Mode of alpha-amylase production by the shochu koji mold Aspergillus kawachii

Aspergillus kawachii produces two kinds of alpha-amylase, one is an acid-unstable alpha-amylase and the other is an acid-stable alpha-amylase. Because the quality of the shochu depends strongly on the activities of the alpha-amylases, the culture conditions under which these alpha-amylases are produced were examined. In liquid culture, acid-unstable alpha-amylase was produced abundantly, but, acid-stable alpha-amylase was not produced. The acid-unstable alpha-amylase was produced significantly when glycerol or glucose was used as a carbon source, similarly to the use of inducers such as starch or maltose. In liquid culture, A. kawachii assimilated starch at pH 3.0, but no alpha-amylase activity was recognized in the medium. Instead, the alpha-amylase was found to be trapped in the cell wall. The trapped form was identified as acid-unstable alpha-amylase. Usually, acid-unstable alpha-amylase is unstable at pH 3.0, so its stability appeared to be due to its immobilization in the cell wall. In solid-state culture, both kinds of alpha-amylase were produced. The production of acid-stable alpha-amylase seems to be solid-state culture-specific and was affected by the moisture content in the solid medium.     

Antioxidative pyranonigrins in rice mold starters and their suppressive effect on the expression of blood adhesion molecules

Antioxidants having a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity in rice mold starters, which are used for the preparation of various Japanese fermented foods, and their effectiveness against the expression of blood adhesion molecules were examined. An antioxidant was isolated from the rice mold starters used for shochu and identified as pyranonigrin-S (PG-S) by (1)H-NMR, (13)C-NMR, and FAB-MS analyses. It was a derivative of pyranonigrin-A (PG-A), which has been isolated as an antioxidant from the rice mold starters. Pyranonigrins PG-A and PG-S were found to exist in spores on rice mold starters which had been prepared by Aspergillus awamori, A. kawachii, and A. saitoi. PG-S exhibited a higher level of DPPH radical scavenging activity than PG-A. PG-A was found to have a significant suppressive effect on the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-alpha (TNF-alpha) (P<0.05).     

The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall-bound beta-glucosidases

We cloned the genomic DNA and cDNA of bglA, which encodes beta-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound beta-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound beta-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal beta-glucosidases classified in subfamily B. We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant beta-glucosidase in the periplasm fraction of the recombinant yeast. A. kawachii can produce two extracellular beta-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound beta-glucosidase. A. kawachii in which the bglA gene was disrupted produced none of the three beta-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound beta-glucosidases in A. kawachii.     

Cloning and sequencing of the xynA gene encoding xylanase A of Aspergillus kawachii.

We have cloned the xynA gene coding for xylanase A, a major component of the xylanase family, from Aspergillus kawachii. The cDNA was isolated from an A. kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein. Nucleotide sequence analysis of the cDNA showed a 981-bp open reading frame that encoded a protein of 327 amino acid residues. The signal peptide was composed of 25 amino acid residues and the N-terminus of the mature protein was pyroglutamic acid. The transformed yeast with a cloned cDNA produced xylanase. The genomic DNA was arranged as ten exons and nine introns.     

Isolation of the antioxidant pyranonigrin-A from rice mold starters used in the manufacturing process of fermented foods

Rice mold starters prepared from Aspergillus species are commonly used for the manufacture of koji in the production of oriental fermented foods. Methanol extracts of rice mold starters fermented by the Aspergillus species, A. awamori, A. kawachii, A. oryzae, A. saitoi, and A. sojae, were examined for their antioxidative activity by using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging system. The extracts of A. awamori, A. kawachii, and A. saitoi exhibited higher activity than those of A. oryzae and A. sojae. An antioxidant was isolated from the extract of A. saitoi and identified as pyranonigrin-A by 1H-NMR, 13C-NMR, and FAB-MS analyses. The antioxidative activity of pyranonigrin-A was approximately equivalent to that of ferulic acid, an antioxidant in cereal grain. It was present in rice mold starters prepared by A. awamori, A. kawachii, and A. saitoi, although there was no pyranonigrin-A in the A. oryzae and A. sojae starters. The results suggest that the content of pyranonigrin-A in rice mold starters has a correlation with the antioxidative activity, and that it is induced in rice mold starters at the sporulation stage.     

Phenolic composition and radical scavenging activity of sweetpotato-derived shochu distillery by-products treated with koji

Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses. Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content. Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato. Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production. Caffeic acid was not detected in raw or steamed roots of "Koganesengan", the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation. The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar. These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions. Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid.     

