Apparent amylose contents of rice grains have been investigated by two measuring methods. The measurement of wavelength at maximum absorbance of iodine absorption spectra of unpolished brown rice can be simply done, and that had high relationship with apparent amylose contents investigated by enzymic chromatographic method. We proposed the calculation formula of the apparent amylose content derived from the value of wavelength at maximum absorbance of iodine absorption spectra of unpolished brown rice.

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Starch in embryo of lotus seed (Nelumbo nucifera GAERTN.) was prepared by wet milling with dilute alkali, and its chemical and physical properties were investigated in comparison with starches of lotus rhizome (lotus root), maize (corn) and potato. The results were as follows. 1. The starch granules of lotus seed measured by scanning electron microscopy (SEM) were small and oval in shape with an average size of 13 sum. These were quite different compared with very large ellipsoidal shape (30×60μm) of lotus-root starch. 2. By X-ray diffractometry, the lotus-seed starch showed A-type patterns. 3. The digestibility of the lotus-seed starch granules by fungal glucoamylase (Nagase) was 100% in 24 hr reaction while that of the lotus root was about 42%. 4. The initiation temperatures of the lotus-seed and -root starches were 79 and 65°C by amylography, 67.5 and 65°C by photopastegraphy, and 69.9 and 56.7°C by differential scanning calorimetry, respectively. The maximum viscosity of the lotus-seed starch measured by Brabender's amylography was 610 B.U., higher than those of the lotus-root starches. 5. The apparent amylose content of the lotus-seed starch determined by the amperometric iodinetitimetry and gel filtration after isoamylase-debranching was 27.7 and 29.9%, respectively, indicating higher amylose contents than the lotus-root starch, 21.7 and 18.5%, respectively.

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The properties of six new characteristic rice cultivars (Hoshiyutaka, Saikai 198, Saikai 194, Saikai 184, Hokuriku 149, Suigen 258) suitable for the cultivation in southwestern Japan, have been investigated concerning the pasting properties and the structures of starch. The pasting properties of their powders and starches were analyzed by a Rapid Visco-Analyzer and similar behavior was observed between respective rice powders and starches. Hoshiyutaka showed the lowest maximum viscosity and breakdown viscosity and Saikai 184 also gave a low breakdown viscosity, having some distinguishable properties from the other cultivars. Apparent amylose contents of six rice starches were in the range of 18.5 to 30.0% but actual amylose contents were lower, being in 16.2-20.8%. Hoshiyutaka and Saikai 184 showed especially high apparent amylose contents, 27.5 and 30.0%, but their actual amylose contents were of 20.8 and 18.1%, respectively. The amyloses from Hoshiyutaka and Saikai 184 had slightly higher molecular weights and a greater number of side chains than those from the other cultivars. The amylopectins of Hoshiyutaka and Saikai 184 showed higher iodine affinities of 1.5 and 2.8 g/100 g and the higher intrinsic viscosities of 149 and 171 (ml/g), respectively, than those (iodine affinities of 0.23-0.56 g/100 g and intrinsic viscosities of 138-143 (ml/g)) from the others, and linked considerable amounts of extremely long chains of similar size to amylose. These distinct structures of Hoshiyutaka and Saikai 184 starches were suggested to have some relation to their characteristic pasting behaviors.

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Candida guilliermondii H-404 isolated from soil produced a large amount of thermostable α-galactosidase intra-(cell-bound) and extracellularly. The cell-bound enzyme was easily solubilized by autolysis and purified from the autolyzate with DEAE-Toyopearl 650M, SP-Toyopearl 650M, and Butyl-Toyopearl 650M chromatographies. The molecular weight of the cell-bound enzyme was 280, 000 by gel filtration, and 65, 000 by SDS-PAGE. The enzyme seemed to be constructed of four identical subunits. The enzyme was most active at pH 5 and at 70°C (10 min reaction) and was stable between pH 3.5-9.5 and below 50°C. The enzyme was strongly inactivated by 1 mM AgCl, 1 mM HgCl₂, and 1 mMPCMB. The cell-bound enzyme well hydrolyzed α-1, 6-galactobiose, but poorly α-1, 4-galactobiose. The enzyme had high transfer activity and wide acceptor specificity. There was no significant difference in the enzymatic properties of the cell-bound and extracellular enzymes.

