Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae.
The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C. It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.
Kimura
Tetsuya
T
Faculty of Bioresources, Mie University, Tsu, Japan. t-kimura@bio.mie-u.ac.jp
Suzuki
Hayato
H
Furuhashi
Hirofumi
H
Aburatani
Takeshi
T
Morimoto
Kenji
K
Sakka
Kazuo
K
Ohmiya
Kunio
K
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000235:genetics* / Q000302:isolation & purification
D004271:DNA, Fungal; D005656:Fungal Proteins; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase