An actinomycete producing pectate lyase (PL) isolated from soil was affiliated as Streptomyces thermocarboxydus by 16S rDNA analysis. S. thermocarboxydus PL I was purified to homogeneity by two steps of column chromatography from the culture filtrate. The molecular mass of the PL I was estimated to be 28 kDa by SDS-PAGE, and using electrospray ionization mass spectrometry, it was determined to be 23,897 Da, low-molecular-weight type. The amino acid sequence of the N-terminal 22 residues of PL I suggest that the enzyme is calcified into Family 3 of polysaccharide lyase. The optimum pH value was 9.0, and the thermal and the pH stabilities were stable up to 50°C and at a pH ranging from 6 to 10, respectively. The enzyme was designated an endo-type because of the detection of oligo-galacturonic acids (GalUAs) as products from the initial reaction. Although S. thermocarboxydus PL I had little action on highly esterified polygalacturonic acid methylglycoside, pectin for the PL I was a better substrate than polygalacturonate. For these three substrates, the PL I showed maximum activity with the addition of 0.6 mM Ca²⁺. S. thermocarboxydus PL I acted on poly- and oligo-GalUAs over di-GalUA and better on the larger GalUAs until at least DP 8. The final products with the PL I were both 4,5-unsaturated di- and tri-GalUA. This is the first report of PL from S. thermocarboxydus.

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Kinetic studies on hydrolysis of the substrates p-nitrophenyl α-D-glucopyranoside (Glcα-O-pNP), methyl β-D-maltoside (Mal β-O-Me), and their 2- or 3-deoxy analogs, were conducted to investigate the interaction between sugar hydroxyl groups and catalytic carboxyl groups of Aspergillus niger α-glucosidase (ANGase). Optimal pH value for the reaction between the enzyme and Glcα-O-pNP (pH 4.4) was different than that for both its 2- and 3-deoxy analogs (pH 5.6), suggesting that ionization of two catalytic carboxyl groups of ANGase is affected by glycon OH-2 and -3 groups on Glcα-O-pNP. Through hydrolysis of the glycosides we elucidated pK_e and pK_es for each carboxyl group of the enzyme using Dixon-Webb semi-log plots. The pK_e and pK_es values were different for Glcα-O-pNP, but identical for the 2- and 3-deoxy analogs. Similar results were obtained in the reaction involving Malβ-O-Me and its 2-deoxy analog as substrates, indicating that the glycon OH-2 and -3 groups of the glucoside are intimately involved in the ionization of two catalytic carboxyl groups of ANGase, while aglycon hydroxyl groups do not appear essential for the ionization of the carboxyl groups.

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Thermomonospora viridis TF-35 maltogenic amylase (TVA) and Taka-amylase A (TAA) converted about 66-68% of soluble starch into maltose (G2) as a main product. High G2 syrup containing 89% G2 (G2-89 syrup) was converted to extremely high G2 syrup containing 94.2% G2 in the presence of TVA. In the case of TAA, G2-89 syrup was converted to G2 syrup containing 86% G2. We compared the transfer action of TVA and TAA. It is suggested that the transglycosyl activity of TVA is higher than that of TAA in the decomposition of G3, and that although TAA degrades G2, TVA does not degrade G2-89 syrup at a high concentration. Because of the above differences in transfer action, the current industrial production of extremely high G2 syrup containing more than 94% G2 can be produced by a cooperative 2-step reaction with the enzyme after obtaining starch hydrolyzates containing about 89% G2 by the simultaneous actions of soybean β-amylase and Flavobacterium odoratum isoamylase from a high concentration of starch liquefite.

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A polysaccharide was extracted from the pericarp of Citrus depressa which was collected Ogimi Village, Okinawa, Japan. The yield of purified polysaccharide was 2.6% (w/w) based on fresh material. The contents of total carbohydrate, uronic acid, ash and moisture of the polysaccharide were 88.0, 78.0, 4.7 and 7.2%, respectively. The degree of methoxylation of the polysaccharide was estimated to be 62.9%. The purified polysaccharide was composed of D-galacturonic acid, D-galactose, L-arabinose, L-rhamnose, D-glucose and D-mannose in the molar ratio of 100 : 9.20 : 1.34 : 1.02 : 0.88 : 0.78 respectively. The molecular mass of the polysaccharide was estimated to be approximately 6.8×10⁴ by gel chromatography. The specific rotation of the polysaccharide was +149° at 25°C, which indicated that the polysaccharide mainly had α-glycosidic linkages. The infrared spectra of the polysaccharide and the de-esterified polysaccharide were in agreement with those of standard pectin and de-esterified standard pectin over wide ranges of wave numbers. Chemical shifts of ¹H- and ¹³C-NMR spectra of the polysaccharide and the de-esterified polysaccharide were also consistent with those of standard pectin and de-esterified standard pectin. NOESY spectroscopy showed that the polysaccharide contained (1→4)-linked D-galacturonic acid residues. The polysaccharide and the de-esterified polysaccharide formed gels in the presence of sucrose under acidic conditions and of Ca²⁺ ions, respectively. These results indicated that the polysaccharide extracted from the pericarp of C. depressa was a pectin.

