Physicochemical properties of root starches from new types of sweetpotatoes [low amylose variety, Kyukei 89376-12 (K89376-12) ; high sweet variety, Kyushu No. 127 (K127) ; Okinawa variety, Miyano No. 36 (M36)] showing distinct root properties after cooking were determined . The structural properties of K89376-12 starch were similar to those of Koganesengan starch except for the amylose content. The result suggests that a difference in the pasting properties between the two starches was attributable to the difference in their amylose contents. On the other hand, the starches from K127 and M36 showed low pasting temperature and large viscosity on cooling, and also high digestibility by a crude glucoamylase. In terms of the apparent amylose content or chain-length distribution of the debranched whole starch, M36 starch showed an ordinary amylose content . M36 amylopectin, how ever, was found to have a large amount of long chains overlapping with the amylose fraction; thus, a low amylose character of M36 starch was revealed. Furthermore, high-performance anion-exchange chromatography revealed that the amylopectins of K127 and M36 were rich in short chains with DP 6- 11. Thus, it was found that the new types of sweetpotatoes possess the respective characteristic starches.

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A thermostable trehalose phosphorylase (EC 2.4.1.64) from a thermophilic anaerobe, Thermoanaero bium brockii ATCC 35047, was purified to an electrophoretically homogeneous state by successive column chromatographies on DEAE-Toyopearl 6505, Butyl-Toyopearl 650 M and Ultrogel AcA 44 . The molecular weight of the enzyme was estimated to be 190, 000 Da by gel filtration, and 88, 000 Da by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity in the range of pH 7.0 to 7.5 for phosphorolysis, and pH 6.0 to 7.0 for synthesis . The optimum temperature of the enzyme was 70°C in both directions. The enzyme was stable from pH 6.0 to 9.0, and up to 60°C . This result showed that our trehalose phosphorylase was the most thermostable in those reported to date . Activity was inhibited by Cue, Hg²⁺, Mg²⁺, Mn²⁺, Pb²⁺, and Zn²⁺ The Kms for trehalose, Pi, D-glucose and R-D-glucose 1-phosphate were 0.97, 0.57, 2.4 and 0.75 mM, respectively.

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Cross-linked modifications of Taka-amylase A (TAA) with various dialdehyde derivatives including starch- and dextran-dialdehyde were examined. A rapid and significant decrease in the amount of free amino groups and enzyme activity of TAA was attained by starch-dialdehyde, whereas dextran dialdehyde caused much lower loss in activity of TAA in spite of a similar extent of amino group modification. The molecular weight of the dextran-modified TAA increased by about 10, 000-20, 000 Da, which was measured by SDS-gel electrophoresis. A comparison of the activity of modified TAA on various substrates indicated a slight but definite increase in susceptibility to maltose and phenyl α-glucoside according to the increase in the extent of modification of TAA. In contrast, the activity of the modified TAA on maltotriose, maltotetraose, and γ-cyclodextrin decreased upon modification. Here, again, TAA modified with dextran-dialdehyde retained higher activity to those oligosaccharides than that modified with starch-dialdehyde. Moreover, inactivation caused by the fluorescent reagent, o-phthalaldehyde, was retarded by previous modification with dextran-dialdehyde, indicating that reaction between TAAand dialdehyde derivatives occurred through non-critical amino groups for enzyme activity.

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Characteristics of wheat flour graded by polishing and the effect of the graded flour substitution with conventionally milled flour on the physical properties of wheat dough and bread were studied. When wheat grains are milled by the conventional method, most of the fibers and minerals are lost. For that reason, a polished-grading method which keeps whole part of grains was introduced. Wheat grains were gradually polished in a:stepwise manner from the outer layer by 10% of the total weight to the core of the original soft-type wheat grain Normn 61. The loaf volumes of all graded flours were distinctly smaller than that of conventionally milled Normn 61 (N61) and the bread crumbs were quite hard. All graded flours clearly increased viscoelastic properties such as compression stress, modulus of elasticity and viscosity coefficient. The graded flours had a higher SH content, whereas there was a lower SS content in dough than conventionally milled hard-type flour. Any fraction of the polishedgraded flours alone did not have good baking properties. When 10% of N61 was substituted with a fraction of the graded flour, A-4 (70-60% of original grains) or A-7 (40-30%), the specific volume increased slightly as compared with that of N61 alone. Moreover, the addition of hemicellulase to the combined flour improved the loaf volume distinctly, and the bread crumbs became quite soft. Microscopic observation of the dough showed that the gluten matrix became thicker and most of the starch granules were sufficiently covered with the matrix, as compared with N61 alone. From these results, a 10% substitution of graded flours A-4 and A-7 to N61 with or without hemicellulase was found to improve some of the viscoelastic and baking properties of wheat dough.