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Transfer action of isomaltodextranase [EC 3.2.1.94] from Arthrobacter globiformis T6 was studied using glucose and maltose as acceptors. This enzyme transferred an α-isomaltosyl residue from dextran to the C-4 and C-6 positions at the non-reducing end of acceptors . Thus, the enzyme transfer red isomaltose to glucose and maltose to produce panose and isomaltotriose, and 4²-α-isomaltosyl maltose (IMM) and 4-α-isomaltotriosyl D-glucose (IMTG), respectively. The transisomaltosyl activity at the C-4 position was 10-13% of that at the C-6 position in HPLC analysis, and in Fuji Film BAS2000 image analyzer analysis using D- [U-¹⁴C] glucose as an acceptor.

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Periodate oxidation of clinical dextran gave dextran-dialdehyde, which had absorption at the 1740cm-1 region of the infrared spectrum and contained 62% of D-glucose residues in oxidized form. Bovine pancreatic trypsin was modified with dextran-dialdehyde. The molecular weight of the modified trypsin was estimated to be 92, 000 based on the protein standards. The isoelectric point (p1) of trypsin decreased from 10.1 to 6.7 upon modification. Ten residues out of 15 free amino groups were modified per trypsin molecule. Carbohydrate content of modified trypsin was 49%. The modified trypsin showed a similar hydrolysis pattern of insulin B-chain with that of the native trypsin.

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Taka-amylase A (TAA) was modified with ο-Phthalaldehyde (OPA), and the modified enzymes, Ml. and M2-TAA were separated by a DEAE-Sephacel column chromatography. Although OPA modification caused a large decrease in enzyme activity, residual activity could be measured by Neocuproine method and glucose oxidase method. Compared with the polymer hydrolyzing activity, M1-and M2- TAA exhibited rather high relative activity for the low-molecular weight substrates such as maltooligosaccharides and their reduced forms. Maltotriose was a poor substrate for native TAA (NTAA), while modified TAA showed good susceptibility. Moreover, N-TAA gave no glucose from maltooligosaccharides whereas modified TAA yielded a notable amount of glucose. Optimum pH was shifted to the acidic regions by 1.0-1.5 pH units with modified enzymes, which was much more evident for the modified enzymes than N-TAA. These results indicated that OPA-modified TAA was enhanced in so-called maltosidase activity.

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The physicochemical properties of starches from 16 kinds of sweetpotato cultivars were investigated . On the general properties of the starches, the average granular size, the crude protein content, the phosphate content and the pattern of X-ray diffractograms, no specific starch was found. The apparent amylose content of the starch was estimated by the blue value method and the dual-wavelength method . The amylose contents ranged from 13 to 19%, and the contents obtained by the two methods were in agreement with each other except for a few starches. The debranched starches roughly separated into three fractions on gel-permeation chromatography (GPC) and the fractions were defined as Fr 1, 2 and 3 on the basis of the degree of polymerization, in which minor differences were found in their chain length distributions. On the other hand, each starch showed characteristic pasting properties examined on amylography, swelling factor and solubility. Statistical analysis revealed that the proportion of Fr 2 on the GPC of debranched starch was significantly (ρ<0.01) correlated with the pasting temperature and the setback. The content of the long chains of amylopectin, which was estimated as the difference between the amylose and Fr 1 contents, was also correlated with some pasting properties . No correlation was found between the amylose content and the pasting properties. It was considered, therefore, that the diversity of the pasting properties of starches from sweetpotato cultivars was more accurately reflected by amylopectin than by amylose.

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The essential ionizable groups of rice α-glucosidase II were identified by kinetics and chemical modification methods. The pKe of ionizable group 1 (on the acidic side) was 3.1, and that of ionizable group 2 (on the alkaline side) was 6.1. The heat of ionization of ionizable group 2, ΔH₂, was -1.7 kcal/mol. The pH of both the pKe₁ and pKe₂ values increased by about 0.5 when the dielectric constant of the reaction medium was lowered by adding 20% methanol. The chemical modification of the active site in rice α-glucosidase II with conduritol B epoxide, an affinity labeling reagent, resulted in inactivation of the enzyme. The inactivation followed pseudo-first-order kinetics. The enzyme was protected from inactivation by a competitive inhibitor, Tris, and the partially inactivated enzymes showed only a decrease in V values and no change in K_m. These results strongly suggest that the essential ionizable groups of rice α-glucosidase II are -C00-and -COOH.

