Two pectate lyases (PLs I and II) were isolated from the culture filtrate of Stereum purpureum, the causative fungus of apple silver-leaf disease. The lyases were isolated using DE52 column chromatography, and their molecular masses were estimated to be 48 kDa by SDS-PAGE. After treatment with endo-β-N-acetylglucosaminidase (Endo H), their molecular masses decreased equally to 38 kDa, indicating that these PLs are glycoproteins. The pI values for PLs I and II were determined to be 4.15 and 4.10, respectively. The amino acid compositions of PLs I and II were very similar, and furthermore the amino acid sequences of the N-terminal 20 residues of the two enzymes were identical. Their optimum pH values, and thermal and pH stabilities were 9.5, up to 50°C, and pH 6-7, respectively. Both PLs I and II required 0.2 mM Ca²⁺ ion for maximum activity. The enzymes were assigned as endo-types because of the detection of oligo-GalUAs as products from the initial reaction. However they mainly formed products less than octa-GaIUA in degree of polymerization. Erwinia carotovora PL mainly formed products more than octa-GalUA. Therefore they were different in their initial reaction products. As in the case of the E. carotovora PL, the final products of S. purpureum PL were 4, 5-unsaturated di- and tri-GalUA.

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To investigate the physiological role of an α-amylase, TVA II from Thermoactinomyces vulgaris R-47, which hydrolyze cyclodextrins and pullulan, a region located upstream of the TVA II gene was cloned and sequenced. Five open reading frames, designated as ORF-1 to -5, were found in a fragment of about 5 kbp. Three of these genes, ORF-1, -2 and -3, were homologous to the genes which encode proteins related to the sugar metabolic systems, such a maltose system of Esch erichia coli and cyclodextrin metabolic system of Klebsiella oxytoca. An expression vector for the ORF-3 protein, which was similar to maltose-binding protein from E. coli (MalE) and a cyclodextrinbinding protein from K. oxytoca (CymE), was constructed, and the expressed ORF-3 protein was purified. The ORF-3 protein was a cyclodextrin-binding protein because of having a higher binding ability for cyclodextrins than for maltose.

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Hydroquinone (HQ) and epicatechin (EC) were glycosylated by transfer reaction by β-fructofranosidases using sucrose (Suc) as a donor substrate. An enzyme from Arthrobacter sp. was the most effective among several enzymes tested. The glycosylation product from HQ, HQ βfructofuranoside (HQF), was synthesized most effectively at a HQ concentration of 0 .4 M and a Suc concentration of 1.0 M. HQF was isolated by gel filtration and its structure was confirmed by ¹³C -NMR spectroscopy. Fructosylation of EC was conducted in the presence of 5% 2-propanol in order to increase the solubility of the acceptor in the reaction system. EC gave two transfer products in a ratio of almost 7 : 2. Of the products, the main one (ECF) was isolated by silica gel chromatography, gel filtration, and preparative HPLC. ₁₃C-NMR spectroscopy suggested glycosylation occurred at the OH group of site 3 in EC. HQF and ECF exhibited inhibition activities against mouse melanoma cell tyrosinase, a key enzyme for melanin synthesis, suggesting their potential application as skin-whitening substances.

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The subsite affinities of isomalto-dextranase (EC 3.2.1.94) from a soil bacterium, Arthrobacter globiformis T6 were evaluated to be>7.3, <-7.2, 6.7, 0.74 and 0.18 kcal/mol for subsites 1, 2, 3, 4 and 5, respectively, from the rate parameters (Km and k0) for the hydrolyses of isomalto-triose, -tetraose, -pentaose, -hexaose, -heptaose and -octaose. The intrinsic rate constant (kint) was calculated to be 3.1×10² s^-1. Subsites 1 and 3, showing large affinity values, were thought to attract the substrate and to form the productive binding. Isopanose and maltotriose without α-isomaltosyl unit in their non-reducing ends acted neither as substrate nor as inhibitor for the isomaltodextranase, while the enzyme hydrolyzed oligosaccharides with α-isomaltosyl unit in their nonreducing ends. From these results, it was suggested that the binding of the α-isomaltosyl unit in the non-reducing end to subsites 1 and 2 was essential for the formation of the productive bindings be tween the substrates and the enzyme.