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The thermophilic anaerobe Thermoanaerobium brockii ATCC 35047 produces a novel phosphorylase, kojibiose phosphorylase, which catalyzes the reversible phosphorolysis of kojibiose to form β-glucose 1-phosphate and D-glucose. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on DEAE-Toyopearl 650S, CM-Toyopearl 6505, Hydroxyapatite, Ultrogel AcA44, Mono Q, and Butyl-Toyopearl 650 M . The enzyme had a molecular weight of 83, 000 by SDS-polyacrylamide gel electrophoresis and a pl of 4 .3 to 4.4 by gel isoelectrofocusing. The enzyme showed the highest activity at pH 5 .5 and 65°C, and was stable from pH 5.5 to 9.7 and up to 65°C. The enzyme activity was inhibited by Hg²⁺ and Pb²⁺⁰. The Km values for kojibiose, Pi, glucose, and β-glucose 1-phosphate were 0 .77, 0.85, 3.52, and 0.77 mM, respectively.

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β-D-Xylosidase from Aspergillus pulverulentus regioselectively induced a β-D-xylosyl transfer reac-tion from 4-0-β-D-xylopyranosyl-D-xylopyranose (xylobiose) to the primary hydroxyl group of D-glucose. The 6-0-β-D-xylopyranosyl-β-D-glucopyranose (primeverose, 1) produced was isolated by chromatography on a column of charcoal-Celite in a 29% overall yield based on the donor. In the same way, when p-nitrophenyl β-D-glucopyranoside was used as an acceptor instead of D-glucose, the enzyme predominated p-nitrophenyl 6-0-β-D-xylopyranosyl-β-D-glucopyranoside (pNP β-primeveroside, 2) to its isomers, pNP 4-0-β-D-xylopyranosyl-β-D-glucopyranoside and pNP 3-O-β-D-xylopyranosyl-β-D-glucopyranoside. Three transfer products were easily separated from one another by Toyopearl HW-40S column chromatography and the desired compound, 2, was obtained in a 13% yield based on the acceptor added. These reactions were efficient enough. to allow one-pot preparations of 1 and 2.

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A simple enzymic method for the preparation of 1, 5-anhydrofructose (1, 5-AF) and its antioxidative activity were investigated. α-1, 4-glucan lyase (EC 4 .2.2.13, GLase) was extracted and purified from red seaweed, Gracilaria verrrucosa. One-hundred-and-fifty grams of 1, 5-AF was obtained from 360 g of waxy maize starch by digestion with the purified GLase and the following purification of the product by ion-exchange resins and gel filtration. The purity of the product was 98 .6% by HPLC using an ionexchange resin (MC1 GEL CK08S). The product, 1, 5-AF, was identified by ^IH-¹³C COSY NMR, and the [α] _D²⁵ was -16.8°. The antioxidative activity of 1, 5-AF was examined by comparison with that of ascorbic acid (VC) for preventing the oxidation of linoleic acid . The antioxidative activity of 1, 5-AF was found to be approximately 1.8-fold that of VC by the methods using thiocyanate and 2-thiobarbituric acid.

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Hetero branched CDs linked by N-acetylglucosamine were synthesized from a mixture of N-acetylchitooligosaccharides and branched CDs by transglycosylation of lysozyme. A transfer product from N-acetylchitooligosaccharides and maltosyl-βCD was identified as 6-O-α-(3²-O-β-b-N-acetylglucosaminyl-) maltosylβCD. The optimum pH at 60°C and the optimum temperature at pH. 4.5 to produce N-Getylglucosaminyl-maltosyl-RCD by lysozyme were pH 3.5-6.0 and 60-70°C, respectively. Lysq zyme was unable to link N-acetylglucosamine to mannosyl-CD, galactosyl-CD and directly to CD rings.

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The modification of amino groups in Taka-amylase A (TAA) using trinitrobenzene sulfonic acid (TNBS), formaldehyde, and pyridoxal 5'-phosphate (PLP) was compared with that using a fluorescent reagent, ο-phthalaldehyde (OPA). Although OPA resulted in potent inactivation of TAA as described in previous papers, the other three reagents described above caused no such inactivation of TAA. HPLC analysis showed that reactions of OPA with Lys residue differed greatly depending on the pH and the presence of Cys residue. Therefore, the different response of TAA for the modifications between OPA and other reagents suggested that Cys residue played an important role in the OPA reaction.

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The branch formations on starches from rice, potato, sweet potato, wheat, corn, sago, and tapioca using rice branching enzyme 1 (RBEI) were examined by high-performance anion exchange chromatography with a pulsed amperometric detector (HPAEC-PAD) after debranching with isoamylase. Irrespective of the difference of the structures of substrate starches, RBEI changed them to an almost similar structure with the two peaks at degrees of polymerization (DP) of 6 and 10 to 11 glucose chains. The longer chain transformation was saturated by 10-h incubation, while DP 6-glucose chain transformation was not saturated after 22-h incubation. When the structure of the substrate starch was not well branched, both DP 6-glucose chains and longer chains were well transferred by RBEI, but when the substrate was already well branched, the transfer reactions of longer chains decreased. The branching reaction seemed to be limited by the density of the glucose chains regardless of the structures of the original starches.

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3- and 6-Monodeoxy derivatives of ρ-nitrophenyl (PNP) α-D-glucopyranoside (1) were prepared from methyl α-D-glucopyranoside and confirmed to retain 4C1 chair conformations in D2O by 1H-NMR. Rice α-glucosidase did not hydrolyze the 3-, 4- and 6-deoxy derivatives of 1, but revealed high hydrolitic activity against the 2-deoxy derivative of 1.

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Endo-β-1, 4-glucanase and β-glucosidase from Aspergillus sp. K-27 were purified to homogeneity. Endo-β-1, 4-glucanase was a monomeric enzyme with a Mr of .21, 000. Its activities for cellooligosaccharides were high in the order of cellohexaose>cellopentaose>cellotetraose, but no activity was recorded in cellobiose or cellotriose. It degraded carboxymethyl cellulose into cellobiose very well; however, almost no action was recorded on Avicel. β-Glucosidase was shown to be composed of two subunits of different molecular weights (Mr 130, 000 and 105, 000). The activities of the enzyme for different disaccharides were in the order of laminaribiose (β-1, 3) >gentiobiose (β-1, 6) >cellobiose (β-1, 4) > sophorose (β-1, 2). Using cellooligosaccharides as substrates, the enzyme was shown to have the largest and smallest Km/ Vmax values for cellotriose and cellobiose, respectively.

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Dextrin dextranase (DDase, EC2.4.1.2) was extracellularly secreted in a culture medium of Acetobacter capsulatum ATCC 11894 containing both glucose (5.45%) and an extremely small amount of dextrin (0.05%) as the essential carbon sources. The enzyme was simply purified by only one-step centrifugation at 4°C and 20, 000×g for 20 min, and immediately dialyzed against 50 mM acetate buffer (pH 4.5) at CC for 2-3 days. The purified DDase gave a single protein band on both Native- and SDS-PAGE. The molecular mass of the enzyme was estimated to be about 152 kDa by SDS-PAGE. The optimum pH and temperature of the enzyme were 5.2 and 38°C, respectively. The enzyme retained its original activity up to 45°C, and was stable in the range of pH 4.1-5.4 at 4°C for 24 h. The enzyme was completely inactivated by 1 mM of Hg2+ Pb2+ or KMn04. The purified enzyme effectively synthesized α-1, 6-glucan from maltooligosaccharides. The average molecular mass of the product dextran was estimated to be about 1270 kDa.

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A nigerose and nigerosylmaltooligosaccharides-supplemented syrup (Taste-Oligo^TM) was developed by the transglucosylation of the enzyme from Acremonium sp. on an industrial scale. Taste-OligoTM has a mellow and rich taste of good body. The properties of Taste-Oligo^TM such as viscosity, osmotic pressure and water activity are almost the same as that of sucrose. Also, this syrup showed high hydroscopicity, high moisture retaining activity and heat stability by the Candy test as compared with sucrose. All the tested disaccharides having α-glucosidic linkage were hydrolyzed by brush border membranes of rat jejunum. The hydrolyzing activity for nigerose was about 86% of that for maltose. Furthermore, the hydrolyzing activity for nigerosylmaltooligosaccharides was nearly the same as that for maltooligosaccharides and sucrose. Additionally, Taste-Oligo^TM was degraded to about 98% glucose by brush border membranes of rat jejunum. From these results, we believe that the calories of nigerose and nigerosylmaltooligosaccharides are 4 kcal/g, the same as for maltooligosaccharides, sucrose and lactose.

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