Lethal and mutagenic effects of ion beams and γ-rays in Aspergillus oryzae.

Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was (12)C(5+) ion beams with an LET of 121keV/µm. The (12)C(5+) ion beams had a 3.6-times higher lethal effect than low-LET (0.2keV/µm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. (12)C(6+) ion beams with an LET of 86keV/µm were the most effective in inducing selenate resistance. The mutant frequency following exposure to (12)C(6+) ion beams increased with an increase in dose and reached 3.47×10(-3) at 700Gy. In the dose range from 0 to 700Gy, (12)C(5+) ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67×10(-3)) at 400Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between (12)C(5+) ion beams and γ-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all cases. Although the incidence of deletions >2bp was generally low, deletions >20bp were characteristic for (12)C(5+) ion beams. γ-rays had a tendency to generate mutants carrying a multitude of mutations in the same locus. Both forms of radiation also induced genome-wide large-scale mutations including chromosome rearrangements and large deletions. These results provide new basic insights into the mutation breeding of A. oryzae using ionizing radiation.

Design of a new, multi-purpose, light-curing adhesive comprising a silane coupling agent, acidic adhesive monomers and dithiooctanoate monomers for bonding to varied metal and dental ceramic materials.

A newly designed, light-curing adhesive was investigated for its bonding effectiveness to porcelain, alumina, zirconia, Au, Au alloy, Ag alloy, Au-Ag-Pd alloy, and Ni-Cr alloy. Four experimental adhesives were prepared using varying contents of the following: a silane coupling agent [3-methacryloyloxypropyltriethoxysilane (3-MPTES)], acidic adhesive monomers [6-methacryloyloxyhexyl phosphonoacetate(6-MHPA),6-methacryloyloxyhexyl3-phosphonopropionate(6-MHPP)and 4-methacryloyloxyethoxycarbonylphthalic acid (4-MET)], and dithiooctanoate monomers [6-methacryloyloxyhexyl 6,8-dithiooctanoate (6-MHDT) and 10-methacryloyloxydecyl 6,8-dithiooctanoate (10-MDDT)]. After all adherend surfaces were sandblasted and applied with an experimental adhesive, shear bond strengths (SBSs) of a light-curing resin composite (Beautifil II, Shofu Inc., Kyoto, Japan) to the adherend materials after 2,000 times of thermal cycling were measured. For the experimental adhesive which contained 3-MPTES (30.0 wt%), 6-MHPA (1.0 wt%), 6-MHPP (1.0 wt%), 4-MET (1.0 wt%), 6-MHDT (0.5 wt%) and 10-MDDT (0.5 wt%), it consistently yielded the highest SBS for all adherend surfaces in the range of 20.8 (4.8)-30.3 (7.9) MPa, with no significant differences among all the adherend materials (p>0.05). Therefore, the newly designed, multi-purpose, light-curing adhesive was able to deliver high SBS to all the adherend materials tested.

Development of a new single-bottle multi-purpose primer for bonding to dental porcelain, alumina, zirconia, and dental gold alloy.

This study investigated the bonding efficacy of a combined primer application which comprised a silane coupling agent, an acidic adhesive monomer, and a dithiooctanoate monomer, as well as the influence of shelf life on bonding. Five experimental primers (coded as Si-P-SS-1 to Si-P-SS-4, and Si-SS as the control) were prepared using 20.0-40.0 wt% 3-methacryloyloxypropyltriethoxysilane (3-MPTES), 0-7.44 wt% 6-methacryloyloxyhexyl phosphonoacetate (6-MHPA), and 0.50 wt% 10-methacryloyloxydecyl 6,8-dithiooctanoate (10-MDDT). After 24-hour storage at 23°C (Initial) and 2-month storage at 50°C (Aged), tensile bond strengths (TBSs) of a resin cement (ResiCem, Shofu Inc., Kyoto, Japan) to primer-treated porcelain, alumina, zirconia, and Au alloy were measured. With the Initial and Aged primers of Si-P-SS-1 to Si-P-SS-3, there were no statistically significant differences in the mean TBSs (MPa) [porcelain: 21.7-29.2; alumina: 21.4-25.3; zirconia: 20.3-24.5; and Au alloy: 23.4-27.6] among these three primers (p>0.05), but they were significantly higher than that of the control primer (p<0.05). The experimental primers Si-P-SS-1 to Si-P-SS-3 demonstrated good potential as multi-purpose primers: they had good shelf lives as single-bottle primer systems and were thus able to exhibit good bond strength to all the adherends tested after 2-month storage under accelerated aging conditions.

Xylose triggers reversible phosphorylation of XlnR, the fungal transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus oryzae.

XlnR is a transcription factor that mediates D-xylose-triggered induction of xylanolytic and cellulolytic genes in Aspergillus. In order to clarify the molecular mechanisms underlying XlnR-mediated induction, Aspergillus oryzae XlnR was fused with the c-myc tag and examined by Western blotting. Phosphate-affinity SDS-PAGE revealed that XlnR was present as a mixture of variously phosphorylated forms in the absence of D-xylose, and that D-xylose triggered additional phosphorylation of the protein. D-Xylose-triggered phosphorylation was a rapid process occurring within 5 min prior to the accumulation of xynG2 mRNA, and removal of D-xylose caused slow dephosphorylation, leading to less-phosphorylated forms. At 30 min after removal, the phosphorylation status was almost identical to that in the absence of D-xylose, and the level of xynG2 mRNA started to decrease. These results indicate that XlnR is highly phosphorylated when it is active in transactivation, implying that D-xylose-triggered reversible phosphorylation controls XlnR activity.

Loss of Aspergillus oryzae amyR function indirectly affects hemicellulolytic and cellulolytic enzyme production.

Aspergillus oryzae AB390, a derivative of A. oryzae OR101, was found to be suitable for soy sauce production, yielding a product light brown in color. Compared to the parent strain, hemicellulase and cellulase activities in the mutant were higher; however, its amylase activity was found to be much lower. To determine the cause of these differences, the enzymatic profile change, as a function of the carbon source in submerged cultures, was examined. Amylase activity in AB390 was hardly detectable and not affected by the carbon source utilized. In the absence of starch where glucose could not be generated, hemicellulase and cellulase activities in both the parent and mutant were the same. A nonsense mutation was found in the upstream region of the putative transactivation domain of the transcriptional activator of the amylolytic genes, amyR in AB390. Complementation of AB390 with the wild-type amyR reduced hemicellulase and cellulase activities and increased amylase activity in soy sauce koji, the mold responsible for giving soy sauce. Northern analysis and two-dimensional (2-D) electrophoresis indicated that the unique enzymatic profile of AB390 was regulated transcriptionally. The results suggested that the loss of amyR function indirectly affected the production of hemicellulolytic and cellulolytic enzymes, likely through a carbon catabolite repression-mediated control.

Characterization of Aspergillus oryzae aspartyl aminopeptidase expressed in Escherichia coli.

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.