Distribution of isoamylase-debranched starch components, X-ray diffraction patterns, gelatinization characteristics by photopastegraphy and differential scanning calorimetry, and starch-granule susceptibility to amylases were investigated for starches of Job's tears (hatomugi, Coix lacryma jobi L. mayuen (Roman) Stapf. and juzudama, Coix lacryma-jobi L.). Starches were prepared from seeds of hatomugi and juzudama cultivated or collected in Japan and Korea. There were two types of grain among hatomugi open-cultivated at Nakazato and Okayama. Therefore, the waxy-type grains were separated from the nonwaxy-type ones by iodine staining of endosperms. The results obtained were as follows; (1) Fundamentally, endosperms of hatomugi had waxy-type starch and those of juzudama had nonwaxy-type starch. Starch of nonwaxy-type Nakazato was resemble to that of juzudama. (2) Starches of hatomugi and juzudama were close resemblance in distribution of starch components and gelatinization characteristics to those of waxy and nonwaxytype of maize, respectively. (3) All starches had A-type pattern of X-ray diffractograms. (4) Starchgranules of juzudama showed a wide range of susceptibility values to amylases. The phenomenon indicated that the starches had a wider range of characteristics than those of domesticated plants. (5) There was a great resemblance in properties of endosperm starches between Job's tears and maize.

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A study of the iodine spectra of a number of glycogens and amylopectins and their β-amylase limit dextrins in the presence of half saturated ammonium sulphate has been carried out. The changes in the wavelength of maximum absorption (λmax) with iodine concentration, reveal differences in the chain-length distribution of mammalian and non-mammalian glycogens. Reduction of the iodine concentration results in an increase in λmax values with mammalian glycogens, a decrease with non-mammalian glycogens but with amylopectins the value λmax is much less dependent on iodine concen tration. The structural significance of these results is discussed.

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A pullulanase (R-enzyme) was purified from potato tubers by fractional precipitation at pH 5, 20% ethanol precipitation, 40% saturated (NH₄)₄SO₄ precipitation, ion exchange chromatography on Whatman DE 32 and finally by affinity chromatography on Sepharose 6B-αCD. The final preparation having a specific activity of 11.4 U/mg was obtained with a yield of 35.6%. The purified enzyme separated on gel filtration on Sephadex G-150 into 2 forms, the apparent molecular weight of which were calculated to be 220, 000 and 87, 000. Two forms converted spontaneously to each other. The pullulanase hydrolyzed pullulan most rapidly, β-LDs more rapidly than their original polysaccharidesbut could not act on oyster glycogen. It released maltosyl and maltotriosyl residues from poly-and oligosaccharides at approximately the same rate. All these catalytic properties are characteristic to cereal pullulanases and close to those of Aerobacter aerogenes (Klebsiella pneumoniae) pullulanase. The potato isoamylase purified in the previous paper could not hydrolyze pullulan at all but instead hydrolyzed oyster glycogen quite well. It favoured polysaccharides with longer branch chains than those with maltose and maltotriosyl stubs. Maltosyl stubs are debranched much more slowly than maltotriosyl stubs by this enzyme. All these properties are identical to those of Pseudomonas isoamylase. Data presented in this paper indicated clearly that potato tubers have a pullulanase and an isoamylase.

