In this study, we purified and characterized the β-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified β-xylosidase (β-Xyl'ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that β-Xyl'ase Le-1 has a monomeric structure in plant cells. The N-terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N-glycans were used as substrates, β-Xyl'ase Le-1 showed optimum activity at about pH 5 at 40 °C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. β-Xyl'ase Le-1 hydrolyzed the β1-2 xylosyl residue from Man₁Xyl₁GlcNAc₂-PA, Man₁Xyl₁Fuc₁GlcNAc₂-PA, and Man₂Xyl₁Fuc₁GlcNAc₂-PA, but not that from Man₃Xyl₁GlcNAc₂-PA or Man₃Xyl₁Fuc₁GlcNAc₂-PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of β-Xyl'ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the β1-2 xylosyl residue by β-Xyl'ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.

We constructed a quintuple protease (alp, npII, pepE, npI, pepA) and double amylase (taaG3, taaG1) gene deletant, KO4, for the development of a heterologous gene expression system for an industrial shoyu koji mold, Aspergillus oryzae KBN616. Multiple gene deletion was performed using the Latour system, a simple and effective chromosome modification method developed in Schizosaccharomyces pombe. Gene deletion was confirmed by Southern blot analysis for protease genes and PCR amplification for amylase genes. Deletion of the five protease genes reduced the extracellular protease activity to approximately 1.0% of the level of the parent strain. Double deletion of the amylase genes completely eliminated all detectable α-amylase activity. These results indicate the construction of a host strain applicable to the efficient production and purification of heterologous proteins.

The gene, designated AsamyR, encoding a transcriptional activator involved in amylolytic gene expression was isolated from a shoyu koji mold, Aspergillus sojae KBN1340, and characterized. The structural gene comprised 1,951 bp with 2 introns. AsAmyR consisted of 595 amino acid residues possessing a high degree of sequence identity with other Aspergillus AmyRs. The AsamyR gene disruptant showed significantly restricted growth on starch agar plates and produced no detectable α-amylase production in SP medium. The multicopy AsamyR strain exhibited approximately two-fold higher amylase activity when compared to that of the control strain, but the increased rate of the amylase activity was not as high as the copy number of the integrated AsamyR gene.

No abstract