α-Amylase, glucoamylase, and acid protease of rice-koji (a culture of Aspergillus oryzae on steamed rice) are the most important enzymes participating in sake fermentation. The parallel fermentation of sake moromi-mash, in which alcoholic fermentation proceeds keeping pace with dissolution of rice starch by amylases, is constructed with rice dissolution by α-amylase, glucose production by glucoamylase, and alcohol production by yeast.
Following three equations were established for the rice dissolution, glucose production, and alcohol production rates, respectively: -dS/dt=k⋅E_a^α⋅S (1) dG/dt=k₁⋅E_ge^-k₂⋅t (2) dA/dt=k₃⋅N (3) where S is rice starch, E_a is α-amylase activity, G is glucose, E_g is glucoamylase activity, A is alcohol, N is yeast number, t is time (day), and k, k₁, k₂, k₃, and α are constants.
Apart from the rate equations, it was found that acid protease had great effects on the parallel fermentation; promotion of rice dissolution, hence enhancement of glucose production and alcohol fermentation.
Concerning the effect of acid protease promoting the rice dissolution by α-amylase and the small value of index α (0.20-0.25) in equation (1), adsorption of α-amylase onto steamed rice was given attention. Steamed rice adsorbed well α-amylase resulting in poor rice digestion; it was called unavailable adsorption. It could be the reason why activity of α-amylase was restricted as shown by small value of index in equation (1). Action of acid protease on steamed rice released the α-amylase adsorption, promoting the efficiency of α-amylase action.
Adsorption of α-amylase onto steamed rice was examined variously and it was inferred that α-amylase and steamed rice combined electrostatically mediated by oryzenin, main rice protein. Isoelectric point of α-amylase is pH 3.7 charging negative at pH 4.5 (pH of sake moromi-mash) and that of oryzenin is pH 6.5 charging positive at pH 4.5. The negative charge of α-amylase is owed with carboxyl residues of acidic amino acids in protein molecule and the positive charge of oryzenin will be owed to basic amino acid residues. Acid protease will attack to the basic amino acid residues to change the protein charge.
Application of a saccharogenic α-amylase of Bacillus subtilis having an isoelectric point of pH 5.2 was examined. The amylase did not adsorbed onto steamed rice and hence expected to act effectively in sake moromi-mash.

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1. α-Amylase and glucoamylase of shochu koji (Aspergillus kawachii) have high stability in low pH 3.0-4.0 which is pH of shochu moromi mash.
2. α-Amylase and glucoamylase of shochu koji have high thermostability and optimum temperature of both enzymes are 70°C at pH 5.0.
3. Raw starch digestion activity of shochu koji has low optimum pH, 3.5.

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We cloned genes coding for two thermophilic α-amylases from Bacillus licheniformis and B. stearothermophilus and one β-amylase from B. polymyxa. The nucleotide sequence determination of these genes revealed the characteristics of their sequences such as promoter and signal sequence were clarified. The thermostability of the thermophilic amylases was discussed on the basis of the hydropathy profile of amino acid sequence deduced from the nucleotide sequence. The recombinant DNA technology was shown to be useful for determining the region responsible for the thermostability or optimal pH of the enzymes. Amino acid sequences of liquefying α-amylases secreted by the bacteria of Bacillus species were found to be similar each other. Interestingly, three homologous sequences at the active sites (catalytic and substrate binding sites) were conserved among not only bacterial amylases but also amylases of fungal and animal origins. The thermophilic amylase genes were expressed in heterologous bacteria such as Escherichia coli and B. subtilis. Most efficient formation and secretion of the enzyme (as high as 0.5g/liter) were observed when B. brevis 47, a protein-producing bacterium, was used as a host. β-Amylases with three different molecular weights were produced by both B. polymyxa and heterologous bacteria having the β-amylase gene on plasmids. These amylases have exactly the same amino acid sequence at the amino terminal region and cross-reacted with the antibody raised for the β-amylase with the largest molecular weight.

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6-O-α-maltosyl- and 6-O-α-maltotriosyl-cyclodextrins (CDs) were produced from α-, β-, and γ-CDs and maltose and maltotriose with Pseudomonas isoamylase. The reaction rate was greater with maltotriose than with maltose, and with increasing size of CDs. Besides these branched cyclodextrins, branched hexaoses, 6¹-, 6²-, and 6³-maltotriosylmaltotriose in the 1:4.5:1 ratio were formed by the condensation of two maltotrioses. Thus, maltotriose was effective as both a side-chain donor and an acceptor as well, but maltose was only effective as a side-chain donor. The specific activities of isoamylase for the formation of maltotriosyl-CDs were greater than those of pullulanase. The branched CDs and the hexaoses were purified by liquid-gel chromatography and HPLC, and their structures were determined by chemical, enzymic and ¹³C-NMR spectroscopic analyses.

