Seven sweet potato (Ipomoea batatas) cultivars were grown in 1994 and 1995 by standard harvesting (SH), early harvesting with mulch (EHM) and late harvesting with mulch (LHM). The effects of flesh color, cultivation method and year on starch and cell-wall material (CWM) contents and starch characteristics [amylose content and gelatinization properties by differential scanning calorimetry (DSC)] were tested. Orange flesh cultivars had a lower ratio of starch to CWM content and a higher amylose content than purple and white flesh cultivars. In almost all cases, cultivation method and year had little impact of starch, CWM and amylose contents. Significant differences in DSC thermograms were seen among cultivation methods, showing the highest values of onset temperature for gelatinization, lowest values in the gelatinization range at EHM and lowest values in gelatinization heat at SH. Yearly effects on DSC characteristics were not observed.

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Two varieties of squash, Ebisu (Cucurbita maxima DUCH.) and Kogiku (Cucurbita moschata DUCH.), were harvested on flowering day and every one or two weeks during the period of June to August 1988. The carbohydrate contents of squash and the properties of starch granules prepared from the squash fruits were examined. The results obtained were as follows: 1) The soluble sugar contents of squash were found to increase during development. The glucose content was about 1% to squash weight and scarcely changed during development. 2) The average granular sizes of Ebisu and Kogiku starches were in the range of 2.9 to 8.8 μm and 3.2 to 8.0 μm, respectively, and tended to increase in the early stages of squash fruit development (1 week after flowering). 3) The amylose contents of Ebisu and Kogiku starches determined by the amperometric titration method were in the range of 16.5-22.5% and 14.1-18.0%, respectively, and tended to be higher in the later stages of develooment than in the earlier stage. 4) The initiation temperature for gelatinization of Ebisu and Kogiku starches determined by differential scanning calorimetry (DSC) were in the range of 62-64°C and 62-66°C, respectively, and it rarely changed throughout the squash fruit development. 5) The maximum viscosity and breakdown of Ebisu and Kogiku starches determined by Brabender amylography were found to increase during development. 6) X-ray diffractography of the squash starches showed B-type patterns throughout the squash fruit development. 7) The susceptibility of the squash starch granules to hog pancreatin was approximately 40-50% to that of normal maize starch granules, and it scarcely changed during development. By scanning electron microscopy (SEM), we observed hollows on the surfaces of squash starch granules, after an attack by pancreatin.

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The condensation reaction of β-cyclodextrin (βCD) and galactosyl-maltooligosaccharides (4-O-β-D-galactopyranosyl-maltooligosaccharides) was investigated using pullulanase and isoamylase. The structures of the condensation products were found to be β-D-galactosyl-(1→4)-α-maltosyl-(1→6)-βCD, β-D-galactosyl-(1→4)-α-maltotriosyl-(1→6)-βCD, β-D-galactosyl-(1→4)-α-maltotetraosyl-(1→6)-βCD and β-n-galactosyl-(1→4)-α-maltopentaosyl-(1→6)-βCD by FABMS, ¹³C-NMR and methylation analysis.

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Haw and lemon pectins gave mainly two fractions (C1, C2 from haw and Li, L2 from lemon) by DEAE-Sephadex A-50 column chromatography. One (C2, L2) showed higher viscosity than the other (C1, L1). On hydrolysis by purified pectinase, the fractions of C2 and L2 were completely hydrolyzed, while the fractions of Ci and L1 left pectinase-resistant fractions (C1-1 and L1-1) in a high molecular weight region. DEAE-Sephadex A-25 column chromatography of the resistant fractions showed three peaks (C1-1-A, -B, -C) for Ci-i and two peaks (L1-1-A, -C) for L1-1. Acidic fractions C1-1-C and L1-1-C were structurally similar to each other, but neutral fractions, C1-1-A and L1-1-A were totally different in constitutional sugar. The structural difference of neutral fractions (C1-1-A and L1-1-A) and presence/absence of acidic fraction corresponding to C1-1-B in the pectin molecule may be related to the viscosity of the pectins.

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Pumpkins (variety; Kogiku) were stored at room temperature and 5°C for 1 or 2 months and changes in the carbohydrate contents of the pumpkin and properties of starch granules prepared from the pumpkin during preservation were examined. 1) The starch contents of pumpkin decreased and soluble sugar contents increased inversely by preservation. The decrease in starch contents and increase in soluble sugar contents tended to be higher at room temperature than at 5°C. 2) The amylose contents of pumpkin starches, determined by amperometric titration and enzymatic and chromatographic methods, increased by preservation at room temperature and 5°C. 3) The starch granules of pumpkin during preservation suffered enzymatic attack, after which the susceptibility of starch granules prepared from stored pumpkin to hog pancreatin was higher than that before preservation.