Cloning and sequence analysis of endoglucanase genes from an industrial fungus, Aspergillus kawachii

Three endoglucanase genes (cel5A, cel5B, and cel61A) were cloned from an industrial fungus, Aspergillus kawachii. Yeasts transformed with these cDNAs showed endoglucanase activity in medium. Cel5A and Cel61A contained a type 1 cellulose-binding domain (CBD1) at the C-terminus of the enzyme. The putative catalytic regions of Cel5A and Cel5B showed homology with various endoglucanases belonging glycosyl hydrolase family 5 (GH5). Cel5B showed high homology with Cel5A in catalytic region, but it lacked CBD1 and linker. The cel5A contained four introns, whereas cel5B contained five introns. The putative catalytic region of Cel61A showed homology with enzymes belonging to GH61. The cel61A contained no introns.     

Purification, characterization, and partial primary sequence of a major-maltotriose-producing alpha-amylase, ScAmy43, from Sclerotinia sclerotiorum

A novel alpha-amylase (alpha-1,4-alpha-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Sclerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal alpha- amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and 55oC with an apparent Km value of 1.66 mg/ml and Vmax of 0.1 micromol glucose x min-1 x ml-1. ScAmy43 activity was strongly inhibited by Cu2+, Mn2+, and Ba2+, moderately by Fe2+, and was only weakly affected by Ca2+ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and beta-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.     

Metabolite profiling and bioactivity of rice koji fermented by Aspergillus strains

In this study, the metabolite profiles of three Aspergillus strains during rice koji fermentation were compared. In the partial least squares discriminant analysis-based gas chromatography-mass spectrometry data sets, the metabolite patterns of A. oryzae (KCCM 60345) were clearly distinguished from A. kawachii (KCCM 60552) and only marginal differences were observed for A. oryzae (KCCM 60551) fermentation. In the 2 days fermentation samples, the overall metabolite levels of A. oryzae (KCCM 60345) were similar to the A. oryzae (KCCM 60551) levels and lower than the A. kawachii (KCCM 60552) levels. In addition, we identified discriminators that were mainly contributing tyrosinase inhibition (kojic acid) and antioxidant activities (pyranonigrin A) in A. oryzae (KCCM 60345) and A. kawachii (KCCM 60552) inoculated rice koji, respectively. In this study, we demonstrated that the optimal inoculant Aspergillus strains and fermentation time for functional rice koji could be determined through a metabolomics approach with bioactivity correlations.     

Production of heterologous polygalacturonase I from Aspergillus kawachii in Saccharomyces cerevisiae in batch and fed-batch cultures

The pg1 gene from the filamentous fungus Aspergillus kawachii, which codifies for an acid polygalacturonase, was cloned into the pYES2 expression vector, giving rise to the pYES2:pg1ΔI construct. Engineered Saccharomyces cerevisiae, transformed with pYES2:pg1ΔI construct, both expressed and exported an active polygalacturonase with a MW of ~60 kDa and an isoelectric point of 3.7, similar to those reported for the wild-type enzyme. The recombinant enzyme has the ability to hydrolyze polygalacturonic acid at pH 2.5. Heterologous PG1 production was studied under controlled conditions in batch and fed-batch systems. A simultaneous addition of glucose and galactose was found to be the most suitable feeding strategy assayed, resulting in a final PG1 production of 50 U/ml. The production process proposed in this study could be applied for the industrial production of a novel and useful polygalacturonase.     

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.     

Kohninia linnaeicola, a new genus and species of the Sclerotiniaceae pathogenic to Linnaea borealis

A new genus and species is described to accommodate a newly discovered fungus pathogenic to Linnaea borealis. The fungus forms true sclerotia on stems and leaves of its host and apothecia arise singly or gregariously on the surface of ripe sclerotia. The new fungus was collected together with a stromatic conidiomal fungus that occurred on the same host. A putative teleomorph-anamorph connection of the observed taxa was ruled out by sequence comparison of the nuclear ribosomal internal transcribed spacer DNA sequences (ITS rDNA). Based on morphology and pathogenicity, the new fungus belongs in the family Sclerotiniaceae, Helotiales, Ascomycota. A phylogenetic analysis of ITS rDNA sequences from 26 taxa of the family Sclerotiniaceae was performed to conclude on the systematic position of the new fungus. The small tuberoid sclerotia, brownish subsessile to substipitate apothecia, four-spored asci, ellipsoid to isthmoid ascospores, inability to grow on PDA culture media and a number of ITS rDNA sequence autapomorphies characterize and distinguish the fungus from other taxa of the Sclerotiniaceae.     