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Efficient production of an isomalto-dextranase from Arthrobacter globiformis T6 has been achieved in Escherichia coli. The combination of an expression vector lacking the region for the signal peptide, cultivation at 25°C, and optimization of E. coli cells allows us to produce the isomalto-dextranase at more than 1000 times the amount under the original conditions, which then enables us to characterize the enzyme in detail. The primary structure of the isomalto-dextranase from A. globiformis T6 has a distant similarity with enzymes belonging to glycosyl hydrolase family 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases. Therefore, the reaction of the isomalto-dextranase for melibiose, a substrate for α-galactosidases, has also been investigated. The isomalto-dextranase did not hydrolyze melibiose or p-nitrophenyl α-D-galactoside. The K_i values for isomaltose and maltose were 2.3 and 7.8 mM, respectively, while melibiose scarcely inhibited the activity of the isomalto-dextranase. Moreover, melibiose was a poor acceptor for the transglycosylation with dextran, and the maximum accumulation of the transglycosylation product was 12-fold less than that for isomaltose. The findings indicated here that the isomalto-dextranase is highly specific for the glucosyl moiety in both hydrolysis and transglycosylation.

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Clostridium thermoamylolyticum glucoamylase gene was overexpressed in Escherichia coli cells. This glucoamylase gene consisted of 2133 bp that encoded a 710-amino-acid protein with a molecular mass of 79,920 Da. The glucoamylase fell into glycoside hydrolase family 15, showing 84% identity and 90% similarity to an amino acid sequence of Clostridium sp. G0005 glucoamylase, and showing 82% identity and 87% similarity to that of Thermoanaerobacterium thermosaccharolyticum glucoamylase. The corresponding sequence to the mature protein was placed under the control of the T7 promoter as a strong and constitutive promoter. The recombinant glucoamylase was purified by a Ni-NTA column. The molecular mass of the mature glucoamylase was 77 kDa by SDS-PAGE, and it was purified 10-fold with a recovery of 65%. The specific activity was determined to be 1.8 U/mg for maltose. The value of K_m for maltose was determined to be 5.4 mM, and the k₀ was 7.1 s^-1. The optimum pH of the enzyme was determined to be 4.5, and more than 80% of the enzyme activity remained between pH 3.5 and 9.0. The optimum temperature was 65°C, and more than 80% of the enzyme activity remained up to 65°C.

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The gene encoding an oligo-1,6-glucosidase was cloned in terms of walking downstream from the glucodextranase gene of the chromosomal DNA of Arthrobacter globiformis I42. An open reading frame consisted of 1731 base pairs that encoded a mature protein of 577 amino acids (Mr, 63,000) was found. Transformed Escherichia coli cells carrying the 1.7-kb fragment overproduced the oligo-1,6-glucosidase under control of the T7 promoter of a pET system. Kinetic analyses of the recombinant protein gave K_m 1.76 mM and k₀ 697 s^-1 for p-nitrophenyl α-D-glucopyranoside and K_m 24.1 mM and k₀ 41 s^-1 for isomaltose. Its deduced amino acid sequence showed 54% similarity to two amino acid sequences of Bacillus cereus oligo-1,6-glucosidase and Bacillus sp. α-glucosidase. The oligo-1,6-glucosidase has four conserved regions shared with α-amylases. The gene cluster consisted of the glucodextranase and oligo-1,6-glucosidase genes, suggesting that both genes could participate in the degradation for utilization of dextran in the bacterium.

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Physico-chemical and mechanical properties of extruded cornstarch-poly(lactic acid) (PLA) with addition of ceric ammonium nitrate (CAN) were investigated. An addition of 0.25% CAN improved bulk density and hardness of the extrudates, but 0.5 or 1.0% CAN addition reduced expansion, and increased bulk density and hardness significantly. Water solubility was increased, but cohesiveness was decreased with increasing CAN concentration. An addition of 0.25% CAN decreased thickness of the extrudate cell walls to about 20 μm in comparison with that without CAN (50-10 μm). This corresponded to a decrease in bulk density of the extrudate. Size exclusion chromatography showed that the addition of CAN accelerated the degradation of starch during extrusion. Furthermore, with 0.5 or 1.0% CAN the extrudates expanded poorly and there was no peak at the void volume. PLA molecules have not been degraded by CAN addition.