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Interaction between a transition-state analogue, gluconolactone (L), and Aspergillus niger β-glucosidase was studied using the stopped-flow method by monitoring the decrease in the enzyme Trp fluorescence intensity, in relation to the subsite structure of the enzyme. The dependence of kapp, which was evaluated from the reaction curve for the binding of L to the enzyme, on [L]_o showed a saturation curve. This result is most reasonably accounted by the two-step mechanism; a fast bimolecular binding process followed by a slow unimolecular process. In the first step, L is transiently bound to subsite 2 and in the second step, it relocates to subsite 1, accompanied by a decrease in fluorescense intensity. Furthermore, the dependence of kapp on [L]_o in the presence of galactose was investigated with the same procedure. The result supports the mechanism on the binding of gluconolactone.

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Functional developments in the area of carbohydrate are progressing quickly and several new enzymes catalyzing useful reactions for carbohydrate production have been found recently. In this study, various aspects on the effective utilization of dextran and related enzymes were examined. Production of dextran and its utilization: Leuconostoc mesenteroides NRRL B-512F is known to synthesize dextran, which has a wide variety of industrial uses. B-512F dextransucrase was obtained in a highly purified form by the use of a convenient affinity purification method, and chemical modification of this enzyme gave peptide sequences including the catalytic residues. Moreover, production of a new cyclic isomaltooligosaccharide (cyclodextran) has been achieved. Cyclodextran has been shown to have a potent anti-cariogenic activity.. The preparation of proteases having high resistance to selfdigestion was developed by cross-linking with dextran-dialdehyde. Periodate-oxidized polysaccharides bound with fluorescent reagent, and the resulting polysaccharides served as the substrate for enzyme activity assay and for the classification of an action pattern of enzymes. Modification of carbohydrases: Chemical modification of Taka-amylase A (TAA) with o-phthalaldehyde (OPA) provided a modified enzyme with better susceptibility to oligosaccharides. OPA was incorporated into the junction of main-domain and C-terminal domain of the TAA molecule. New methods for carbohydrate and enzyme analysis: For micro-analysis of uronic acids, new methods for determination of the carboxyl group and for fluorescent labeling of uronic acid were developed. Periodate oxidation enabled sensitive detection and labeling of the sugar-chain in glycoprotein. A new slab-gel plate with multiple lanes provided a convenient and rapid polyacrylamide gel electrophoresis to analyse enzyme characteristics. A forced affinity chromatography technique in the presence of a highh concentration of ammonium sulfate was developed as a new method for the purification of various carbohydrases.

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It has long been known that ungerminated barley contains only β-amylase while α-amylase appears at germination and that there are two types of β-amylases, active and inactive. Many scientists have so far paid much attention to the activation mechanism of the inactive β-amylase. However, their papers were found to have some problems left unresolved. One of the main reasons seems to be due to the failure to isolate inactive β-amylase in a native state and no experiments on it in vitro. Therefore, I attempted to isolate salt-soluble inactive β-amylase and two types of inactive enzymes, heteropolymer type (MW. 280, 000) and homopolymer type (MW. 160, 000) were isolated. From the activation of these enzymes in vitro, it was shown that both reductive cleavage of disulfide bonds and proteolytic cleavage of peptide bonds are necessary for the complete activation of all the types of inactive β-amylases in ungerminated barley. On the other hand, the activation of these amylases during germination was found to proceed mainly through protein disulfide reductase and malt proteinase. On the basis of these results, it was concluded that active β-amylase synthesized in the early stages of barley ripening is polymerized by disulfide bonds and/or peptide bonds into inactive enzymes in the later stages of ripening and that, during germination, the inactive enzymes are again depolymerized into active β-amylase which hydrolyzes starch with α-amylase to supply energy for germination. It is interesting that the reversible reactions between the sulfhydryl and disulfide groups are concerned with the activation and inactivation of β-amylase in barley. In the 1970s the distribution of fl-amylase was reported in microorganisms. We also isolated several microorganisms producing β-amylase from soil. Bacillus cereus BQ10, one of the isolates, was treated by UV irradiation for enhancement of β-amylase productivity. The BQ10-S1, UV-mutant was found to produce about 30 times more β-amylase than the wild strain. Furthermore, a rifampin-resistant, asporogenous mutant, BQ10-S1 Spo-, was obtained by NTG treatment. The amount of β-amylase produced by this asporo-genous mutant reached about 500 times that of the wild strain. The molecular weight of the enzyme was 60, 000 and the optimum pH and temperature were around neutral pH and 55°C. The gene of β-amylase was isolated and cloned in E. coli, and all the base sequences and amino acid sequences were determined. The homology of the amino acid sequences between other plants and microbial β-amylases was compared. About 50% homology was found among those from B. polymyxa, B. circulars, Cl. thermosulfurogenes, etc., and about 30% among those of soybean, barley, sweet potato, etc. Amino acid residues at the catalytic site of β-amylase, which have long remained unelucidated, were examined with B. cereus crystallized β-amylase in enzymatic and X-ray crystallographic studies and found to be not sulfhydryl but two glutamic acid residues. The hydrolytic activity of raw starch by BQ10-S1 Spo-was also found to be due to a "starch-binding domain (SB)" which was not found in plant β-amylases.

