Expression of Aspergillus oryzae phytase gene in Aspergillus oryzae RIB40 niaD(-)

Aspergillus oryzae RIB40 niaD(-) was transformed using a plasmid constructed with the A. oryzae phytase gene and pNAN8142 vector. The culture broth of the transformant, which was grown in a medium containing starch as a carbon source and polyvinylpyrrolidone showed phytase activity of a maximum of 2.0 units ml(-1) at 37 degrees C, pH 5.5.     

Cloning and expression analysis of two catalase genes from Aspergillus oryzae

Fungi contain distinct genes encoding the same class of enzyme that are differentially regulated according to conditions. We cloned two catalase genes, catA and catB, from Aspergillus oryzae. The catA gene predicts a 747-amino-acid polypeptide sharing 81% identity with Aspergillus fumigatus catalase (catA) and 77% with Aspergillus nidulans catalase (catA). The catB gene predicts a 725-amino-acid polypeptide sharing 82% identity with A. fumigatus catalase (catB) and 75% with A. nidulans catalase (catB). However, the catA and catB genes share little homology (41%) with one another, suggesting that each gene belongs to a distinct gene family. Overexpression studies demonstrated that both genes encode a functional catalase. Promoter assays indicated that the catA gene is developmentally regulated as it was preferentially expressed in solid-state cultures undergoing sporulation. However, its expression was not affected by hydrogen peroxide treatment. Conversely, the catB gene was highly expressed under all culture conditions tested, and it was induced by hydrogen peroxide treatment. These results suggest that the catB gene may be mainly used for detoxification of oxidative stress while the catA gene may have another role such as chaperoning proteins in the spore.     

Stable repeated-batch production of recombinant dye-decolorizing peroxidase (rDyP) from Aspergillus oryzae

Recombinant Aspergillus oryzae expressing a dye-decolorizing peroxidase gene (dyp) was cultivated for repeated-batch production of recombinant dye-decolorizing peroxidase (rDyP) using maltose as a carbon source. High-level rDyP activity in limitation of carbon and nitrogen sources was maintained stably for 26 cycles of repeated 1-d batches of A. oryzae pellets without any additional pH control. Cultures maintained at 4 degrees C for 20 d resumed rDyP production following a single day of incubation. One liter filtrated crude rDyP containing 4600 U rDyP decolorized 5.07 g RBBR at the apparent decolorization rate of 17.7 mg l(-1) min(-1).     

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27°C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.     

High-throughput genotyping of filamentous fungus Aspergillus oryzae based on colony direct polymerase chain reaction

High-throughput genotyping of Aspergillus oryzae was achieved using an FTA card for the extraction of a genomic DNA template for polymerase chain reaction from a fungal colony growing on an agar plate. This method was then applied to detect other fungal species from agar slants and food materials. This method offers a convenient tool for the genotyping of filamentous fungi without using an organic solvent or specialized equipment.     

Production and partial purification of proteases from Aspergillus oryzae grown in a medium based on whey protein as an exclusive nitrogen source

Several attempts have been made to incorporate whey proteins into curd to increase cheese yield. For some types of cheese, degradation of whey proteins that have been incorporated into the curd would be required to obtain acceptable flavor and texture. On the basis of the high potential for protease synthesis in Aspergillus oryzae, sodium nitrate as a nitrogen source in a minimal medium for fungi, known as Czapek-Dox medium, was replaced with whey protein isolate to induce the protease to hydrolyze whey protein using A. oryzae AHU7146. A solid-phase medium adjusted to pH 6 was suitable for this purpose when incubation was carried out at 25°C for 2 wk. The application of column chromatography enabled the resolution of 3 proteolytic components (1, 2, and 3). With respect to optimal temperature and zymographic analysis, component 1 was similar to component 3. In contrast, component 2 was less abundant than the other components and exhibited activity in the alkaline pH region. The degradation of β-lactoglobulin and α-lactalbumin in whey protein isolate solution by the crude enzyme was primarily attributed to the action of components 1 and 3, based on HPLC analysis and the N-terminal amino acid sequences; however, zymography demonstrated evident proteolysis due to component 2. Because heat-denatured whey protein aggregates were digestible by the crude enzyme, the proteolytic system from A. oryzae has the potential as an additive to stimulate the ripening of cheese enriched with whey protein.     

Construction of a thiamine pyrophosphate high-producing strain of Aspergillus oryzae by overexpression of three genes involved in thiamine biosynthesis

We have found a gene (thiP) encoding thiamine pyrophosphokinase (TPK) in the Aspergillus oryzae genome. No riboswitch-like region was found in the upstream region of thiP, although it was repressed probably by thiamine pyrophosphate (TPP) as well as thiA and nmtA, which are strictly regulated by TPP-riboswitch sequence. To improve the productivity of TPP in A. oryzae, we constructed the strain in which thiA, nmtA and thiP were overexpressed simultaneously. The resulting strain accumulated intracellular TPP 4-fold higher than did the control strain.     