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Two active components (PPA Iand PPA II) of porcine pancreatic α-amylase [EC 3.2.1.1] from the crystalline preparation were highly purified by a novel ion-exchanger, TSK-Gel DEAE-Toyopearl® 650S (DEAE-Fractogel® TSK 6505) chromatography. The PPA I and II purified showed a single band in polyacrylamide gel electrophoreses with or without sodium dodecylsulfate. PPA II migrated to the anode more rapidly than PPA Iin the polyacrylamide gel electrophoresis (pH 9.0). Isoelectric points of PPA I and II were estimated to be 6.5 and 6.1, respectively. Activation by chloride in concentration above 50 mM was more marked on PPA I than on PPA II. Except the differences described above, the enzymatic properties of PPA I and II examined were almost same as shown below: 1) optimum pH and temperature for enzyme action on starch were 6.9 and 53°C, respectively, 2) thetwo components were more stable at alkaline pH in the presence of Ca²⁺ than in the absence of Ca²⁺, 3) both activities of PPA I and II were markedly inhibited by Hg²⁺, Cu², Ni²⁺, Zn²⁺, N-bromosuccinimide, 5, 5′-dithiobis-(2-nitrobenzoic acid) and some microbial α-amylase inhibitors to nearly the same extent, and 4) K_m, and k₀ values of PPA I and II for short chain amylose (DP= 17), and Ki values for α-amylase inhibitors calculated from Lineweaver-Burk plots were coincident with each other, respectively.

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Some properties of edible canna starch produced in Taiwan were studied. Compared to potato starch it has larger granular size (50.8×34.5 μm), higher resistance to enzymatic digestion and showed the same B type X-ray diffraction pattern. It gelatinized at higher temperature than potato starch and its paste retrograded more extensively than that of potato starch. It has higher amylose content (27.1%) but its amylose (DP≅3, 100) is considerably smaller than potato starch amylose (5, 500) whereas no significant difference was observed between unit chain profiles of amylopectins of both starches . Edible canna starch contains almost the same amount of P (52 mg%), but different from potato starch, contains much more amount of Ca (25 mg%) and considered to be Ca-starch . When this Ca is substituted for K, the resulting K-starch showed the pasting properties such as high maximum viscosity (760 B.U.) and large breakdown (580 B.U.), quite high transmittance and high swelling power. On the other hand, the original Ca-starch showed depressed gelatinization, i.e., it showed low (450 B.U.) but stable viscosity, low transmittance and low swelling power . In total, edible canna K-starch is close to potato starch but its Ca-starch has pasting properties similar to those of cereal starches.

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The major amylolytic enzyme from Hypocrea peltata was purified by consecutive column chromatography procedure, and characterized as a glucoamylase [EC 3.2.1.3]. The specific activity was brought to 26.8 units of soluble starch-saccharifying activity per mg of enzyme protein, and the enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme was estimated to be about 73, 000 by SDS polyacrylamide gel electrophoresis. The optimum pH and temperature of the enzyme were pH 4.5-5.0 and 60°C, respectively. The enzyme was stable over the range of pH 4.0-7.0 at 4°C for 24 hr, and was completely inactivated by heating at 80°C for 10 min. The enzyme was completely inactivated by 1 mM Hg², and partially by 1 mM Ag⁺ and Fe²⁺. The action of the enzyme on glycogen, amylopectin, amylose, 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. The α-configuration of the anomeric carbon atoms is produced in the hydrolysis products of soluble starch. 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.

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Binding of isomaltose with glucoamylase from Rhizopus niveus was studied statically in the comparison with maltose, by monitoring the intensity change of the enzyme fluorescence. The dissociation constants and the maximum fluorescence intensity decreases of the binary com plexes with the enzyme were determined for isomaltose and maltose, in the presence and absence of gluconolactone, a transition state analogue. The dissociation constant of the enzymeisomaltose complex was found to be larger by a factor of ten than that of the enzyme-maltose complex . It was confirmed that isomaltose and gluconolactone bind to the enzyme independently and form the enzymeisomaltose-gluconolactone ternary complex, whereas gluconolactone and maltose bind to the enzyme competitively. Considering that gluconolactone binds to Subsite 1 of the enzyme active site and that substrates must occupy Subsite 1 in their productive binding modes, these findings suggest that isomaltose binds to the enzyme almost nonproductively, resulting its much slower hydrolytic rate compared with maltose .