Loss of Rim15p in shochu yeast alters carbon utilization during barley shochu fermentation

ABSTRACT

Rim15p of the yeast Saccharomyces cerevisiae is a Greatwall-family protein kinase that inhibits alcoholic fermentation during sake brewing. To elucidate the roles of Rim15p in barley shochu fermentation, RIM15 was deleted in shochu yeast. The disruptant did not improve ethanol yield, but altered sugar and glycerol contents in the mash, suggesting that Rim15p has a novel function in carbon utilization.     

Use of acid-tolerant xylanase for bleaching of kraft pulps

A xylanase from Aspergillus kawachii, active at pH 2.0–2.5, was found to be suitable for improvement of bleachability. Due to the low working pH of the xylanase, the metal cations were also removed and thus the metal removal stage using chelating agents could be omitted. The use of acid-tolerant xylanase is especially beneficial prior to ozone or chlorine dioxide bleaching stages due to the minimization of pH adjustment steps during bleaching.     

Purification and partial characterization of an acidic polygalacturonase from Aspergillus kawachii

An endo-polygalacturonase, named PGI, was purified to homogeneity from the culture filtrate of Aspergillus kawachii IFO 4033 grown in a glucose-tryptone medium. The molecular mass of PGI was estimated to be 60 kDa by SDS-PAGE and 40 kDa by gel filtration on Sephacryl S-100. The isoelectric point was 3.55 as determined by isoelectic focusing. PGI exhibited binding properties to ConA-Sepharose suggesting that the protein is glycosylated. The N-terminal amino acid sequence was also determined as S-T-C-T-F-T-D-A-A-T-A-S-E-S-K. The remarkable property of PGI was its high activity in the pH range 2.0-3.0 towards soluble and insoluble substrates, while being inactive at pH 5.0. Enzyme stability at low pHs was markedly enhanced by different compounds, such as proteins, polysaccharides, simple sugars and the substrate pectin. PGI was very efficient to extract pectin from lemmon protopectin and to macerate carrot tissues at pH 2.0. These properties make PGI an interesting biocatalyst for industrial applications under highly acidic conditions.     

Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P-450 cDNA from Fusarium oxysporum

A cDNA clone for the nitrate/nitrite-inducible cytochrome P-450 (P-450) of the fungus Fusarium oxysporum (tentatively termed P-450dNIR) was isolated by an immunoscreening method. Sequence determination revealed a polypeptide of 403 amimo acid residues (Mr = 44,371), which was shown to contain the full-length sequence of the fungal P-450. The amino terminus region of the predicted sequence contained neither the signal-like, hydrophobic domain that is commonly observed in microsomal P-450s nor the tagging prosequence that is essential for localization of mitochondrial P-450s. Further, the sequence exhibited higher homologies against those of soluble bacterial P-450s, in particular P-450s of Streptomyces, rather than those of eukaryotic P-450s including yeast and fungal P-450s. These results are highly indicative that P-450dNIR is the first soluble P-450 derived from eukaryotic organisms. The unique features might be related to the novel function of P-450dNIR, which is involved in a dissimilatory reduction of nitrite by the fungus. P-450dNIR was classified into a new family, P-450LV, and the corresponding gene of the fungus was named CYP55.     

Haradamyces foliicola anam. gen. et sp. nov., a cause of zonate leaf blight disease in Cornus florida in Japan

A fungus causing zonate leaf blight diseases in various evergreen and deciduous woody plant species in Japan was characterized by a discoid multicellular propagule arising from a hyaline sclerotium-like structure in the leaf tissue and dark-coloured microconidia produced enteroblastically from the terminal cells on the surface of the discoid propagules. Myrioconium-like microconidiophores also producing microconidia were occasionally produced in culture. No teleomorphic characteristics were observed on the fungus. Molecular analysis based on the partial nu-rDNA sequence data revealed that the fungus was phylogenetically related to the Sclerotiniaceae, Leotiomycetes, and Ascomycota. Because the morphology and sequence data of this fungus does not coincide with those of any known anamorphic fungi, Haradamyces foliicola is proposed here as a new anamorphic genus and species for this fungus.     

Draft Genome Sequence of Aspergillus oryzae Strain 3.042

Aspergillus oryzae is the most important fungus for the traditional fermentation in China and is particularly important in soy sauce fermentation. We report the 36,547,279-bp draft genome sequence of A. oryzae 3.042 and compared it to the published genome sequence of A. oryzae RIB40.