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We found a new enzyme, neopullulanase, and proved that the enzyme catalyzes both hydrolysis and transglycosylation at α-(1→4) - and α-(1→6)-glucosidic linkages by one active center. A series of experimental results using neopullulanase indicated that the four reactions described above could be catalyzed in the same mechanism. On the basis of the common catalytic mechanisms and the structural similarities among the enzymes which catalyze the four reactions, we proposed a general concept for an enzyme family, α-amylase family. The substrate specificity and the transglycosylation activity of neopullulanase were altered by site-directed mutagenesis on the basis of information from a threedimesionalstructure predicted by computer-aided molecular modeling. From the standpoint of industrial application, we developed a new way of producing isomalto-oligosaccharide syrup using the transglycosylation reaction of neopullulanase. We also expanded the concept of α-amylase family into branching enzymes and constructed chimeric enzymes of starch branching enzymes I and II isof orms from maize endosperm. The results indicated that the N- and C-terminuses may be involved in determining substrate preference, catalytic capacity, and chain length transfer.

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A crystalline α-glucosidase (ANGase) purified from Aspergillus niger showed high activity to maltooligosaccharides, and no or less to heterogeneous substrates and α-glucans, implying that ANGase is a so-called “maltase”- type enzyme. The essential ionizable groups in the active site were identified to be -COO- and -COOH by the kinetic studies and the chemical modification technique using water-soluble carbodiimide. ANGase was composed of two different subunits, namely Pl and P2, which were separable only by reversed-phase chromatography. The amino acid sequences of P1 (227 amino acids) and P2 (719 amino acids) were determined by the EDMAN degradation method. ANGase had seven Cys of which only one residue was free, meaning that six Cys form three S-S bridges. One set of S-S bridges in P1 and two of those with one free Cys in P2 were identified with a chemical modification analysis using fluorescent SH reagents. Two subunits were not linked with disulfide bond, which suggests that P1 and P2 are tightly bound by non-covalent linkage to make a three-dimensional structure. The genomic nucleotide structure of the ANGase analyzed showed that the DNA sequences coding P1 and P2 were in a single open reading frame in the this order. Between them there were 14 extra amino acids deduced from DNA sequence, suggesting that the limited proteolysis may remove them and contribute to form a mature protein. This is one of the unique post-translational modifications of ANGase. Another post-translational modification is the binding of sugar chains to Asn and Ser/Thr in ANGase. ANGase contained about 25% carbohydrate composed of mannose, glucose, galactose and N-acetylglucosamine. We purified seven N-linked high-mannose type oligosac-charides and determined their structures. Four sugar chains had one galactofuranosyl residue, abbreviated as Gall, bound to Man (A) via α- (1→2) -linkage. Three of them were novel oligosacchα-rides. More than 70% of the Asn-linked sugar chain in ANGase was found to contain Gall, implying that the transferring step of this residue to Man (A) is the main biosynthesis route for making N-linked oligosaccharide in A. niger. Nine kinds of Ser/Thr-linked sugar chains were cleaved from ANGase by the β-elimination method. Five of them were isolated, and their structures were determined. They were found to be classified into two types of carbohydrates, such as mannooligosaccharides and glucosylmannooligosaccharides. Two of them are the novel O-linked sugar chains, which are only found in microbial glycoprotein. Using a mechanism-based irreversible inactivator is the effective method to learn the reaction mechanism and to identify the catalytic amino acid residue. Conduritol B epoxide, abbreviated as CBE, has been known as a mechanism-based inactivator for α-glucosidase. The inactivation of ANGase by CBE followed pseudo-first-order kinetics with forming a reversible enzyme-inhibitor complex before covalent inactivation. A competitive inhibitor, Tris, decreased the inactivation rate. The incorporation of one mole of CBE per mole of ANGase completely abolished the enzyme activity. A dissociated carboxyl group (-COO-) in the active site was suggested to attack the C-1 of CBE. The sequence analysis of CBE-labelled peptide showed that the Asp (224) of P2 was identified to be one of catalytic amino acids. From the homology of whole amino acid sequences more than 20 α-glucosidases and their variety in substrate specificity, α-glucosidases can be classified into two groups at least, family-I and family-II. ANGase is a member of family-II, and the sequence motif containing Asp (224) of P2 was conserved completely in all α-glucosidases of this group. The I-type enzymes belong to the α-amylase family. Using the CBE labelling method, we have identified the catalytic amino acid of Asp of honey bee α-glucosidase (isoenzyme I), which is classified to family-I.

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