanases