Tissue-specific expression of the isoforms of rice α-amylase and their expression in suspension cultured cells were investigated, together with regulation by various sugars. Six isoforms, Y, A, G, H, I and J, were identified: Isof orm A, Isof orm Y and Isof orms A-J were mainly detected in germinating seeds, young plant tissues, and suspension-cultured cells, respectively. From the results of amino acid sequencing of the purified isoforms, isoform A and isoform H were identified as the gene products of RAmy1A and RAmy3D, respectively (Plant Physiol., 110, 1395-1404 (1996)) . Since metabolic regulation of the expression of rice a-amylase has been reported, we analyzed this phenomenon by following the changes in levels of each isoform in cells in suspension cultures in the presence of various sugars . The expression of the RAmy3D-encoded protein (isoform H) was completely repressed by so-called metabolic sugars, while expression of the RAmy1A-encoded protein (isoform A) was repressed to a less extent. The results indicate that high expression of the RAmy3D-encoded protein is controlled much differently from that of the RAmy1A-encoded protein.

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Acidobacterium capsulatum, an acidophilic, mesophilic and chemoorganotrophic bacterium, constitutively produced the acid α-glucosidase. The enzyme, which was successively purified to homogeneity by CM-Sepharose, Sephacryl S-300 and Mono-S ion-exchange chromatography, was a monomeric protein, whose molecular weight was estimated to be 65, 000 by gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum.activity at pH 4.5 and 30°C, being stable in the pH 3.5 to 7.0 region and in the range of 10 to 50°C. No activity was detected above pH 7.5 or above 60°C. Its isoelectric point was 7.0. The enzyme hydrolyzed pnitrophenyla-glycoside, oligosaccharides containing α-1, 3 (nigerose), α-1, 4 (maltose), α-1, 6 (isomaltose), and α-1, β-2 linkages (sucrose), and soluble starch and produced α-configurational glucose. These findings indicate that the A. capsulatum enzyme represents a novel type of α-glucosidase exhibiting a broad substrate specificity. Amino terminal analysis by a protein sequencer provided the sequence of the first eighteen residues as Ser-Ala-Thr-Gly-Ala-Pro-Trp-Trp-Lys-Asn-Ala-Val-Ile-Tyr-Glu-Val-Tyr-Pro.

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A nonreducing trisaccharide was formed in a reaction mixture containing trehalose and the cell-free extract of a thermophilic anaerobe, Thermoanaerobium brockii ATCC 35047. This saccharide was isolated from the mixture by trehalase digestion, alkaline treatment and preparative HPLC. From the results of partial acid hydrolysis, methylation analysis, and ¹³C-NMR analysis, the chemical structure of the saccharide was determined to be 2-O-α-D-glucopyranosyl α-D-glucopyranosyl α-D-glucopyrano- side (selaginose). Two enzymes related to Selaginose synthesis were partially purified by DEAEToyopearl column chromatography from the cell-free extract of T brockii. One was a trehalose phosphorylase (EC 2.4.1.64), which catalyzes the reversible phosphorolysis of trehalose into β-glucose-1-phosphate and glucose. The other synthesized kojibiose from β-glucose 1-phosphate and glucose. The latter enzyme was considered to be a novel phosphorylase. The hypothetical mechanism of selaginose synthesis was proposed as follows: trehalose+Pi→glucose+β-glucose 1-phosphate, β-glucose 1-phosphate +trehalose→selaginose+Pi.

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Gal-α(1→3) -GalαpNP was regioselectively synthesized from para-nitrophenyl α-D-galactopyranoside (GalapNP) in 8.0% yield by the transglycosylation reaction, using α-galactosidase from Penicillium funiculosum. After conversion to an acetamidophenyl group, the para-nitrophenyl group was removed by using cerium ammonium nitrate (CAN). The heptaacetyl galactosyl disaccharide was converted to Gal-α (1→3) -Gal by deacetylation. To synthesize the trisaccharide, the heptaacetylgalactosyl disaccharide was converted to the trichloroacetimidate derivative, then coupled with a selectively protected GlcNAc derivative to give a trisaccharide derivative, which was then deprotected to provide Gal-α (1→3) -Gal-β(1→4) -GlcNAc.

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The production of extracellular polysaccharides by 15 screened strains, which used glucuronic acid as the carbon source in their cultures, was examined. Among the strains tested, GLCA 9B strain, classified into a facultative anaerobe of Aeromonas sp., produced 5 g/L of extracellular polysaccharide under the nitrogen-limited culture. Carbon sources of glucuronic acid, galacturonic acid, and glucose gave no significant difference in their sugar compositions of the product, extracellular polysaccharides. GLCA 9B polysaccharide, produced by glucuronic acid culture, contained galactose (55%) and mannose (29%) as the major components. Rhamnose, arabinose, and glucose were other neutral sugar components (total 3%), and glucuronic acid (6%) and galacturonic acid (7%) were detected as the acidic sugar components of these polysaccharides. Methylation analysis showed that galactose had linkages at 1, 3-position and 1, 4, 6-position, and mannose had linkage at 1, 2, 4-position. Because acidic polysaccharide of GLCA 9B had high molecular weight and high viscosity, practical uses of this polymer in various aspects were expected.

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The gelatinization characteristics of the native and heat-moisture treated (HMT) starches from katakuri, potato, and edible canna and their elasticity at near the sol-gel transition point were investigated. The swelling power and solubility of each HMT starch were greatly suppressed in comparison with those of each native starch. The X-ray diffractograms showed that the heat-moisture treatment converted the B type pattern to the A type in potato and edible canna starches, whereas katakuri starch did not and indicated the A type with only 2 diffraction peaks, at 4a and 6a. The viscograms of HMT starches indicated greatly suppressed viscosity without breakdown. The digestibility of the HMT starch with a-amylase was found to be more susceptive than that of the native starch. The concentration dependence of the mechanical properties of native and HMT starches at near the sol-gel transition point was analyzed based on the scaling law derived from the percolation theory. The gelation concentration of the HMT katakuri starch was higher than that of the native starch. The critical concentration of the native and HMT katakuri starches for gelation were estimated to be 1.5 wt% and 2.5 wt%, respectively. The HMT katakuri starch formed softer gels below the critical concentration than the native starch did, and it formed firmer gels above it. It was recognized that the scaling law could be applied to the native and HMT katakuri starches and determined that the critical exponents were 3.2 and 3.7, respectively; but it could not be applied to the native and HMT potato starches or to the HMT edible canna starch.

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L-Arabinose is a common component in a plant cell wall and is widely distributed in the plant kingdom. It is a main component of cereal hemicellulose, such as corn, wheat, rye, and rice, pectic substances of beet and apple pulps, and some plant gums. The sugar occurs in the free state in the heartwood of coniferous trees, but its small amounts have been found also in several foods and beverages made from cereals and other plant sources such as bread, miso, beer and tea. L-Arabinose is produced by the mild acid hydrolysis of some plant gums, corn fiber, and beet pulps. The taste of L-arabinose is quite similar to sucrose, but approximately half the sweetness . Naturally occurring arabinose is an L-form, and it is not metabolized in animals; thus it is a noncaloric sugar . Furthermore, it strongly inhibits intestinal sucrase uncompetitively and consequently inhibits the absorption of sucrose from the small intestine. The addition of 2-3% of L-arabinose to sucrose causes about a 60% reduction of the digestion of sucrose in the small intestine. The nondigested sucrose and L-arabinose, possibly metabolized by intestinal microbes, produce short-chain fatty acids and thus function similar to dietary fiber. L-Arabinose, by this function, reduces the increase of the levels of blood sugar, insulin, triglycerides, and cholesterol by the ingestion of sucrose . Therefore it has great merits as a sweetener and a food additive to improve the obesity and to maintain good health .

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Corticium rolfsii AHU 9627 secretes strong raw-starch-saccharifying enzyme (RSSE) that is superior to other enzymes. The RSSE consisted of five forms of glucoamylase (G1-G5) and a small amount of α-amylase. These glucoamylases, showed nearly identical characteristics, except that G4 and G5 were unable to hydrolyze raw starch. A cDNA coding for C. rolfsii glucoamylase G2 was cloned. This clone (CG 15) contains an entire coding region for a polypeptide of 579 residues, which has catalytic domain and starch-binding domain like other glucoamylases from filamentous fungi. Some differences were observed in the starch-binding domain and in the linker region between catalytic domain and the starch-binding domain. The obtained cDNA was introduced into Saccharomyces cerevisiae AH 22. The transformants acquired starch-saccharifying ability. The amount of secreted glucoamylase, however, was very small (0.001 U/mL). Therefore the cDNA was introduced into Aspergillus pryzae for better production. Through optimization of the culture conditions, the amount of recombinant glucoamylase obtained in the culture supernatant reached 100 mg/L (3.5 U/mL). The glucoamylase G2 secreted from A. oryzae (G2A0) had almost the same specific activity as native G2 from C. rolfsii. Thermal and pH stabilities of G2A0, however, were significantly lower than those of native G2. To clarify domain relationships and to compare their properties with those of other glucoamylase, two chimeric gluco-amylases whose domains were interchanged with that of Aspergillus awamori var. kawachi gluco-amylase (GAI) were made, and their enzymatic characteristics were investigated. The chimeric glucoamylases showed the pH and thermal stabilities similar to those of glucoamylases from which their catalytic domain derived. Native G2, GAI secreted from A. oryzae, and two chimeric gluco-amylases showed a similarly good rate of hydrolysis of raw potato starch, although G2A0 showed a significantly lower rate of hydrolysis.

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α-Amylases from Thermoactinomyces vulgaris R-47 (TVA I and TVA II) hydrolyze pullulan to produce panose, and isopullulanase (IPU) from Aspeygillus niger ATCC 9642 hydrolyzes Pullulan to produce isopanose. The structure/function relationships of these pullulan-hydrolyzing enzymes were studied. The crystal structure of TVA II has been determined. TVA II is composed of domain A/B, which has a (β/α) ₈ barrel structure with a small component called domain B; domain C, which contains the C-terminus; and domain N, which contains the N-terminus. Although the role of domain N for the enzyme activity has been unclear, it is unique in the structures of the α-amylase family. We modified some amino acid residues in region II, one of the four regions conserved in α-amylase family enzymes, of the TVA I by means of site-directed mutagenesis. The action pattern of the mutated enzyme for pullulan was greatly altered and it produced maltotriose from pullulan. The ipuA gene encoding IPU has been isolated. Although IPU does not hydrolyze dextran, IPU showed a high amino acid sequence similarity with dextranases. The ipuA gene was expressed in Aspeygillus oryzae, and the substrate properties of the recombinant IPU were identical with those of the native enzyme.

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The enzyme-substrate complex structure of α-amylase is modeled by a 3-D computer-simulated docking, and the reactivity of α-and β-amylases is analyzed to elucidate the difference in their stereoselectivity. The mechanism by which α-amylase perfectly produces only α-anomers in hydrolysisis explained. It is revealed that Asp206 is at the bottom of the enzyme cleft to hold the carbonium cation intermediate after the cleavage of glucosidic linkage Cl-Oglyc-C4′, and that G1u230 and Asp297 are on the upper side to catch the water molecule . Therefore the water molecule (OH^-) needed for the hydrolysis is allowed to come only from the top of the cleft to attack Cl⁺ of the intermediate. Consequently, this attack occurs from the same side that the leaving Oglyc-C4′ was on because the direction of the original glucosidic linkage (as indicated by the V-shape of the glucosidic linkage Cl-Oglyc-C4′) points to the cleft top. For β-amylase, the enzyme-substrate crystal structure indicates that the original glucosidic linkage to be hydrolyzed points toward the cleft bottom (the side opposite that from which the water molecule attacks), resulting only in R-anomers . The significance of that directional change of glucosidic linkage introduced by the 180° rotation of the dihedral angle from its normal value is discussed.