Uniform culture in solid-state fermentation with fungi and its efficient enzyme production

Solid-state fermentation (SSF) has attracted a lot of interest for carrying out high-level protein production in filamentous fungi. However, it has problems such as the fermentation heat generated during the culture in addition to the reduced mobility of substances. These conditions lead to a nonuniform state in the culture substrate and result in low reproducibility. We constructed a non-airflow box (NAB) with a moisture permeable fluoropolymer membrane, thereby making it possible to control and maintain uniform and optimal conditions in the substrate. For the NAB culture in Aspergillus oryzae, temperature and water content on/in the whole substrate were more consistent than for a traditional tray box (TB) culture. Total weight after the culture remained constant and dry conditions could be achieved during the culture. These data demonstrate the possibility of growing a uniform culture of the whole substrate for SSF. The NAB is advantageous because it allows for the control of exact temperature and water content in the substrate during the culture by allowing vapor with latent heat to dissipate out of the box. In addition, several enzymes in the NAB culture exhibited higher production levels than in the TB culture. We believe that culturing in the constructed NAB could become a standard technique for commercial SSF.     

Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae

Isoamyl alcohol oxidase (IAAOD) is a novel enzyme that catalyzes the formation of isovaleraldehyde, which is the main component of mureka that gives sake an off-flavor (Yamashita et al. Biosci. Biotechnol. Biochem., 63, 1216–1222, 1999). We cloned the genomic DNA sequence encoding IAAOD from a koji mold, Aspergillus oryzae, using a PCR-amplified DNA fragment corresponding to the partial amino acid sequences of the purified protein as a probe. The cloned gene comprises 1903 bp of an open reading frame with three putative introns and encodes 567 amino acids with a presumed signal peptide consisting of 24 amino acids at the N-terminus. Moreover, nine potential N-glycosylation sites were present. Homology search on amino acid sequence showed that IAAOD has a region significantly similar to those conserved in FAD-dependent oxidoreductases. Southern hybridization analysis revealed that the cloned gene exists as a single copy in the A. oryzae RIB 40 chromosome. The cloned gene was overexpressed under the control of the amyB promoter in A. oryzae. The isovaleraldehyde-producing activity in the culture supernatant of one transformant was over 800 times as high as that of transformant with the control vector. This result demonstrates that the cloned gene encodes IAAOD. We named this novel alcohol oxidase gene “mreA”.     

Cloning of Aspergillus oryzae Aovps5 gene,homologous to vacuolar protein sorting associated gene VPS5 and construction of the disruptant

Aovps5 gene was isolated from Aspergillus oryzae as a homologue to S. cerevisiae VPS5 gene which encodes a polypeptide consisting of 451 amino acids that is nearly 32% homologous to Vps5p. Three Aovps5 gene disruptants were generated and they showed higher activity of tripeptidyl peptidase, which is mainly detected in vacuoles, in their culture medium. Higher amount of nitrogenous constituent was found in the filtrate of wheat gluten degraded by addition of culture medium of these disruptants than that of wild type strain. These results suggest that disruption of Aovps5 may contribute to production of fermented foods.     

Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease

Angiotensin-I converting enzyme inhibitory activities were measured after hydrolysis of casein by 9 different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest angiotensin-I converting enzyme inhibitory activity per peptide. The A. oryzae peptide also showed the highest antihypertensive effect in spontaneously hypertensive rats when the systolic blood pressure was measured 5 h after oral administration of 32 mg/kg of various enzymatic hydrolysates. Significant antihypertensive effects were observed with dosages of 9.6, 32, and 96 mg of the A. oryzae peptide/kg of body weight (BW), and the effects were dependent on these peptide dosages.Analysis of peptide length showed the A. oryzae hydrolysate was the shortest of all tested casein hydrolysates; the peptide mixture had an average value of 1.4 amino acids (AA) in the sequence. To further characterize the A. oryzae hydrolysate, we analyzed the AA sequence of the whole peptide mixture. Various AA were detected at the first AA position, however, an increased number of Pro residues were observed at the second and third position of the A. oryzae hydrolysate. No strong signals were detected after the fourth AA position of the A. oryzae hydrolysate. These results suggest that the casein hydrolysate of A. oryzae, which expressed potent antihypertensive effects in spontaneously hypertensive rats, mainly contain short peptides of X-Pro and X-Pro-Pro sequences.     

Cloning and optimized expression of a neutral endoglucanase gene (ncel5A) from Volvariella volvacea WX32 in Pichia pastoris

A cDNA fragment encoding a mature neutral endoglucanase with 366 amino acids was cloned from Volvariella volvacea WX32. Online analysis of amino acid sequence homology showed that the endoglucanase, designated as NCel5A, belongs to glycoside hydrolase family 5. The recombinant plasmid, pPIC9K-ncel5A, was transformed into Pichia pastoris GS115 by electroporation. Screening of multiple copies of the gene ncel5A in transformants was performed on YPD plates containing geneticin G418. One transformant expressing the highest recombinant NCel5A (rNCel5A) activity, numbered as GSNCel4-3, was chosen for optimizing expression conditions. In optimized conditions, the expressed rNCel5A activity was up to 4612 U/ml. SDS-PAGE and enzyme activity assays demonstrated that the rNCel5A, a glycosylated protein with an M.W. of about 42 kDa, was extracellularly expressed in P. pastoris. The rNCel5A showed the highest activity at pH 7.5 and 55°C and was stable at a broad pH range of 6.0-9.0 and at a temperature of 55°C or below.