A study on the situation of water in various starches which adsorbed and desorbed various moisture by means of the vapor pressure method, was undertaken by the wide line method of proton magnetic resonance (PMR). The results were as followed : 1. The half width of the PMR spectra of various starches was reduced by increasing of moisture contents, and especially, it became very narrower in the range of moisture contents more than 20%. 2. There were no significant differences between the half width in moisture adsorption and that in moisture desorption in the same moisture contents . 3. The relation between the half width and moisture contents gave two straight lines which intersected on half-logarithm graph. In the point of intersection of the graph, the moisture contents of various starches, such as tapioca, potato, corn, sweet potato, wheat and rice were 19. 1, 17. 3, 16. 2, 15. 7, 14.4 and 13.7%, respectively and the values of half width of potato, tapioca and other starches in the moisture content of 0% which were estimated by extrapolation were 45, 33 and 35 ppm, respectively.

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The objective of this study was to make SEM observations of starch granules of several different maize endosperm mutants following the early germination period and to compare the relative patterns and digestibility by starch-degrading enzymes. Mature kernels of the ae endosperm mutant in four inbred lines and in a single cross hybrid and of five endosperm mutants in an inbred and a single cross hybrid, and their normal counterparts were used. Enzymatic erosion of the starch granules was visible in the disected endosperm of the normal maize following germination 4, 6 and 8 days after planting. The types of the enzymatic erosion were similar to those of the in vitro attack by a-amylase on normal starch granules. Namely, numerous pin holes were visible on the surfaces of the granules and the pores penetrated into the granules and the inner portion, which appeared to be terraced or step-shaped indicative of layered internal structures. Some types of enzymatic erosion suggested the possibility of enzymatic attack other than that of a-amylase. Starch granules of the germinating Oh43, C103, W64A, and Oh43XC103 ae endosperm mutant lines scarcely showed enzymatic erosion on their surfaces. These observations were similar to the in vitro amylase attack of ae starch granules. On the other hand, enzymatic erosion of starch granules of the B37 ae endosperm was similar to the germinating kernels of the normal B37. The types of enzymatic erosion of endosperm starch granules of the germinating su₂ su₂o₂, wx, and 0₂ mutants were similar to those of their normalcounterparts.

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It was found that l-menthol was firmly included in polysaccharide materials such as starch granules and cellulose powder. The inclusion occured when the dried polysaccharides were contacted with menthol molecules in the presence of methanol. The maximum amount of menthol included by the polysaccharides used came to about 5, 0 mg/g complex. The inclusion of menthol was inhibited by the moisture both in polysaccharide and methanol. The included menthol could be most efficiently extracted with methanol among several organic solvents. The inclusion complexes were very stable against dry beatings (e. g., at 140°C under reduced pressure), but the included menthol was gradually losed from the complex on standing in damp atmosphere.

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The major amylolytic enzyme present in Meicelase, a commercial crude cellulase preparation from Trichoderma viride; was purified by consecutive column chromatography, and characterized as a glucoamylase [EC 3.2.1.3]. The specific activity was brought to 18 .3 units of soluble starch-saccharif ying activity/mg of enzyme protein, and the enzyme showed a single band on polyacrylamide gel disc electrophoresis. Some properties of the purified glucoamylase were investigated. The molecular weight of the enzyme was estimated to be about 75, 000 on the basis of SDS polyacrylamide gel electrophoresis. The optimum pH and the optimum temperature for the activity of the enzyme were pH 5.0-5.5 and 60°C, respectively. The enzyme was stable over the range pH 5.0-7.0 at 4°C and was completely inactivated by heating at 90°C for 10 min. Hg²⁺ completely inhibited the enzyme, but other metal ions tested had little effect on the activity at the concentration of ions used (1 mM) . The action of the enzyme on glycogen, amylopectin, soluble starch, short chain amylose (DP =17.3), maltose, isomaltose and panose was studied. Glucose was the sole hydrolysis product found in digests of these substrates. At the same substrate concentration (0.075%, w/v) and enzyme concentration, the relative initial rates of glucose production from amylopectin, soluble starch, short chain amylose and maltose were about in the proportions 8 : 8 : 8 : 1. The K_m and V_max values at 30°C and pH 5.0 were calculated for the enzyme acting on glycogen, amylopectin, amylose, soluble starch, short chain amylose and maltose. By using the authentic anomers of D-glucopyranose under conditions limiting mutarotation, it was found that the condensation reactions catalyzed by glucoamylase require a donor substrate of specific configuration. The purified enzyme, which hydrolyzes amylaceous substrates to β-D-glucopyranose, was found to catalyze the rapid synthesis of maltose and isomaltose specifically from β-D-glucopyranose. The configurational inversion accompanying the condensations indicates that the D-glucopyranosyl portion of β-D-glucopyranose is interchanged with hydrogen at the C₄ or C₆ carbinol site of a second D-glucose molecule (glucosyl transfer).