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The action of cyclization of Bacillus macerans cyclodextrin glucanotransferase (BME) was studied by use of various kinds of surfactants. And it was elucidated by use of reducing end labelled maltosaccharidesthat cyclization proceeds from the non-reducing end of α-1, 4 glucan. Surfactants which have straight carbon chains as hydrophobic moiety were extremely effective in promoting the enzyme action forming α-cyclodextrin (α-CD). On the other hand, surfactants which have more bulky hydrophobic moiety than straight carbon chains were extremely effective for β-CD formation (β-CD forming surfactants). By use of the dextrin, which was prepared from defatted waxy corn starch by the treatment with bacterial liquefying α-amylase and β-amylase and from which only traces of CDs were formed by the action of BME without sodium lauryl sulfate (SDS), the enzyme action was changed so as to form mainly α-CD with α-CD forming surfactants and mainly β-CD with β-CD forming surfactants . And therate of α-CD formation from soluble starch in addition of SDS was 1.6 times as reference (without SDS). Hydrolyzing activity was effectively depressed by the addition of SDS. From these results the authors infer that cyclization proceeds on 6₅-helices, 7₆-helices to form a and β-CD from the non-reducing end of α-l, 4 glucans and so that the action pattern of BME depends not only on the specificity of the enzyme itself, but also on the conformation of the substrate as modified by helicogenic complexing agents .

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Naegeli amylodextrin obtained by intensive acid hydrolysis of waxy maize starch, was fractionated in quantity into three fractions by step-wise precipitation using a solvent system consisting of pyridine, methanol and water. It has been our experience that all the fractions of amylodextrin co-precipitate in a solvent system without pyridine and the only way to separate them is gel permeation chromatography. There are two main groups of polysaccharides in amylodextrin. One is DP 12-16 linear a-(14) polysaccharide and the other is DP 25-30 branched α-(14) polysaccharide in which two chains of approximately the same DP are linked by an α-D-(16) glucosidic linkage adjacent to the reducing end. These materials have been regarded as low molecular weight models of native starch. The chemical and physical structure of two of the fractions obtained by the method discussed here were identical with these polysaccharides . Since large quantities of the homogeneous fractions can be prepared by relatively simple steps, we think this method should be very useful for studying the chemical, physical and biochemical properties of the starch molecule .

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New insight has been obtained into the catalytic capabilities of cyclodextrin glycosyltransferase, an enzyme long believed to attack only maltosidic linkages and to catalyze only glycosidic linkage exchange. The effective conversion of α-maltosyl fluoride to α-maltosaccharide 1-fluorides and cyclodextrins reveals the enzyme's additional ability to cleave C-F glycosylic bonds and to catalyze a class of irreversible reactions providing de novo glycosidic linkage synthesis. Cyclodextrin formation by Bacillus macerans CGTase, heretofore observed only with starch and dextrins, was more pronounced with α-maltosyl fluoride than with maltotetraose; conversion of the dimeric fluoride to α-, β-, and γ-cyclodextrins by Bacillus megaterium CGTase was as great (90%) as with the best of CGTase substrates. Although glycosidic exchange reactions occur in digests with α-maltosyl fluoride, the very effective conversion of this small molecule to higher DP products is the consequence of novel reactions of “total chain lengthening” (α-G₂F+α-G₂Fα-G₄F+HF, etc.) and “total cyclization” (e.g., α-G₇Fβ-CD +HF) not found with traditional substrates. The results establish CGTase to be a glycosyl mobilizing enzyme, and illustrate the inadequacy of the long accepted notion that glycosyltransferases require donors with a glycosidic bond.