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1, 5-Anhydro-D-fructose (1, 5-anhydro-D-arabino-hexo-2-close, 1, 5-AF) reacted well with the amino group and seemed to be useful as a reagent for modification of the amino compounds. The rate of browning of 1, 5-AF by reaction with glycine was higher than those of glucose and xylose at 50°C and pH 7.5. Forty-five percent of the amino group of glycine was modified by the incubation of a lyophilized mixture of 1, 5-AF and glycine at 30°C for 48 h, whereas the degree of modification by glucose was less than 5%. This modification was applied to lysozyme. A mass spectrum indicated that the sugar moiety was incorporated into the lysozyme. The optimum pH values of the modified and unmodified lysozyme on glycolchitin were pH 4.5 and 5.0, respectively. On the other hand, the optimum pH of the modified enzyme on freeze-dried cells of Micrococcus lysodeikticus shifted from pH 7.8 to 6.0. Wheat proteins, gliadin and glutenin, were also modified. The modified gliadin had higher emulsification ability and solubility than the unmodified ones.

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Each xylanase shows a unique aglycone specificity in transglycosylation, though most xylanases are classified under only two families based on the amino acid sequence and the structure of the protein. We investigated the aglycone specificity of a xylanase from Hypsizigus marmoreus using many kinds of aromatic compounds as acceptors. Xylanase was stable in the presence of the aromatic compounds (-100 mM) used in this experiment except for phenol. Some compounds such as vanillyl alcohol accelerated the degradation of xylan by xylanase and in the presence of them xylanase produced more reducing sugars in addition to β-glycosides than in the absence of those acceptors. Among the aromatic compounds having alcoholic hydroxyl groups, cinnamyl alcohol-having a double bond was the best acceptor and the amount of transglycosyl product was 30.8% against the whole product. Xylanase also catalyzed the transglycosylation in the presence of diphenol compounds and aromatic compounds that have both the phenolic and the alcoholic hydroxyl group. Xylanase could catalyze the transglycosylation to the alcoholic-OH of the acceptor rather than to the phenolic-OH.

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The effects of emulsifiers on starch retrogradation and/or firming were investigated by kinetic treatment of static viscoelastic parameters for non-glutinous rice starch gels (30%). The anti-firming ability of the sucrose mono ester of various fatty acids (C8 through C18) increased with increasing fatty acid chain length, and possessed maximal efficiency at palmitate (C 16) ester. However, the ability of food grade emulsifiers consisting of a mixture of stearate and palmitate (70: 30) and having various hydrophilic residues was not proportionally affected to an amylose complexing ability, but strongly influenced by hydrophlle-lipophile balance (HLB) values. Among sucrose esters having varied HLB values, the best anti-firming ability was obtained when HLB value was 11. These results suggest that the anti-firming efficiency of emulsifiers on starch gels might be attributed to the multiple effects between the amylose or amylopectin forming complexes with the lipophilic portion (fatty acid), and the hydration of the hydrophilic portion with water molecules in the starchwater systems.

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Two strains of acid-tolerant and starch-degradable yeast were isolated from rivers flowing in Kusatsu and Manza, considering an effective reuse of great mass of starch-based biodegradable plastic. The isolated strains could grow in acid media of pH 1.8 and were identified as Candida fluviatinis and Candida intermedia. The yeasts could assimilate many carbon sources and ferment glucose and sucrose in acid media to alcohol.

