Molecular Cloning, Characterization, and Expression Analysis of the xynF3Gene from Aspergillus oryzae

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzaeKBN616, used for making shoyu koji.The structural part of xynF3was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3was 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 xynF3gene was successfully overexpressed in A. oryzaeand 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°C. It had a Km of 6.5 mg/ml and a Vmaxof 435 μmol•min−1•mg−1when birch wood xylan was used as a substrate. Expression of the xynF3gene was analyzed using an Eschelichia coliβ-glucuronidase gene as a reporter. The result indicated that xynF3is expressed in the medium containing wheat bran as a carbon source.