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Starch granules were prepared from 8 kinds of acorn; Kunugi (Quercus acutissima Carruth), Konara (Q. serrata Thunb), Naragashiwa (Q. aliena Blume), Shirakashi (Q. myrsinaefolia Blume), Matebashii (Lithocarpus edulis Nakai), Tuburajii (Shiia cuspidata Makino), Arakashi (Q. glauca Thunb), and Sutajii (Shiia Sieboldii Makino, Castanopsis cuspidata Schottky var. Sieboldii Nakai). Granular sizes, contents of the apparent amylose (26-28%), and chain length distributions of amylopectin of the acorn starches are similar to those of maize starch. Chain length distributions measured by HPAEC-PAD showed that amylopectinns of Sudajii and Tsuburajii starches had low amounts of chains with DP 9-17 in comparison to maize starch and the other acorn starches. Each acorn starch showed different gelatinizing temperature by DSC; those of Arakashi and Tuburajii starches had lower values similar to potato starch and that of Kunugi had higher values similar to sweet potato starch. On the heats of gelatinization the acorn starches were different from rice and maize starches and similar to potato and sweet potato starches. Peak viscosities of the acorn starches by RVA were higher than rice and maize starches and similar to sweet potato and wapoto starches.

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The formation of amylose complexes with eight lysophosphatidylcholines; 1-capryl lysophosphatidylcholine (LPC-C 10 : 0), 1-lauroyl LPC (LPC-C 12 : 0), 1-myristoyl LPC (LPC-C 14 : 0), 1-palmitoyl LPC (LPC-C 16 : 0), 1-stearoyl LPC (LPC-C 18 : 0), 1-oleoyl LPC (LPC-C 18 : 1), 1-linoleoyl LPC (LPC-18 : 2) and 1-arachidoyl LPC (LPC-C 20 : 0), was conducted in aqueous solution using a commercial amylose with an average degree of polymerization (DP) of 18. The chain distributions of amylose precipitated by forming a complex in aqueous solution (2°C) were analyzed by high-performance anion exchange chromatography with a pulsed amperometric detector (HPAEC-PAD). The smallest DPs of amylose forming complexes with LPC-C 14 : 0, LPC-C 16 : 0, LPC-C 18 : 0, LPC-C 18 : 1, LPC-18 : 2 and LPC-C 20 : 0 were DP 27, DP 29, DP 29, DP 31, DP 31 and DP 35, respectively. The principal amylose chains found in complexes with LPC-C 14 : 0, LPC-C 16 : 0, LPC-C 18 : 0, LPC-C 18 : 1, LPC-18 : 2 and LPC-C 20 : 0 were DP 34-36, DP 32-34, DP 33-35, DP 34-36, DP 36-38 and DP 36-38, respectively.

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Structures and properties of enzymes for bacterial glycogen metabolism have been investigated. Branching enzyme (BE, EC 2.4.1.18), which is responsible for α-1,6 glucosidic linkages of glycogen, was found to catalyze cyclization of amylose. It was suggested that the ratio of branching to cyclization reactions is dependent on the size and concentration of substrate. We also demonstrated that the thermostable BE from Bacillus stearothermophilus efficiently catalyzes cyclization of B chains of amylopectin to produce highly-branched cyclic dextrin, Cluster Dextrin™. In spite of its high molecular weight and relatively long unit chains, Cluster Dextrin™ is highly soluble in water and shows characteristic properties for food and non-food applications. Moreover, glycogen-like polysaccharide with an extremely high molecular weight (>10,000,000) could be synthesized by using α-glucan phosphorylase with BE. Properties of other enzymes for glycogen metabolism are also described. Comparison of the primary structures of ADP-glucose pyrophosphorylases (AGPs) derived from genome projects and the experimental results using the enzyme from B. stearothermophilus suggest a remarkable variety of AGP.

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I. The development of trehalose production from starch. Two novel enzymes, malto-oligosyltrehalose synthase (MTSase, EC 5.4.99.15) and malto-oligosyltrehalose trehalohydrolase (MTHase, EC 3.2.1.141) were isolated from bacterial strains belonging to the genus Arthrobacter. It was found that trehalose was produced from starch by the joint reaction of both enzymes. We improved the enzyme-producing strain and established a method for the enzyme production. In order to increase the reaction yield of trehalose, several enzymes such as isoamylase and cyclomaltodextrin glucanotransferase were added into the MTSase/MTHase reaction. We succeeded in the conversion of starch into trehalose in a high reaction yield of more than 85% by the multi-enzyme reaction. In 1995, Hayashibara Co. started a mass production of trehalose crystal powder, and now the production amounts to twenty thousand tons a year. II. The application of trehalose. Trehalose has many good properties to improve the qualities of foods. The hydration activity of trehalose is applied to keep foods from damage by moisture or freezing. This saccharide has inhibitory actions on starch retrogradation, protein denaturation, fat oxidation and the deterioration of other nutrients such as vitamins or superoxide dismutase-like components. The interaction with minerals is applied to prevent Ca insolubilization by phosphate or Mg elution from vegetables and meat. This saccharide also masks unpleasant taste and odors in foods. The acid production from trehalose by oral microbials is low compared to that from sucrose. Experiments using an ovariectomized murine model of osteoporosis suggested that ingestion of trehalose might be effective in the prevention of osteoporosis. In addition to these applications for foods, trehalose is a useful ingredient for cosmetics and pharmaceuticals.

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