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The present study was undertaken to evaluate the Bioavailability of calcium-bound phosphoryl oligosaccharides (POs-Ca). POs-Ca was not hydrolyzed by artificial gastric acid. The gradual decrease of POs-Ca injected into the rat ligated jejunum loop was observed, suggesting that POs-Ca was hydrolyzed and absorbed in the small intestine. The overall rate of plasma glucose response after oral administration of POs-Ca to rats was similar to the case of administration of control saccharides, glucose and high-maltotetoraose syrup (TETRUP(R)-H). However, the glucose concentration of POs-Ca group at 0.25 h after administration was significantly lower than those of other groups. In the case of POs-Ca administration, plasma Ca concentration showed slight but significant increase during 0.25-0.5 h concurrent with the increase of plasma glucose . On the other hand, plasma P concentration decreased and then increased gradually from 2 h after the administration. Body weight gain, food intake and food efficiency of rats fed the diet containing 10% POs-Ca for 15 days were not different from those of rats fed control diet, and no diarrhea was observed in the POs-Ca group. The serum TG concentration of POs-Ca group was significantly lower than that of the control group. Other serum biochemical indices and organ (liver and cecum) weight were not significantly different among groups. From these results, it was assumed that the POs-Ca orally administered would be hydrolyzed into glucose, Ca, and P and then absorbed completely in the small intestine, therefore not causing acute diarrhea.

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Two endo-dextranases (dextranases I and II) were highly purified from cell-free culture broth of Arthrobacter globiformis W31 by consecutive column chromatographies. The purified enzymes were judged to be homogeneous on Native- and SDS-PAGE as well as isoelectric focusing, and their pIs and molecular masses to be 4.3, 107 kDa (dextranase I) and 4.5, 70 kDa (dextranase II). Both purified enzymes had an identical N-terminal amino acid sequence of NH2-S-V-A-P-L-A-S-TP-T-L-T-T-W-. Optimum pH values of both enzymes were pH 6.0. The anomeric configurations of the enzymatic hydrolysis products from dextran T-2000 were a-form, indicating that the aglucosidic linkages in the substrates are retained. The enzymes split dextran T-2000 in an endolytic fashion to produce predominantly isomaltotriose, a series of isomaltooligosaccharides having a DP of more than 4 and an appreciable amount of unknown oligosaccharides. Furthermore, neither enzyme had any action on isomaltose, isomaltotriose or even isomaltotetraose.

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β-Galactosidase from Thermus thermophilus A4 (A4-β-Gal) is stable at 70°C for 20 h. It belongs to the glycosyl hydrolase family (GF) 42, and enzymes in GF-42 are stable under extreme circumstances such as high temperature or high salt concentration. In order to obtain the structural basis of its reaction mechanism and stability, we conducted X-ray crystallography of the enzyme. We identified the structure of free and galactose-bound A4-β-Gal at 1.6 A and 2.2 A resolution, respectively. The A4-β-Gal has a “flower pot-like” trimeric structure. The monomer structure of A4-β-Gal is composed of three domains. N-terminal domain (domain A) contains a galactose binding site and has a TIM barrel fold. We identified the residues interacting with the galactose. Since Trp182, a residue related to molecular symmetry, constitutes a part of the active site pocket, the trimeric conformation is necessary for A4-β-Gal to retain enzyme activity. We also identified G1u141 and G1u312 as catalytic residues for A4-β-Gal on the basis of the structural evidence. Glu 141 and G1u312 are located close to the C-termini of the fourth and seventh β-strands of the barrel structure, indicating that GF-42 belongs to 4/7 superfamily.

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Glycogen debranching enzyme (GDE) from Saccharomyces cerevisiae possesses two differentcatalytic activities, 4-α-glucanotransferase/amylo-l, 6-glucosidase, on a single polypeptide chain.To investigate this bifunctional enzyme on the molecule level, GDE gene from S. cerevisiae wascloned and expressed into Escherichia coli, and the best conditions for the enzyme expression wereexamined. When the cultivation temperature of recombinant E. coli strains was lowered to 25°C and the isopropyl-β-D-thiogalactopyranoside (IPTG) concentration used for induction was decreased to as low as 0.02 mM, a total of about 33 mg of recombinant GDE can be isolated from alitre culture. We developed an affinity chromatography using βcyclodextrin immobilizedSepharose-6B (β-CD Sepharose 6B) for purifying GDE. The method requires only a single-steppurification and renders an 87% recovery of the enzyme. Moreover, the purified recombinant GDE is a homogeneous protein and possesses the same characteristics as those of S. cerevisiae. To elucidate the structure-function relationship of yeast GDE, the catalytic residues of the enzyme were determined by sitedirected mutagenesis. Asp535, G1u564 and Asp670 on the Nterminal half, and Asp1086 and Asp1147 on the Cterminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes. Five mutant enzymes, D535 N, E564Q, D670N, D1086N and D1 147N, were constructed. All the purified mutant enzymes possessed either the transferase activity or glucosidase activity. Three mutants, D535N, E564Q and D670N, lost transferase activity but retained glucosidase activity. On the contrary, the other mutants, D1086N and Dl 147N, lost glucosidase activity but retained transferase activity. Furthermore, kinetic parameters of the remaining activity of mutants did not vary markedly from those of wildtype enzyme. These results indicate that the residues, Asp535, GIu564 and Asp670, on the Nterminal half are the catalytic residues of transferase activity and the Asp 1086 and Asp 1147 on the C-terminal half involve glucosidase activity, and provide direct evidence that the transferase and glucosidase of yeast GDE are independent each other.

