HAP-01, the first chromogenic substrate for Aspergillus oryzae acid protease.

The first chromogenic substrate for Aspergillus oryzae acid protease, 'HAP-01', was successfully developed after evaluating the substrate specificity of the enzyme. Furthermore, with HAP-01, digestion-triggered chromophore release was employed as a novel chromogenic technique.

Analyzing the relationship between the inorganic element profile of sake dilution water and dimethyl trisulfide formation using multi-element profiling.

Dimethyl trisulfide (DMTS) is the main component of hineka, an off-flavor generated in sake during storage. Genshu, or undiluted sake, is usually diluted with water during warimizu, the process of adjusting the alcohol content of sake. In this study, we evaluated how the inorganic element composition of sake dilution water affects the DMTS-producing potential of the sake (DMTS-pp, determined as the DMTS concentration in sake stored at 70°C for 1 week after dilution) using inductively coupled plasma-mass spectrometry (ICP-MS). Partial least squares (PLS) regression analysis was conducted with the ICP-MS data as the explanatory variable and DMTS-pp as the response variable, and the selection of inorganic elements for the construction of the PLS model was performed using variable importance in projection scores. The findings confirmed that some of the compounds containing the inorganic elements extracted from the PLS regression analysis contribute to DMTS-pp.

Characterization and identification of partial amino acid sequence of a novel elastase inhibitor, Asnidin from Aspergillus nidulans.

A novel elastase inhibitor from Aspergillus nidulans NBRC 4340, Asnidin, was isolated, and biochemical properties and partial amino acid sequence were examined. Column chromatography using diethylaminoethyl (DE) 52-Cellulose and reversed-phase HPLC were used to purify the inhibitor. Purified Asnidin was found to be homogeneous as indicated by reversed-phase HPLC and TOF-MS (Time of Flight Mass Spectrometry). Asnidin has a molecular weight of 4,181.63 as determined by TOF-MS. The elastolytic activities of elastases from A. fumigatus, A. flavus, and human leukocytes but not chymotrypsin, and elastases from snake venom and bacteria were inhibited by Asnidin. The fibrinogenase and collagen type IV hydrolytic activities of the elastase from A. fumigatus were inhibited by Asnidin. Asnidin was found to be stable under heat treatment and over a wide pH range. The elastolytic inhibitory activity of Asnidin was inhibited by dithiothreitol (DTT), while no inhibition was observed with ethylenediaminetetraacetic acid (EDTA-2Na) and benzamidine. Since there is a possibility of Asnidin becoming another drug in the arsenal of weapons against aspergillosis or interstitial pneumonia, further studies are warranted.

X-ray structure analysis and characterization of AFUEI, an elastase inhibitor from Aspergillus fumigatus.

Elastase from Aspergillus sp. is an important factor for aspergillosis. AFUEI is an inhibitor of the elastase derived from Aspergillus fumigatus. AFUEI is a member of the I78 inhibitor family and has a high inhibitory activity against elastases of Aspergillus fumigatus and Aspergillus flavus, human neutrophil elastase and bovine chymotrypsin, but does not inhibit bovine trypsin. Here we report the crystal structure of AFUEI in two crystal forms. AFUEI is a wedge-shaped protein composed of an extended loop and a scaffold protein core. The structure of AFUEI shows remarkable similarity to serine protease inhibitors of the potato inhibitor I family, although they are classified into different inhibitor families. A structural comparison with the potato I family inhibitors suggests that the extended loop of AFUEI corresponds to the binding loop of the potato inhibitor I family, and AFUEI inhibits its cognate proteases through the same mechanism as the potato I family inhibitors.

High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae.

AFUEI, an elastase inhibitor produced by Aspergillus fumigatus strongly inhibits the elastolytic activity of A. fumigatus etc. To purify AFUEI, we constructed a strain that overproduces AFUEI by introducing the gene encoding AFUEI (Genbank accession no. AB546725) under control of the amyB promoter into the heterologous host Aspergillus oryzae. A. oryzae TF-4 displayed strong elastase inhibitory activity and produced considerably more AFUEI than that of A. fumigatus. Furthermore, AFUEI could be purified using culture broth and single ultrafiltration (UF) treatment, allowing for the effective production of AFUEI for use in clinical trials.