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Glucodextranase was purified to homogeneity by means of affinity column chromatography, hydro-phobic, anion exchange and molecular-sieve HPLC from the culture of Arthrobacter globiformis T-3044 isolated from soil. The Mr of the enzyme was estimated to be 120 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme had an optimum pH for activity at pH 6.0, and was stable at pH 6.0 to 7.5 at 25°C for 24h. It was stable up to 40°C and was almost completely inactivated at 50deg;C for 15 min. The enzyme produced only β-D-glucose from dextran. The enzyme also hydrolyzed starch at about 15% of the hydrolysis ratio to dextran. This ratio was about 15 times higher than that of I42 GDase. The enzyme is characterized by a high activity toward starch.

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The binding of α-amylase inhibitor isolated from white kidney bean Phaseolus vulgayis (abbreviated to PHA) to two active components of porcine pancreatic α-amylase [EC 3.2.1.1] (abbreviated to PPA I and II) were studied by isothermal calorimetry at pH 6.9 and at various temperatures ranging from 20 to 40°C. The net enthalpy changes of binding were determined after correction for the buffer ionization heat. Thermodynamic quantities for the binding of PHA to the enzymes were derived as fuctions of temperature from inhibitor constants and isothermal calorimetric experiments. At 25°C, the thermodynamic quantities were ΔG=-51.8 kJ moL^-1, ΔH=5.2 kJ mol^-1, ΔS=0.19 kJ K^-1 mol^-1 and ΔCp=-1.18 kJ K^-1 mol^-1 for PPA land ΔG=-53.9 kJ mol^-1, ΔH=1.9 kJ mol^-1, ΔS=0.19 kJ K^-1 mol^-1 and ΔCp= -1.25 kJ K^-1 mol^-1 for PPA II. From these results, it was concluded that, like many other protein-protein interactions studied calorimetrically so far, the binding of PHA to PPA I and II are also characterized by a predominant contribution from hydrophobic effects.

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The effects of calcium (Ca) gluconate, Ca-lactate and sodium (Na) gluconate on some rheological properties of wheat flour dough, on the interaction between gluten and starch in dough, and on loaf volume were studied. The dough containing Ca-gluconate (0.3-0.5% w/w flour basis) and Ca-lactate (0.1%) increased the loaf volume significantly. In the farinograph test, Ca-gluconate and Ca-lactate decreased the dough development time, whereas Na-lactate increased it. In the rheometer test, Cagluconate- containing dough had a higher modulus of elasticity and viscosity coefficient than the control. Ca-lactate and Na-gluconate caused a decrease in the two parameters. Scanning electron microscopy showed that Ca-gluconate-treated Boughs contained thick fibrils not observed in the control Boughs. DSC measurements showed that the gelatinization temperature and the enthalpy of wheat flour dough increased upon the addition of Ca-gluconate. Ca-gluconate and lactate decreased the SH content of dough, and also tended to make the diameter of gas cells slightly smaller. The observed effects of Ca-gluconate can be explained on the basis of cross-link formation in the gluten proteins.

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Thermoactinomyces vulgaris R-47 produces two a-amylases, TVA I and TVA II, and both enzymes catalyze the same transglycosylation reaction. However, TVA II accumulates the transglycosylation products more than TVA I. The time course of the accumulations between the two TVAs was compared using pullulan and [U-→¹⁴C]glucose. The reactions consisted of two stages, which are the formation of the transglycosylation products and the degradation of the accumulated products, whether TVA I or TVA II was used. The rates of product formation by TVA I were similar, 1 to 2.4-fold, to those by TVA II, whereas TVA II accumulated 3.5 to 7.5 times as much products as TVA I. Thus, we infer that the rates of product degradation by TVA I were larger than those by TVA II. The difference in the degradation rates was not due to the hydrolytic activity, ie., differences in the kcal/Km values between TVA I and TVA II, but due to the inhibition of TVA II by panose, the hydrolysate of pullulan.

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Some aspects of structual correlation to functions of unique plant and microbial polysaccharides, and their potential utilization have been described.

