Visualization of nuclei in Aspergillus oryzae with EGFP and analysis of the number of nuclei in each conidium by FACS.
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Aspergillus oryzae has been reported to form conidia with multinuclei. In order to analyze nuclei in living cells, we developed an expression system of the A. nidutans histone H2B protein tagged by EGFP (H2B::EGFP). In both A. oryzae niaD300 and A. nidulans FGSC89 transformants expressing H2B::EGFP, fluorescence was detected in nuclear regions of hyphae and conidia. While a conidium contained only one fluorescent spot in the A. nidulans transformant, approximately 66% of conidia had two, 24% had one, and 10% had three or more in the A. oryzae transformant. The conidia expressing H2B::EGFP were put through FACS (fluorescence-activated cell sorting) analysis and two sharp peaks, corresponding to one and two nuclei in each conidium, were noted in the A. oryzae transformant. In addition, the A. oryzae uninucleate conidia that were successfully isolated by FACS reproduced conidia with almost the same number distribution of nuclei as that of the original. Conidia of five A. oryzae strains used in sake brewing were scored for the number of nuclei, showing that a varied number of nuclei existed in each conidium and some strains had a small number of uninucleate conidia. (+info)
On the involvement of electron transfer reactions in the fluorescence decay kinetics heterogeneity of proteins.
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Time-resolved fluorescence study of single tryptophan-containing proteins, nuclease, ribonuclease T1, protein G, glucagon, and mastoparan, has been carried out. Three different methods were used for the analysis of fluorescence decays: the iterative reconvolution method, as reviewed and developed in our laboratory, the maximum entropy method, and the recent method that we called "energy transfer" method. All the proteins show heterogeneous fluorescence kinetics (multiexponential decay). The origin of this heterogeneity is interpreted in terms of current theories of electron transfer process, which treat the electron transfer process as a radiationless transition. The theoretical electron transfer rate was calculated assuming the peptide bond carbonyl as the acceptor site. The good agreement between experimental and theoretical electron-transfer rates leads us to suggest that the electron-transfer process is the principal quenching mechanism of Trp fluorescence in proteins, resulting in heterogeneous fluorescence kinetics. Furthermore, the origin of apparent homogeneous fluorescence kinetics (monoexponential decay) in some proteins also can be explained on the basis of electron-transfer mechanism. (+info)
Intake, digestion, and digestive characteristics of Neotyphodium coenophialum-infected and uninfected fescue by heifers offered hay diets supplemented with Aspergillus oryzae fermentation extract or laidlomycin propionate.
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Tarentaise heifers fitted with a rumen cannula (539 +/- 7.5 and 487 +/- 15.7 kg avg initial BW in Exp. 1 and 2, respectively) were used in two Latin square metabolism experiments having 2 x 2 factorial treatment arrangements to determine the effects of supplementation with Aspergillus oryzae fermentation extract (AO) or laidlomycin propionate (LP) on intake, digestion, and digestive characteristics of Neotyphodium coenophialum-infected (IF) or uninfected (FF) tall fescue (Festuca arundinacea) hay diets consumed ad libitum. Heifers were housed in individual stanchions in a metabolism facility with ambient temperatures controlled to range between 26.7 and 32.2 degrees C daily. Total feces and urine were collected for 5 d following a 21-d dietary adaptation period. In situ DM and NDF disappearance and ruminal fermentation characteristics were also determined. In Exp. 1, DMI was 24% greater (P < 0.01) by heifers offered FF than by those offered IF (6.7 vs 5.4 kg/d). Heifers fed 2 g/d AO tended (P = 0.09) to consume 4% more DM than those fed a diet without AO. Degradable DM and NDF fractions of IF were greater (P < 0.01) than those of FF, but AO supplementation did not affect situ disappearance (P > or = 0.42). In Exp. 2, DMI was 18.9% greater (P < 0.01) by heifers offered FF than by those offered IF (6.6 vs 5.5 kg/d). Heifers fed LP (50 mg/d) consumed 10.6% less (P < 0.05) DM than those not fed LP (5.7 vs 6/5 kg/d). Digestibility of NDF tended to be greater (P = 0.08) and digestibility of ADF was greater (P < 0.05) from FF than from IF. Conversely, apparent N absorption (%) was greater (P < 0.05) from IF than from FF. Heifers fed LP had lower (P < 0.05) ADF digestibility than those not fed LP. In situ degradable DM and NDF fractions were greater (P < 0.01) from IF than from FF. Diets supplemented with LP had higher (P < 0.01) indigestible DM and NDF fractions than those without LP. Propionic acid and total VFA concentrations were greater (P < 0.05) from heifers offered FF than from those offered IF and from heifers fed LP than from those not fed LP. Therefore, it appears the major effect of N. coenophialum was a reduction in forage intake and total-tract fiber digestibility in certain situations. Response to the feed additives was similar whether heifers were offered IF or FF and no evidence was apparent that either additive would improve performance substantially by animals consuming low-quality fescue hay diets. (+info)
Metabolic engineering of the morphology of Aspergillus oryzae by altering chitin synthesis.