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.

The promoter activity of isovaleryl-CoA dehydrogenase-encoding gene (ivdA) from Aspergillus oryzae is strictly repressed by glutamic acid.

We cloned the isovaleryl-CoA dehydrogenase (IVD)-encoding gene from Aspergillus oryzae. The promoter of ivdA was subjected to beta-glucuronidase (GUS) reporter assays in which certain amino acids were used as a major carbon source. L-leucine most strongly induced GUS-activity, while in the case of L-glutamate, significantly low activity was found, indicating that ivdA transcription was strongly repressed by glutamic acid.

Thiamine-regulated gene expression of Aspergillus oryzae thiA requires splicing of the intron containing a riboswitch-like domain in the 5'-UTR.

Exogenous thiamine regulates Aspergillus oryzae thiA, which is involved in thiamine synthesis. One of the two introns in its 5'-untranslated region (5'-UTR) contains motifs (regions A and B) highly conserved among fungal thiamine biosynthesis genes. Deletion of either region relieved the repression by thiamine and thiamine inhibited intron splicing, suggesting that regions A and B are required for efficient splicing. Furthermore, transcript splicing was essential for thiA gene expression. These observations suggest a novel gene expression regulatory mechanism in filamentous fungi, in which exogenous thiamine controls intron splicing to regulate gene expression. Interestingly, regions A and B constitute a part of a thiamine pyrophosphate-binding riboswitch-like domain that has been quite recently found in the 5'-UTR of thiA.

Molecular breeding of the Mureka-non-forming sake koji mold from Aspergillus oryzae by the disruption of the mreA gene.

Mureka-non-forming sake koji molds were constructed from an Aspergillus oryzae industrial strain by the disruption of the mreA gene using a host-vector system with the ptrA gene as a dominant selectable marker. All of the mreA gene disruptants obtained retained the advantages of the host strain in terms of the brewing characteristics, while their isoamyl alcohol oxidase (IAAOD) activities were significantly lower than that of the host strain. Sake brewing was successfully carried out using the koji prepared with the disruptants, followed by storage of the resultant non-pasteurized sake (nama-shu). The isovaleraldehyde (i-Val) concentration in the sake brewed the host strain increased rapidly and reached the threshold values for mureka, 1.8 ppm and 2.6 ppm after storage at 20 degrees C for 42 d and 63 d, respectively, while those of the disruptants were less than 0.5 ppm even after storage at 20 degrees C or 30 degrees C for 63 d. In the sensory evaluation of the sake stored at 20 degrees C or 30 degrees C for 63 d, all members of the panel recognized the strong mureka flavor of the sake brewed with the host strain, while they did not detect this flavor in the sake brewed with the disruptants. Thus, we concluded that the mreA gene disruptants can be used for the production of sake in which mureka is not formed.

Transformation of Aspergillus sp. and Trichoderma reesei using the pyrithiamine resistance gene (ptrA) of Aspergillus oryzae.

A pyrithiamine (PT) resistance gene (ptrA) was cloned from a PT resistant mutant of Aspergillus oryzae and was useful as a dominant selectable marker for transformation of all A. oryzae wild type strain as well as A. nidulans. For further study, we examined whether or not ptrA could be used as the transformation marker in other species of filamentous fungi. Two types of plasmid, which contain ptrA as a selectable marker, were constructed, and the transformation experiments were done with them. One is an integrative plasmid, pPTRI, and another is the autonomously replicating plasmid pPTRII, which contains AMA1. PT-resistant transformants were obtained in the cases of A. kawachii, A. terreus, A. fumigatus, and Trichoderma reesei as hosts with pPTRI and pPTRII. Furthermore, a beta-glucuronidase (GUS) gene was introduced into A. kawachii and A. fumigatus using pPTRII. Almost all the transformants turned blue on GUS assay plates. These results indicate that ptrA can also be used for some other filamentous fungi besides A. oryzae and A. nidulans.