Shigetoshi Sudo

Molecular cloning and determination of the nucleotide sequence of a gene encoding an acid-stable α-amylase from Aspergillus kawachii

Abstract Complementary and genomic DNAs encoding an acid-stable α-amylase (asAA) were cloned from Aspergillus kawachii IFO4308 and their nucleotide sequences were determined. The structural gene (asaA) consisted of a 1,920 bp open reading frame that encoded 640 amino acids. The gene consisted of 9 exons and 8 introns. The signal peptide was composed of 21 amino acid residues. The amino acid sequence from the N-terminus to the 479th residue shoeed 97% homology with the Aspergillus niger acid α-amylase and that from the 480th residue to the C-terminus, including the raw-starch-affinity site, i.e., the TS region, the amino acid sequence of which was rich in threonine and serine, was characteristic of the asAA. A yeast transformant carrying the cDNA produced and secreted an acid-stable α-amylase which was capable of digesting raw starch.

Comparison of Acid-Stable α-Amylase Production by Aspergillus kawachii in Solid-State and Submerged Cultures

The reasons why Aspergillus kawachii IFO4308 (shochu white-koji mold) produced larger amounts of acid-stable α-amylase (asAA) in solid-state culture (SSC) than in submerged culture (SC) were studied. In both cultures, asAA began to be produced in the middle of the cultivation (t0) and in both cases, the production was approximately represented by a growth associated-type equation, asAA = k(m−m0), where m and m0 are the amounts of mycelia at t (≧t0) and t0, respectively. The proportional constant between the amount of asAA produced and mycelial growth, k (mg-asAA/mg-dry mycelia) was 0.038 on average in SSC; this value was almost the same as the maximum k (0.034) obtained in a submerged medium suitable for asAA production (AP medium). In SSC, t0 was shorter and the mold could grow more rapidly (logarithmically) after t0 than in SC, resulting in larger amounts of (m−m0). Furthermore, in SSC k rose more readily to a ultimate value after t0. These SSC characteristics were concluded to be the major reasons for the higher asAA production than in SC. On the other hand, it was considered that in SSC, similar to SC, asAA was produced when the content of storage glycogen (CSG) decreased, the triggers of asAA production being the low glucose concentration in the medium in the case of SC and the rapid mycelial re-growth in the case of SSC. asAA production is believed to be controlled not only by catabolite repression but also by an asAA production control mechanism.

Characteristics of Acid-Stable α-Amylase Production by Submerged Culture of Aspergillus kawachii

Abstract A culture medium having a suitable composition for acid-stable α-amylase (asAA) production by submerged culture of Aspergillus kawachii IFO4308 (shochu white-koji mold) is described. Using the proposed submerged medium, approximately 130 mg of asAA protein per liter of the medium were produced at 30°C for 5 d cultivation. The mold had glycogen as the storage polysaccharide and when the content of storage glycogen (CSG) began to decrease in the presence of an inducer such as dextrin, the production of asAA always began. A high level of asAA was produced after production began and when CSG decreased virtually to zero, however, when CSG started to increase, as AA production tended to be repressed. CSG was affected by both the glucose concentration of the medium and the growth rate of the mycelia. The amount of asAA produced was approximately proportional to the amount of mycelia which had grown after asAA production began.

Growth of submerged mycelia of Aspergillus kawachii in solid-state culture

Abstract Submerged mycelial growth of Aspergillus kawachii IFO4308 in solid-state culture (SSC) was studied. From the result of Northern blot analysis, acid-stable α-amylase was found to be produced mainly by the submerged mycelia rather than the aerial mycelia. The submerged mycelia showed better growth in SSC using rice as the solid substrate ( koji ) than in agar plate culture in spite of low concentrations of dissolved oxygen in koji . Good growth in SSC suggested the existence of an effective oxygen transfer mechanism in koji which governed the mycelial growth. When koji was submerged in water, small bubbles were generated. This phenomenon indicated the formation of vacant spaces in koji during SSC. The submerged mycelia showed better growth in the koji having a larger number of vacant spaces. Considering these facts it was concluded that the vacant spaces participate in effecting an oxygen transfer mechanism in koji as air vents because the diffusivity of oxygen in an air is larger than in koji itself.

The Concentration of Ethyl Carbamate in Commercial Ume (Prunus mume) Liqueur Products and a Method of Reducing It

The ethyl carbamate concentration of commercial ume liqueur products was studied, and a method of reducing it was examined from the viewpoint of antioxidation. The average ethyl carbamate concentration across 38 ume liqueur products was 0.12 mg/l (0.02–0.33 mg/l). When potassium metabisulfite was added to a concentration of 0–1,000 ppm during production, the generation of ethyl carbamate was reduced in a concentration-dependent manner, but when the amount of potassium metabisulfite added was below the maximum level allowed under the Japanese Food Sanitation Act, the reduction was only 27%. When ume liqueurs were produced under deoxygenated conditions created using an oxygen absorber, the ethyl carbamate concentration was reduced by up to 47% as compared with the control group, probably due mainly to a reduction in free hydrogen cyanide. When ume liqueur was produced in an oxygen atmosphere, the ethyl carbamate concentration increased by up to 50% as compared with the control group. Thus, oxygen may be involved in the generation of ethyl carbamate in ume liqueur production.

Lignin Is Linked to Ethyl-Carbamate Formation in Ume (Prunus mume) Liqueur

Ethyl carbamate concentrations in oak barrel-aged ume (Prunus mume) liqueurs were measured, and possible explanations for elevated levels were examined. The average concentration was 0.30 mg/L, significantly higher than in ume liqueurs not aged in oak (0.08 mg/L). Oak powder extracts were prepared from both untoasted and toasted oak powder by extraction with aqueous ethanol, and these were used to make ume liqueurs. Relative to a no-oak control, the ethyl carbamate concentrations were 3.8 and 11 times higher in the ume liqueur made with the untoasted and toasted oak powder extracts respectively. The extracts were loaded onto a C18 column, washed with water, and eluted with methanol. The 13C-NMR spectra for the main constituents of the methanol elution fractions were consistent with those for lignin or fragments thereof. The methanol fractions were added to ume liqueur which was stored for 3 months. Relative to a control, the ethyl carbamate concentrations in the 3-month old liqueurs were found to be 1.2 and 4.6 higher for the untoasted oak-powder and the toasted oak-powder respectively. Ethyl carbamate was formed when lignin was added to a 40% aqueous ethanol solution that contained potassium cyanide. These observations suggest that lignin or fragments thereof promote the formation of ethyl carbamate.

Effects of storage conditions of rice on the rice and Sake qualities.

玄米及び白米をガス透過性の低い袋に詰め, 脱酸素, 炭酸ガス, 窒素ガス及びこれらの混合ガスで袋中の気相を交換したものを30, 15℃で1年間貯蔵し, 経時的に試料を採取して成分の変化を分析すると共に, 1年貯蔵米を用いて小仕込みを行った。30℃, 8ヶ月玄米を貯蔵したときの発芽率は気相の変化によって変った。玄米貯蔵を精白した場合, 還元糖や遊離脂肪酸は, 貯蔵期間が長くなる程増加したが, 遊離アミノ酸は減少した。無機りんの生成は増加するものとしないものがあり, クロールは内部に移動していた。陽イオンはカリのように内部に移行するものと, 移動はするがあまり明瞭でないものがあった。小仕込み試験ではもろみが急進しやすくなり, 製成酒は精白米使用の方が窒素成分が少なかった。