Hisashi Fukuda

Production and Properties of Phytase and Acid Phosphatase from a Sake Koji Mold,Aspergillus oryzae

We identified three types of acid phosphatase (ACP-I, ACP-II, and ACP-III) produced by Aspergillus oryzae in a submerged culture using only phytic acid as the phosphorous substrate. The optimum pH for the activities of the three enzymes was in the range of 4.5 to 5.5. Analysis of the substrate specificities of these enzymes revealed that ACP-I and ACP-III were acid phosphatases, and ACP-II was a phytase. These enzymes were produced during different periods of mycelial growth: ACP-II was produced during the early phase of cultivation (around 24 h), and ACP-I was produced between 24 to 72 h. ACP-III was detected after the production of ACP-I and ACP-II had ceased. The release of phosphate from phytic acid was expected to be due to the cooperative hydrolysis of these enzymes.

Production of cellulose- and xylan-degrading enzymes by a koji mold, aspergillus oryzae, and their contribution to the maceration of rice endosperm cell wall

The production of cellulose- (CEL), xylan- (XYL), and pectin-degrading enzymes (PEC) by a koji mold, Aspergillus oryzae, was studied, and their contributions to the maceration of the rice endosperm cell wall were investigated with regard to the utilization of available rice in the sake mash. The sake koji mold showed higher CEL and XYL productivities, whereas the miso and soy sauce koji molds showed higher PEC productivity. Statistical analyses indicated that CEL and XYL contribute predominantly and synergistically to the maceration of the rice endosperm cell wall. A. oryzae produced at least three kinds of CEL (Cel-1, 2, 3) and two kinds of XYL (Xyl-1, 2) when cultured in a wheat bran medium. In the solid-state culture, the production of Cel-3 and Xyl-2 was markedly stimulated by decreasing the moisture content of the solid substrate, although the production levels of Cel-1 and Xyl-1 were almost the same. These data suggest that the production of Cel-3 and Xyl-2 is strongly influenced by culture conditions, and that water activity is one of the dominant factors in the regulation of their production.

Critical importance of phytase for yeast growth and alcohol fermentation in Japanese sake brewing

The high phytase producing mutant of Aspergillus oryzae (KL-38) previously isolated was employed for koji making, and the produced koji rice then supplied for sake brewing. The alcohol fermentation was improved compared to that with the parent strain (A. oryzae BP-1). The effects of two phytase isozymes (Phy I and Phy II) produced by A. oryzae on yeast growth and inorganic phosphate liberation were investigated using a synthetic medium containing phytic acid as a sole phosphate source. Yeast growth and the liberation of inorganic phosphate were both enhanced by the combination of Phy I and Phy II at a ratio of 1 to 3, which was compatible with the production ratio in KL-38. Based on these results, phytase plays important role in sake brewing, and that the maximum inorganic phosphate liberation from phytic acid can be obtained by a suitable combination of Phy I and Phy II.

Properties of cellulose-degrading enzymes from Aspergillus oryzae and their contribution to material utilization and alcohol yield in sake mash fermentation.

Four cellulose-degrading enzymes were identified in a solid-state culture of Aspergillus oryzae. The three major enzymes were purified and named Cel-1, Cel-2, and Cel-3, respectively. The molecular weights were determined to be 62, 120, and 34 kDa, respectively. The optimum temperature of Cel-3 activity was higher than that of the other enzymes. An acidic pH was found to be more suitable for Cel-1 activity than for the other enzymes, and Cel-3 was more stable under acidic conditions than the other two. These properties and the results of a protein homology search for N-terminal amino acid sequences suggest that Cel-1 and Cel-3 correspond to the previously isolated endo-1,4-beta-glucanase CelB and CelA, respectively. The analysis of substrate specificity suggested that Cel-2 is likely to be beta-glucosidase. The effect of Cel-1, Cel-2, and Cel-3 on the sake mash fermentation was determined and it was found that Cel-2 markedly improved material utilization and alcohol yield in sake mash fermentation.

Contribution of ethanol-tolerant xylanase G2 from Aspergillus oryzae on Japanese sake brewing

We purified three xylanase isozymes (XynF1, XynF3 and XynG2) from a solid-state Aspergillus oryzae RIB128 culture using chromatography. The results of our sake-brewing experiment, in which we used exogenously supplemented enzymes, revealed that only XynG2 improved the alcohol yield and the material utilization. The alcohol yield of the XynG2 batch displayed an increase of 4.4% in comparison to the control, and the amount of sake cake decreased by 4.6%. The contribution of XynG2 was further confirmed through our brewing experiment in which we used the yeast heterogeneously expressing fungal xylanase isozymes. Interestingly XynG1, an enzyme with a XynG2-like sequence that is more vulnerable to ethanol, did not improve the sake-mash fermentation. The stability of XynG2 in ethanol was prominent, and it retained most of its original activity after we exposed it to 80% ethanol for 30min, whereas the stability of the other isozymes in ethanol, including XynG1, was much lower (20-25% ethanol). We concluded, therefore, that the improvement of material utilization achieved with XynG2 is primarily attributable to its characteristically high stability in ethanol, thereby, effectively degrading rice endosperm cell walls under high-alcohol conditions such as a sake-mash environment.