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This paper from the 1995 award address of the Society is composed of the following three research topics that were carried out at the Laboratories of Biological Chemistry, and Molecular Enzymology and Protein Engineering, Faculty of Agriculture, Tokyo University of Agriculture and Technology, from 1983 to 1995. A summary of these is given below. 1) Subsite structure and substrate specificity. The subsite structures of Thermoactinomyces vulgaris α-amyalse (TVA I), and native porcine pancreatic α-amyalse II (PPA II) and PPA II modified with DTNB were determined by the SUGANUMA-HIROMI method. It was obvious that the characteristics of the TVA I subsite structure caused its hydrolysis of (α1→6) glucosidic linkages of isopanose and 62-α-maltosylmaltose as well as (α1→4) glucosidic linkage of maltotriose (G3) on the same active site. The difference between the subsite structures of native and modified PPA Its reflected the change of their substrate specificities; the rate of hydrolysis of G³ by PPA II was lower than that of the modified PPA II and the cleavage point of G³ was changed from its reducing end to its nonreducing end by the DTNB modification. The thermal stability of Bacillus acidopullulyticus pullulanase (BAPase), increased by the addition of sugar alcohol, was dependent on its molecular weight; the sugar alcohols larger than maltotriitol (0.2 M) stabiliyzed BAPase completely for 7 hr at 60t. 2) Cloning of TVA genes and the structure and fucntion of TVAs. Genes of TVA (tva I, tva II) were cloned in Escherichia coli MV1184 using two different methods, the probe DNA method and the shotgun cloning method. TVA I was the same enzyme as described previously, and TVA II was a different gene product from TVA I, whereas these enzymes have the α-amylase and pullulan-hydrolyz-ing (panose-forming) acivities. Their primary structure, amylase family conserved region, subsrtrate specificity and kinetic parameters for low and high molecular weight substrates were compared. The modification of TVA II was tried using site-directed mutagenesis, and enzyme activities of most of mutant enzymes decreased, except for the activity of one mutant (H2O2N). Crystals of TVA II were obtained by the sitting drop vapor diffusion technique and diffraction data to 2.9 A resolution were collected using the R-AXIS llc system (Rigaku). The crystal belongs to an orthorhombic system with cell dimensions of a119.5 Å, b=120.6 Å, and c=114.6 Å and a space group P2¹2¹2¹. 3) Oligosaccharide syntheses by pullulanase, TVA II and isomalto-dextranase. Branched cyclodextrins were synthesized from maltooligosaccharides and cyclodextrins (CDs) using the reverse reaction of BAPase. Under the conditions of 70-75% of substrates and 100-200 units of BAPase/g of CD at 60-70t, the yield of branched CDs was higher than 40%. TVA II transfered panosyl residues from pullulan to the C-4 and C-6 positions of glucose (acceptor). Also isomaltodextranase transferred isomaltosyl residues from dextran to the C-6 and C-4 positions of glucose (acceptor) as well as to sucrose, trehalose and primary alcohols.

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1. Starch granules and phytoglycogen were prepared from kernels during development of several endosperm starch modifying genes [waxy (wx), dull (du), amylose-extender (ae), sugary-2 (su-2), sugary-1 (su-1)] and their normal counterpart in the inbred Oh43 maize (Zea mays L.) background. Starch contents of normal and wx kernels were higher and those of su-1 kernels were lower than those of du, su-2, and ae kernels. Amylose contents of normal, du, su-2, and ae starches increased with development. Amylopectins of ae, du and su-1 were novel types. The average chain length of phytoglycogen was shorter than those of amylopectin. The X-ray diffraction patterns and photopaste-grams of starches were very similar at different stages of development for each genotype. Exhaustive degradation of starch granules by crude glucoamylase showed that ae starches were more resistant to amylase than the other starches. 2. β-Limit dextrins of starches of mature kernels of normal and double- and triple-mutant combinations of the wx gene with other starch modifying genes and su-1 phytoglycogen in the inbred Oh43 maize background were prepared. The β-limit dextrins were successively debranched by isoamylase and pullulanase, and followed by quantitative gel filtration. The ae, wx and du; wx mutant amylopectins were novel. The A : B chain ratios for amylopectins were in the range of 1.1 to 1.4 and were similar to the wx amylopectin. The A : B chain ratio for su-1 phytoglycogen was 1.0. The phytoglycogen did not contain long B chains. 3. Gelatinization characteristics of starch granules and retrograded starches were investigated by differential scanning calorimetry (DSC). Onset, peak, and conclusion temperatures of gelatinization (T_o, T_p and T_c respectively) and heat of gelatinization (ΔH) of starch were determined from DSC thermograms. Noticeable effects of each of wx, ae, and su-2 genes on T_o, T_p, T_c and ΔH were observed in starches of their respective mutant combinations. Apparent epistatic effects of the genes for gelatinization temperature were observed. Thermograms of retrograded starch shifted to lower temperature ranges and were broader compared with the native starch. The ΔH of retrograded starch increased with increasing storage duration. 4. Starch granules were prepared from mature kernels of many foxtail millet (Setaria italica BEAUV.) collected in different places of Asia and Europe and grown in the field of Kyoto University, and their properties were investigated.

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