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Morphology and alpha-amylase production during submerged cultivation were examined in a wild-type strain (A1560) and in strains of Aspergillus oryzae in which chitin synthase B (chsB) and chitin synthesis myosin A (csmA) have been disrupted (ChsB/G and CM101). In a flowthrough cell, the growth of submerged hyphal elements was studied online, making it possible to examine the growth kinetics of the three strains. The average tip extension rates of the CM101 and ChsB/G strains were 25 and 88% lower, respectively, than that of the wild type. The branching intensity in the CM101 strain was 25% lower than that in the wild type, whereas that in the ChsB/G strain was 188% higher. During batch cultivation, inseparable clumps were formed in the wild-type strain, while no or fewer large inseparable clumps existed in the cultivations of the ChsB/G and CM101 strains. The alpha-amylase productivity was not significantly different in the three strains. A strain in which the transcription of chsB could be controlled by the nitrogen source-regulated promoter niiA (NiiA1) was examined during chemostat cultivation, and it was found that the branching intensity could be regulated by regulating the promoter, signifying an important role for chsB in branching. However, the pattern of branching responded very slowly to the change in transcription, and increased branching did not affect alpha-amylase productivity. alpha-Amylase residing in the cell wall was stained by immunofluorescence, and the relationship between tip number and enzyme secretion is discussed. (+info)
The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds.
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The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin. (+info)
Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae.
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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. (+info)
Functional expression in Aspergillus oryzae of p15, a protein with potent neurite-inducing activity in PC12 cells.
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We previously reported that a fungal protein, p15, induces neurite outgrowth and differentiation of rat pheochromocytoma PC12 cells through the activation of the Ca2+ signaling pathway. We report here the secretory production of p15 in Aspergillus oryzae. Analysis of culture supernatant of A. oryzae transformed with the gene encoding the p15 precursor tagged with a hemagglutinin (HA) epitope demonstrated that the transformant secreted a protein with an apparent molecular mass of 17.5 kDa, which is a little larger than the expected size of mature p15-HA. By heat denaturation and ion exchange chromatography, p15-HA was easily purified from the culture supernatant with sufficient abundance. Although purified p15-HA was less active than the native p15 obtained from the culture broth of a producing fungal strain, it had neurite-inducing activity in PC12 cells in a dose-dependent manner, providing a system to study the action mechanism of p15. (+info)
A simple method for enrichment of uninucleate conidia of Aspergillus oryzae.
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Aspergillus oryzae produces multinucleate conidia, which makes the obtaining of homokaryons labor-intensive. Analysis of conidia by flow cytometry clarified the relationship that conidia of lower nuclear number were smaller in size. Based on this, we have developed a simple way to enrich uninucleate conidia with a membrane filter. Our results also suggest that the method is useful for elimination of heterokaryons. (+info)