Shochu brewing characteristics and properties of a trichothecin-resistant shochu yeast mutant

An antibiotic-resistant strain of Saccharomyces cerevisiae was isolated from shochu yeast. Three mutants were used for shochu brewing and gave higher ethanol productivities than the parent. The mutants were resistant to cycloheximide, cerulenin, trichothecin and other organic compounds such as lauric acid. In the presence of 20% (v/v) ethanol, the viability of the mutants was 87–96%, but that of the parent was 77%. Zymolyase treatment for 3 h, decreased the viability of the parent by 44% but that of the mutants only by 11–32%. Thus the higher ethanol productivity of these mutants is related to their high ethanol tolerance and resistance to various organic compounds.

A new transformation system of Saccharomyces cerevisiae with blasticidin S deaminase gene

A new method for transformation of Saccharomyces cerevisiae that allows selection was developed. As the frequency of spontaneous blasticidin S resistant mutants from diploid type yeast strain (X-2180AB) was 5.2×10−6, which was a thousandfold less than that from haploid type yeast strain (X-2180B), it was considered that the mechanism of spontaneous blasticidin S resistant mutations was related to recessive gene. Industrial yeasts, which were diploid, were transformed with blasticidin S deaminase gene from Aspergillus terreus to blasticidin S resistance. Expression of blasticidin S deaminase gene allowed selection of transformants from industrial yeasts.

Saké brewing characteristics and multidrug resistance of trichothecin-resistant yeast mutants

Trichothecin-resistant mutants were isolated from saké yeast. These mutants were subjected to saké brewing, and showed a higher ethanol productivity than did the parents. They showed multidrug resistance, and resistance to organic compounds. We considered that the higher ethanol productivity of the mutants was related to their resistance to organic compounds and to their ethanol tolerance.

Enhancing monoterpene alcohols in sweet potato shochu using the diglycoside-specific β-primeverosidase

Monoterpene alcohols (MTAs) are characteristic flavour-imparting compounds in sweet potato shochu (Japanese distilled spirit) that are liberated following hydrolysis by specific enzymes during fermentation. In the present study, we evaluated the effect of an exogenously added diglycoside-specific β-glycosidase (β-primeverosidase) on aroma formation during shochu brewing using various sweet potato species to address whether MTAs are predominantly present as diglycosidic precursors in raw materials. The results showed that the amount of MTAs produced from enzyme-treated mash was dramatically increased by 2- to 9-fold compared with untreated controls, and the increase varied with sweet potato species. In addition, levels of methyl salicylate, 1-octene-3-ol and ethyl benzoate were also elevated by enzyme treatment. These results indicate that a large amount of MTAs and other volatile aroma compounds are stored in the form of disaccharide β-glycosides such as β-primeverosides in sweet potato. This enzyme may therefore be useful for controlling aroma formation during shochu manufacturing, and may ultimately contribute to diversifying shochu quality.

Discrimination of wine from grape cultivated in Japan, imported wine, and others by multi-elemental analysis

Differences in mineral concentrations were examined among three types of wine in the Japanese market place: Japan wine, imported wine, and domestically produced wine mainly from foreign ingredients (DWF), where Japan wine has been recently defined by the National Tax Agency as domestically produced wine from grapes cultivated in Japan. The main objective of this study was to examine the possibility of controlling the authenticity of Japan wine. The concentrations of 18 minerals (Li, B, Na, Mg, Si, P, S, K, Ca, Mn, Co, Ni, Ga, Rb, Sr, Mo, Ba, and Pb) in 214 wine samples were determined by inductively coupled-plasma mass spectrometry (ICP-MS) and ICP-atomic emission spectrometry (ICP-AES). In general, Japan wine had a higher concentration of potassium and lower concentrations of eight elements (Li, B, Na, Si, S, Co, Sr, and Pb) as compared with the other two groups of wine. Linear discriminant analysis (LDA) models based on concentrations of the 18 minerals facilitated the identification of three wine groups: Japan wine, imported wine, and DWF with a 91.1% classification score and 87.9% prediction score. In addition, an LDA model for discrimination of wine from four domestic geographic origins (Yamanashi, Nagano, Hokkaido, and Yamagata Prefectures) using 18 elements gave a classification score of 93.1% and a prediction score of 76.4%. In summary, we have shown that an LDA model based on mineral concentrations is useful for distinguishing Japan wine from other wine groups, and can contribute to classification of the four main domestic wine-producing regions of Japan.