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A new calcium independent α-amylase has been developed for starch liquefaction. This amylase Termamyl LCTM gives the same performance in the absence of free calcium compared to Termamyl^TM, in the presence of 40 ppm free calcium. The improvement is a result of 5 single amino acid substitutions and one N-terminal alteration. Several new alkaline α-amylases were identified in Nature. We have isolated two amylases that display the desired activity profile but lack the overall stability necessary for a detergent a-amylase. A two amino acid deletion in the region from position 180-184 increased the overall stability both with respect to temperature and calcium sensitivity, to a satisfactory level comparable to that of Termamyl^TM.

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The Cyclization reaction of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from alkalophilic Bacillus sp. A2-5a, Bacillus macerans, and Bacillus stearothermophilus on amylose was reinvestigated by the use of high-performance anion exchange chromatography . When CGTase from alkalophilic Bacillus sp. A2-5a (A2-5a CGTase) was incubated with synthetic amylose, cyclic α-1, 4-glucans with degree of polymerization (DP) from 6 to more than 60 were produced in the initial stage of the reaction. The molecular mass and the cyclic structure of these glucans were determined by TOF-MS and ¹³C NMR. The larger cyclic α-1, 4-glucans produced in the initial stage of the reaction of A2-5a CGTase were subsequently converted into smaller cyclic α-1, 4-glucans and into the final major product, α-CD. CGTase from B. macerans (B, macerans CGTase) also produced larger cyclic α-1, 4-glucans, which were then converted into smaller cyclic α-1, 4-glucans and into the final major product α-CD. B. macerans CGTase, however, converted larger cyclic α-1, 4-glucans into smaller ones more slowly than A2-5a CGTase did. On the other hand, most of the α-1, 4-glucans produced by the action of B. stearothermophilus CGTase were α-, β-, and β-CD, and only a few larger cyclic α-1, 4-glucans were detected. From these results, a new model of the cyclization reaction of CGTase was proposed .

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Hetero-branched cyclodextrins (CDs) were synthesized by transglycosylation or the reverse action of several enzymes such as βββ-galactosidases, α-galactosidases, α-mannosidases, lysozyme and R-Nacetylhexosaminidase. Their structures were analyzed by methylation, FAB-MS, and NMR spectroscopies. β-Galactosidase and a-galactosidase from microorganisms synthesized hetero-branched CDs, of which the galactose residues were linked at side chains of the branched CDs. But these enzymes could not synthesize galactosyl-CDs, directly linked to the CD ring. However, α-galactosidase from coffee bean and α-mannosidase and N-acetylhexosaminidase from jack bean could bind galactosyl, mannosyl and N-acetylglucosaminyl residues directly to the CD rings, respectively, by transglycosylation or reverse action. The effects of the side-chain residues in branched CDs, on solubility, hemolytic activity, and inclusion reactions with 6-O-α-D-glucosyl-CDs, 6-O-α-D-galactosyl-CDs, and 6-O-α-Dmannosyl-CDs, were examined.

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Herein we propose a new strategy for utilizing amino sugar resources that involves the enzymatic removal of N-acetyl groups from the amino sugar structures instead of hydrolysis of the sugar chains . We chose chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum, as a tool for enzymatic deacetylation of amino sugars. We have purified the enzyme from a culture filtrate to electrophoretic homogeneity (944-fold with a recovery of 4.05%). The optimum temperature of the enzyme was 60t, and the optimum pH was 11.5-12.0 when glycol chitin was used as substrate . The enzyme retained 96% of its activity in the presence of 100 mM sodium acetate . The enzyme was active toward chitin oligomers whose degree of polymerization are more than two, and toward partially N-deacetylated water-soluble chitin. The enzyme could convert (GlcNAc) 36 into fully deacetylated corresponding chitosan oligomers. Conversely, (GlcNAc) 2 was partially deacetylated into 2-acetamido-4-0- (2-amino-2-deoxy-β-D-glucopyranosyl) -2-deoxy-D-glucose [GlcNGlcNAc]. The enzymatic deacetylation method has advantageous characteristics over chemical methods: (1) It never causes unexpected side reactions; (2) It is highly reproducible; (3) Unique compounds such as GlcNGlcNAc can be produced. These basic data on characterization of the enzyme will give us important information for its utilization in glycotechnology.

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