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We confirmed that starch granules of potato, Chinese yam (tubers of Dioscorea batatas Decne), banana (Musa cavendish L.), lily (bulbs of Lilium spp.), gingko (seeds of Ginko biloba L.), high-amylose maize, East Indian lotus (tubers of Nelumbo nucifera Gaertn), Japanese chestnut (seeds of Castanea crenata Sieb. et Zucc.), kuzu (Japanese arrowroot, Pueraria lobata Ohwi), sweet potato were respectively in decreasing order more resistant to the attack of pancreatin than were those of normal maize, wheat, rice, red maize (Maize morado), and taro (roots of Colocasia antiquorum Schott). Among endosperm mutants of maize (Zea mays L.), starch granules of amylose-extender mutant were much more resistant to the action of amylases than were those of the normal counterpart. Starch granules of sugary-Ji and sugary-2 mutants were digested much faster than those of the normal counterpart by amylases. Starch granules of waxy, shrunken-2 and brittle-2 mutants tended to be digested faster than those of the normal. When opaque-2 was combined with each one of the endosperm mutants, it was observed that the starch granules of the double mutants were digested by amylases to an extent very comparable to their respective nonopaque single-mutant counterpart. The differences among the endosperm mutants in susceptibility of starch granules to the action of amylases disappeared following gelatinization of starch granules with alkali. Observations under a scanning electron microscope (SEM) revealed that starch granules resistant to the action of amylase showed after the action of amylases shapes and surfaces similar to the intact granules. On the other hand, starch granules susceptible to amylase showed numerous pin holes on the surface layer and the pores penetrated into the inner layers of the granule during the attack with amylases. For some of the granules the inner portion which appeared to be terraced or step-shaped could be seen. This may be indicative of layered or stratified internal structures of the granules. The other characteristic observations by SEM were striated structures on the surfaces of starch granules attacked by amylase, for example those of banana, lotus and lily.

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As an award address, the following three different works which had been achieved by our group since 1959 were presented. I) Glucose isomerization in alkaline aqueous solution To enhance the sweetness of glucose, the LOBRY de BRUYN-ALBERDA van EKENSTEIN reaction was studied. After examing the factors which affected on ketose formation, sugar degradation and coloration, we found that the reaction conditions-higher temperature and shorter reaction time-gave better results in higher fructose formation up to 35%, and lower sugar degradation (only 2-3%), ⁴⁾ compared with the conventional results of lower temperature and longer reaction time. This was proved by continuous isomerization using a semi pilot plant .⁷⁾ II) Fine structure of starch molecule Waxy maize starch was extensively treated by porcine pancreatic α-amylase to prepare αlimit dextrin. Then the fundamental branching structures were determined by the combined action of several enzymes, such as pullulanase, glucoamylase, β-amylase and porcine pancreatic a-amylase. Sixty five percent of α-1, 6 bond existed in amylopectin were obtained in singly branched oligosaccharides and the rest of the branches (35%) were in multiply branched oligosaccharides. These facts indicated the heterogeneity of branching in amylopectin molecules.¹²⁾ By chemical and physical studies of the structure of NAGELI amylodextrin, we proposed parallel double helix in crystalline part of amylopectin.¹⁵⁾ III) Maltohexaose forming amylase Maltohexaose forming amylase was discovered unexpectedly²¹ and identified as a novel exoamylase, ²² after glucoamylase, β-amylase and Ps, stutzeri maltotetraose forming amylase . This enzyme was purified and characterized its enzyme chemical properties.²³ The enzyme produced about 35-40% of maltohexaose from starch by exo-attack. By being improved the stability and productivity, the enzyme will be used in large scale production of maltohexaose.

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