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Branched α- and β-cyclodextrin (CD) were synthesized by the transglycosylation of Bacillus acidopulluliticus pullulanase under the co-existence of α-maltosylfluoride (α-G₂F) and α- or β-CD. The branched CDs were isolated by High Performance Liquid Chromatography. It was concluded by the enzymatic and chemical methods that the sugars were maltosyl-α- or β-CD.
The amount of G₂-α-CD produced was affected by the concentration of α-G₂F and α-CD. From the proportion of the amount of G₂-α-CD produced to that of α-G₂F consumed, the optimum condition of G₂-α-CD formation was found to be α-G₂F (40mM) and α-CD (90mM).

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Regio- and stereo-selective synthesis of β-D-(1→2) linked glucopentaose 1 and α-cyclodextrin 2 were described. In the case of 1, a stepwise approach was employed in order to control the stereochemistry of glycosidic linkages. In the case of 2, an interamolecular cyclization of the key intermediate 7 was performed to afford the completely protected α-cyclodextrin 6, which was deprotected smoothly to give the target molecule 2.

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The bacterium which produces a γ-cyclodextrin forming enzyme was isolated from soil. The isolated strain No. 313 was identified as Bacillus subtilis. The enzyme catalyzed the formation of γ-cyclodextrin, and neither α-nor β-cyclodextrin were detected in the enzymatic hydrolyzates.
γ-cyclodextrin forming enzyme of Bacillus subtilis No. 313 was purified about 35-fold and shown to be a single, homogeneous protein by polyacrylamide gel electrophoresis after ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Chromatofocusing by Polybuffer exchanger PBE 94, Sephacryl S-200 gel filtration and CM-Toyopearl 650M. The molecular weight and isoelectric point were 64, 000 and pH 7.1. The enzyme was most active at pH 8.0 and 65°C;, and stable up to 50°C; at pH 7.0 and in the range of pH 6.0 to 8.0 at 50°C; on 30min incubation. The apparent K_m values for β-and γ-cyclodextrin were 6. 67 and 0.65mM, respectively.

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Three kinds of cyclomaltodextrin glucanotransferases (CGTases) produced by Bacillus ohbensis, Bacillus macerans and Bacillus circulans were respectively purified to a single protein and the CGTase from B. ohbensis was successfully crystallized. Comparative studies of three CGTases revealed that the CGTase from B. ohbensis had the smallest molecular weight (35, 000) and low affinity on starch adsorption while in other properties, i.e., optimum pH, pH stability, optimum temperature and etc., the three enzymes showed similar properties.
CGTase from B. macerans had advantages on the production of α-cyclodextrin (CD) but the reaction conditions must be more strictly controlled. CGTase from B. ohbensis can be favorably used for the production of β-and γ-CD. Futhermore, only CGTase from B. ohbensis produced negligible amount of α-CD.

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Cyclodextrins (CDs) are homogenous cyclic molecules composed of six, seven or eight glucose units and called α-CD, β-CD and γ-CD, respectively. In Japan, CD is being successfully used in various industries because of its ability to make inclusion compounds with various kind of substances, and is especially useful for the stabilization of the unstable materials to oxidation or exposure to heat and light. However, the high cost of CDs has limited their use for food processing and other important application. To solve this problem, Japanese companies started to investigate the method of the mass-production of CD at low cost. As a result, they have succeeded the production of CD in large-scale at reasonable cost. In their manufacture, the most advanced technologies and equipment are used to produce the CD in excellent quality. Therefore, CD has become widely use for stabilizing labile compounds, emulsifying oils, masking flavors or odors, increasing solubility and converting viscous or oily compounds into powders.
In Japan, 4 manufacturers, which have producing capacity of 130tons/month, are producing several kinds of CDs. Some examples of actual applications according to function are as follows;
1) Stabilization of volatile substances: Flavors, spices and black and green tea essences.
2) Antioxidation and protection against the photodecomposition: Fish paste products, liposoluble vitamins, fried cakes, meat products and natural dyestuffs.
3) Modification of physical properties: 1. Increase solubility of medicines, 2. Reduces bitterness of medicines and canned oranges, 3. Masks unpleasant odors of seafood products and mutton products, 4. Reduces deliquescence of powdered sugar, 5. Improves texture of boiled rice, 6. Accelerates hardening of rice cakes.
4) Utilization of surface activity: Minced seafood products and dressings.
5) Utilization as material for making powdered foods: Nuts, natural seasonings, flavors, spices and teas.

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