The molecular structure of Taka-amylase A [EC 3.2 .1.1, Aspergillus oryzae] has been determined by X-ray crystallographic method at 3.0Å resolution . The features of the three dimensional structure and the structure similarities to other enzymes are described . The differ ence Fourier maps for the complex crystals of Taka-amylase A with substrates and inhibitors revealed their binding sites. Some experiments to confirm the homogeneity of the sample of Taka-amylase A were carried out. In order to clalify the catalytic mechanism of the enzymes on atomic level, the improvement of the X-ray structure analysis and the difference Fourier study of various complex crystals are in progress.

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Soybean β-amylase is a monomeric enzyme consisting of a single polypeptide chain (molecular weight 57, 000). Zsozyme patterns of β-amylase in seeds are characteristics to the varieties of soybean and the related species. Five isozymes were purified and crystallized from commercial defatted soybean meal, and their chemical, physical, immunological and enzymatic properties were investigated comparatively. Large single crystals of the enzyme were analyzed by X-ray diffraction method with two isomorphous derivatives with K₂PtCl₄ and p-chloromercuribenzene sulfonate. By the selective modification of five SH groups in the molecule with organic mercu rials and alkylating reagents, the second reactive one was found to be related to the enzyme activity, but it participates neither to the catalytic nor substrate-binding action of the enzyme . The site of five SH groups on the polypeptide chain were determined by cleaving the chain by the cyanide method and by evaluating the molecular weights of the resulting peptide fragments by SDS-polyacrylamide gel electrophoresis. As to the action of β-amylase on the substrate, the substrate-binding affinities of five subsites of the soybean enzyme were determined, and the first site was found to be the strongest, which may determine the mode of action of β-amylase.

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A rifampin-resistant, asporogenous mutant, BQ10-S1 Spo-, having highly potent β-amylase productivity was isolated from Bacillus cereus BQ10-S1. A sporogenous revertant, BQ10-S1 Spo+, was also obtained from BQ10-S1 Spo-. Bacteriological characteristics of these mutants and their ways of β-amylase production were compared. The relationship between high β-amylase productivity and sporulation of rifampin-resistance was discussed.

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A new process for maltose, a sugar well-known for years but hardly explored, developed by Hayashibara Co., Ltd, and its properties are discussed. 1. Starch debranching enzymes were formed by 28 genera of bacteria and streptomycetes including Escherichia, Pseudomonas, Bacillus, Micrococcus, Lactobacillus, Nocardia, and Streptomyces. 2. As to sources β-amylase, screening of microorganisms, as well as wheat bran, sweet potato, and soy bean, resulted in the discovery of Bacillus strains, as satisfactory B. megaterium, B. circulars, and B. cereus. 3. High purity (70-100%) maltose is producible at low cost by hydrolyzing liquefied starch slurry with a-amylase free β-amylase and a combination of debranching enzymes, then, further hydrolyzing maltotriose, formed in the hydrolysate, with an enzyme derived from higher plants, molds, or bacteria and purifying followed by crystallization. 4. Maltose is widely used as an intravenous sugar supplement, sweetness-reduced-sweetener in food process, as well as low caloric sweetener (maltitol) and so on.

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Recent development of various maltose production methods coupled with findings of new amylases has been enlarging maltose application field both for food and medical use. Authors reviewed this progress and introduced various maltose production methods from the industrial point of view. Maltose production method or procedure can be divided largely into two parts; one is saccharification process where hydrolyze starch to maltose utilizing amylase and the rest is separation or purification process to give high purified maltose more than 95% maltose content utilizing several purification methods such as adsorption and separation, precipitation by organic solvent, separation by membranes and crystallization. New enzymes from microorganisms like α-amylase, β-amylase and α-1, 6-glucosidase are discovered in these 10 years. Okada and co-workers first clarified microbial β-amylase, Harada and co-workers found interesting isoamylase from Pseudomonas sp ., authors discovered α-amylase from Streptomyces which can produce maltose more than 75% from starch, but it can be said that combination of saccharification and purification is necessary to produce high purity of maltose in industrial scale. Feasibility of all the maltose production methods are discussed.

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Recent topics from our laboratory on the determination of human serum amylase were reviewed. 1) Serum amylase activity after pancreaticoduodenectomy or total pancreatectomy returned to the normal level after initial fall within a few weeks. 2) Hepatic implication in the metabolism of family A of salivary amylase (glycoprotein) indicated the possible mechanism of hyperamylasemia in hepatic diseases. 3) A radioimmunoassay for human pancreatic amylase was developed. The biological and immunological activities in serum amylase were in a significant correlation in general. In postoperative hyperamylasemia of unknown origin, however, there was discrepancy between the biological and immunological activities. 4) The results of differential determination of serum amylase isozymes using amylase inhibitor from wheat showed a significant correlation with those determined by electrophoresis.

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Since the discoveries of specific oligosaccharide forming amylases, such as Exo-maltotetrao hydrolase, Exo-maltohexaohydrolase etc., a large scale preparation of maltooligosaccharides become feasible. The several possible methods to prepare maltooligosaccharides are discussed in this paper as well as the fractionation method such as charcoal column chromatography, gel filtration chromatography. Quantitative and qualitative analyses of various oligosaccharides are discussed with some examples of the analysis and characteristics of each method.

