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Hong S.-B.,National Academy of Agricultural Science | Yamada O.,Japanese National Research Institute of Brewing | Samson R.A.,Fungal Biodiversity Center
Applied Microbiology and Biotechnology | Year: 2014

Black koji molds including its albino mutant, the white koji mold, have been widely used for making the distilled spirit shochu in Northeast Asia because they produce citric acid which prevents undesirable contamination from bacteria. Since Inui reported Aspergillus luchuensis from black koji in Okinawa in 1901, many fungal names associated with black koji molds were reported. However, some species are similar and differentiation between species is difficult. Fungal taxonomists tried to arrange a taxonomic system for black koji molds, but the results were not clear. Recently, multi-locus sequence typing has been successfully used to taxonomy of black Aspergillus. According to β-tubulin and calmodulin gene sequences, black koji molds can be subdivided in three species, A. luchuensis, Aspergillus niger, and Aspergillus tubingensis. Aspergillus awamori, Aspergillus kawachii, Aspergillus inuii, Aspergillus nakazawai, and Aspergillus coreanus are synonyms of A. luchuensis, Aspergillus batatae, Aspergillus aureus (or Aspergillus foetidus), Aspergillus miyakoensis, and Aspergillus usamii (including A. usamii mut. shirousamii) are synonyms of A. niger and Aspergillus saitoi and A. saitoi var. kagoshimaensis are synonyms of A. tubingensis. A. luchuensis mut. kawachii was suggested particular names for A. kawachii because of their industrial importance. The history and modern taxonomy of black koji molds is further discussed. © 2013 Springer-Verlag Berlin Heidelberg. Source

Thongekkaew J.,Ubon Ratchathani University | Ikeda H.,Japanese National Research Institute of Brewing | Iefuji H.,Japanese National Research Institute of Brewing
Biochemical and Biophysical Research Communications | Year: 2012

To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from Saccharomyces cerevisiae (α factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27. kDa, respectively. The fusion enzyme was stable at 80°C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization. © 2012 Elsevier Inc. Source

Thongekkaew J.,Ubon Ratchathani University | Ikeda H.,Japanese National Research Institute of Brewing | Masaki K.,Japanese National Research Institute of Brewing | Iefuji H.,Ehime University
Enzyme and Microbial Technology | Year: 2013

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application. © 2013 Elsevier Inc. Source

Kume K.,Hiroshima University | Koyano T.,Hiroshima University | Kanai M.,Japanese National Research Institute of Brewing | Toda T.,Cancer Research UK Research Institute | Hirata D.,Hiroshima University
Nature Cell Biology | Year: 2011

Microtubules are central to eukaryotic cell morphogenesis. Microtubule plus-end tracking proteins (+TIPs) transport polarity factors to the cell cortex, thereby playing a key role in both microtubule dynamics and cell polarity. However, the signalling pathway linking +TIPs to cell polarity control remains elusive. Here we show that the fission yeast checkpoint kinase Cds1 (Chk2 homologue) delays the transition of growth polarity from monopolar to bipolar (termed NETO; new-end take-off). The +TIPs CLIP170 homologue Tip1 and kinesin Tea2 are responsible for this delay, which is accompanied by a reduction in microtubule dynamics at the cell tip. Remarkably, microtubule stabilization occurs asymmetrically, prominently at the non-growing cell end, which induces abnormal accumulation of the polarity factor Tea1. Importantly, NETO delay requires activation of calcineurin, which is carried out by Cds1, resulting in Tip1 dephosphorylation. Thus, our study establishes a critical link between calcineurin and checkpoint-dependent cell morphogenesis. © 2011 Macmillan Publishers Limited. All rights reserved. Source

Takahashi K.,Japanese National Research Institute of Brewing | Goto-Yamamoto N.,Japanese National Research Institute of Brewing
Journal of Chromatography A | Year: 2011

Free medium-chain fatty acids (MCFAs) can negatively influence the fermentation process and taste quality in alcoholic beverages. Ethyl hexanoate is important in providing a fruit-like flavour to drinks, particularly in Japanese sake. In this study, we developed a direct injection method for a gas chromatography-flame ionization detector following the semi-purification of chemical components, such as esters, alcohols and MCFAs in alcoholic beverages. Evaluation of MCFAs by this method gave a limit of detection on the order of sub-ppm and relative standard deviations less than 10% in standard solution. Good repeatability and recovery rates against MCFAs and ethyl hexanoate were also obtained in non-distilled real alcoholic beverages. Because this method enabled us to simultaneously quantify the concentrations of MCFAs and ethyl hexanoate, the proportion of ester against MCFAs was proposed as a quality control index. This method could be suitable for routine analysis in the alcohol beverage industry. © 2011 Elsevier B.V. Source

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