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Porcine pancreatic α-amylase was purified free of proteolytic activity by the method of Schramm and Loyter using specific glycogen-complex precipitation in the presence of a proteinase inhibitor, phenylmethylsulfonylfluoride. The purified amylase was shown, by SDS-gel electrophoresis, to consist of a single peptide chain having a molecular weight of 53, 000. Treatment of the purified amylase with a pancreatic extract containing proteinase activity had little effect on the amylase activity but produced several low molecular weight proteins on SDS-gel electrophoresis. The effects of trypsin and α-chymotrypsin were tested. Trypsin had no effect on the amylase; α-chymotrypsin, however, cleaved the amylase to give four distinct proteins with molecular weights of 53, 000, 39, 000, 27, 000, and 13, 000, but had little effect on the amylase activity. Reduction and carboxymethylation of the α-chymotrypsin-hydrolyzed amylase gave low molecular weight peptide fragments of molecular weights between 27, 000 and 13, 000. It is concluded that α-chymotrypsin produces limited hydrolysis of the α-amylase molecule into peptide fragments of approximately 13, 000 daltons. These fragments are held together by disulfide linkages to give a dimer (27, 000 daltons), a trimer (39, 000 daltons), and a tetramer (53, 000 daltons). The α-chymotrypsin cleavage does not adversely affect the amylase-active site which retains a high percentage of its activity. Limited chymotrypsin hydrolysis of the α-amylase molecule in commercial, crystalline preparations may account for the formation of two 25, 000 dalton, amylase-active proteins when the preparation is reduced with dithiothreitol.

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Gel filtration profile and ultracentrifugation analysis showed that the molecular weight of the commercial glycogen or the glycogen extracted with trichloroacetic acid (TCA) from whole oyster tissue was smaller than that of the glycogen extracted with dimethyl sulfoxide (DMSO). The degradation extents of glycogens extracted with DMSO and TCA on pullulanase treatment were 6.3% and 10.8 and that of commercial glycogen was 24.8%. Gel filtration profile or paper chromatogram of the pullulanase digest of the glycogen extracted with DMSO (DMSO-glycogen) was similar to those of the glycogen extracted with TCA (TCA-glycogen) and commercial glycogen. These results suggested that TCA-glycogen or commercial glycogen was degraded to some extent during the extraction of glycogen and became more susceptible to pullulanase keeping fundamental structure of the glycogen naltered. Electron microscopic study showed that the particle size (diameter) of DMSO-glycogen ranged 20-180 nm while the particle size of TCA-glycogen or commercial glycogen ranged 10-130 or 10-40 nm, respectively.

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Glycogen was extracted from oyster (whole tissue), abalone (adductor muscle) and scallop (adductor muscle) with dimethyl sulfoxide (DMSO). The average chain lengths (CL) of the glycogens from oyster, abalone and scallop were 12.3, 12.9 and 12.9, respectively. ?A-Amylolysis limits (%) of these glycogens were 32.3, 32.3 and 26.3, respectively. The gel filtration profiles of the glycogens from oyster and abalone showed two peaks indicating the existence of two kinds of glycogens different in size. These glycogens were degraded by 3.6-9.8% with an excess amount of pullulanase, and the A-chains of these shellfish glycogens were shown to consist of G2-G10 in which G3, G4 and G6 were preponderant.Glycogens were completely debranched by isoamylase and the degrees of polymerization (DP) of the most preponderant fractions of the degradation products were 11-13. Electron microscopic study showed that the diameters of the particles of the glycogens of oyster, abalone and scallop were 20-180, 20-230 and 20-120 nm, respectively.

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A sensory investigation of the effect of solution viscosity on sweetness is reported in this paper. A trained taste panel compared the sweetness of 5% sucrose in water to a solution of 5% sucrose containing 2% pregelatinized starch. When the starch was tapioca or waxy corn there was no sigiificant difference between the samples with and without starch although there was a tendency to rank the starch containing solution as less sweet. When the starch was potato there was a statistically significant reduction in sweetness of the starch containing solution. These results are discussed and a possible mechanism for sweetness reduction is postulated.

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To determine the structure of retrograded starch, 10% solutions of normal-and waxy corn starches were treated by repeated freeze-thaw cycles to obtain extremely retrograded starch samples . These starch samples were examined for the determination of the degree of gelatinization, iodine binding, acid and enzyme hydrolysis etc. The residual carbohydrates after acid and enzyme treatment were completely different from each other in molecular weight and shape, determined by the gel filtration of Toyopearl HW-50. We proposed three different domains (A, B and C) existing in retrograded starch, which are schematically illustrated in the text .

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