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Sake contains unfermentable oligosaccharides such as isomaltose, nigerose, kojibiose, panose, isomaltotriose besides glucose. These oligosaccharides occupying 10-25% of total sugarsin commercial sake appear at the comparatively early stages of the sake fermentation.Threshold values for the oligosaccharides were shown. Transglucosidase was applied to sake manufacture to enforce the unfermentable oligosaccharidesin the resulting sake. A part of steamed rice was saccharified with an enzyme preparation. containing α-amylase, acid protease, and transglucosidase to yield a saccharified slurry mainly consistedof the unfermentable oligosaccharides and the slurry was added to main fermentation mash. Theunfermentable sugars increased to give about 60% of the total sugars. Unfermentable sugar preparationwas produced by saccharifying starch with an enzyme preparation containing a-amylase or?A-amylase and transglucosidase. Glucose contaminated to the preparation was removedd by activecarbon treatment. The sugar preparation was added to a synthetic sake to improve the taste. Analytical method of the oligosaccharides by a high performance liquid chromatographywas examined.

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1. Origin and composition of carbohydrates in beer were reviewed. i) Carbohydrates in beer originate mostly from starchy raw materials, such as malt, starch and rice. Hop, one of essential brewing materials, however, is not significant as a source of beer carbohydrates.uA ii) Reflecting the composition of the raw materials, major carbohydrates in beer are degradation products of starch, which are distributed in a wide range of polymerization degree. These dextrins consist of a number of isomers, which are either linear or branched.uB iii) Beer also contains many sorts of minor constituents. Those are as follows; (1) pentoses and their dextrins, (2) oligosaccharides consisting of various kinds of sugars, such as galactose and fructose and (3) oligosaccharides which are considered to be formed either by transglycosidation or by erimerization during brewing process. 2. Recently, a variety of beer has being more and more produced in the world. Corresponding to such variation, it becomes necessary to classify the beers by their constituents. For this purpose, the classification of 128 brands brewed in 19 countries was attempted according to their carbohydrate composition, fermentable sugars and dextrins in beer. It could be proved that carbohydrate composition is a useful index for beer classification. 3. Carbohydrate composition in beer can be regulated by selection of raw materials and by modification of brewing procedures. Among these methods, the effect of (1) utilization of malt amylases, (2) utilization of an external enzyme and (3) addition of sugars to wort and beer on the carbohydrate composition and quality of beer were investigated in detail.

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Automated analysis of oligosaccharides in soy sauce was performed according to gel filtration method with Biogel P-2 by using JLC-6AH liquid chromatograph apparatus. The contents of oligosaccharides in Koikuchi and Usukuchi shoyu, almost of which consisted of disaccharides, were about 0.5 g/1.00 ml. On the other hand, the contents of oligosaccharides in Tamari and Shiro shoyu, almost of which consisted of di-, tri-, and tetrasaccharides, were about 1.0 and 3.0 g/100 ml, respectively. In Koikuchi shoyu mash, there existed di-, tri-, and tetrasaccharides during first 20 days of aging. After stage of alcohol fermentation, the tri-, and tetrasaccharides almost disappeared from the mash. Utilization of oligosaccharides by several microorganisms were studied. Nalophilic yeasts contributed to consumption of higher oligosaccharides. Each fraction separated by gel filtration was chromatographed on anion exchange resin with borate buffer. As the results, several peaks were detected in each chromatographic pattern. The sugar composition of the di-, and trisaccharide fractions were determined as glucose, galactose, arabinose, xylose and mannose. Among them, glucose was main component. The tetrasaccharide fraction from Tamari shoyu was composed of considerably amount of rhamnose. In the case of Shiro shoyu, tetrasaccharide fraction consisted of a lot of xylose and arabinose. The oligosaccharides added into shoyu made little effect on sensory evaluation.

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It has been said that cyclodextrins will be successfully used in various fields of industries because of its ability to make inclusion compounds on complexes with various kinds of substances, especially unstable materials against oxidation, heat and light. However, cyclodextrins have not been widely used for processing foods and others up to now. The main reason not to be used may be owing to high price of cyclodextrins production. To solve this problem, we have been working on production of cyclodextrin producing enzyme and cyclodextrins and established a component of cultivating medium for producing the Bacillus macerans enzyme. The enzyme in a crude enzyme preparation (broth) was adsorbed on moist heated starch granules to make starch adsorbed enzyme which was most useful for production of cyclodextrins. A new method of α-cyclodextrin production was also established. The principle of the method is based on the action of glucoamylase, taka amylase and yeast; taka amylase hydrolyzes β- and γ-cyclodextrins effectively to maltooligosaccharides which are hydrolyzed to glucose by the action of glucoamylase. And glucose is converted to ethanol by yeast. As the result, after being treated with the above mentioned enzyme system, the reaction mixture of cyclodextrin producing enzyme on starch is composed of ethanol and a-cyclodextrin, which is easily crystallized by concentrating the mixture. Cyclodextrins syrup which contains α-cyclodextrin as a main component was also produced for use in the food industries and several examples of its utilization were reported.

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The research of oligosaccharides which are not only noncariogenic, but also anti-cariogenic (inhibitory against the cariogenicity of sucrose) must be valuable to the prevention of caries induced by sucrose. Ten years ago, we had developed sucrose-coupled oligosaccharides (coupling sugarR) from starch and sucrose by the transfer action of cyclodextrin glycosyltransferase. The coupling sugar has the anti-cariogenic properties, and has been introduced for use in the various foods such as cookies, candies and jams, as substitutional sweetner for sucrose. On the basis of this success, various sucrose derivatives were synthesized to get more powerful anti-cariogenic saccharides and examined for the anti-cariogenic properties. Of the saccharides tested, isomaltosylfructoside and xylosylfructoside were not readily fermented by Streptococcus mutans and did not serve as the substrate for glucan synthesis, furthermore, they had very strong inhibitory effects on the synthesis of insoluble glucan from sucrose.

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