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The genes of three a-glucosidases (HBG I, HBG II and HBG III) were isolated from the cDNA library of honeybee, Apis mellifera L. The nucleotide sequences of HBG I, II and III were consisted of 1974, 1910 and 1916 base pair and encoding 588, 580 and 567 amino acid residues, respectively. The putative primary structures showed high homology ranging from N- to C-terminals, and three enzymes belonged to a-glucosidase family I, in which four conservative regions of aamylase family were observed in their sequences. To obtain the recombinant enzymes, we tried to express the cDNAs in Pichia pastoris of heterologous host cells. Although recombinant HBG I was not produced, the recombinant HBG II and III of 2.4 and 1.2 U/mg were respectively expressed and secreted into culture supernatant. The active recombinant enzymes purified had the same properties as those of native ones except sugar content. To investigate the catalytic residues in HBGs, four mutated enzymes (D206N, E259Q, E269Q and D33 1N) of HBG III were constructed, and their specific activities were found to be 0.0004, 4.9, 0.004 and 0.0002 U/mg, respectively. E259Q remained half activity of wild type and those of the others disappeared, implying that three catalyticresidues of HBGs were D212, E281 and D343 of HBG I, D202, E271 and D333 of HBG II, D206, E269 and D331 of HBG III. The homology modeling showed that three enzymes had Ndomain ((β/α)₈ barrel), subdomain, and C-domain(β-sheet structure mainly) like oligo-l, 6-glucosidase from Bacillus cereus.

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Although Mg is abundant within the cell, most is chelated to various organic molecules; only a small fraction of intracellular Mg is in the free (ionized) form, Mgtt Free Mg²⁺ regulates manyenzyme activities in cells. The effect of free Mg²⁺ concentration on the activities of spinach hloroplast fructose-1, 6-bisphosphatase (FBPase) and ribulose 1, 5-bisphosphate carboxylase (rubisco) was examined. Free Mg²⁺ concentrations in the assay mixtures were directly measured by a Mgtsensitive dye, mag-fura-2. FBPase was activated by a physiological concentration range of free Mgt, but the activation of rubisco was not observed. These results suggest that in illuminated chloroplasts, the increase in free Mg²⁺ activates FBPase, and this may be a physiological factor to stimulate CO₂ fixation.

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In many cell types, newly synthesized acid hydrolases acquire mannose 6-phosphate residues which serve as high affinity ligands for binding to Man-6-P receptors and targeting to lysosomes. A key step in this pathway is the ability of the enzyme UDP-G1cNAc: Lysosomal enzyme acetylglucosaminyl phosphotransferase to selective recognize and phosphorylate acid hydrolases. We have previously demonstrated that phosphotransferase recognizes conformational dependent protein determinants present on the acid hydrolases and absent in most nonlysosomal glycoproteins. It was recently reported that the secretory glycoprotein DNase I contains Man-6-P residues and we found that the human, bovine and mouse forms of this enzyme are phosphorylated to a low extent when expressed in COS cells. The poor phosphorylation of human and bovine DNase I is due to the lack of two critical lysines (position 27 and 74) present in the mouse enzyme whereas the poor phosphorylation of mouse DNase I appears to be due to the presence of “inhibitory” amino acid (Val23) that block the phosphotransferase recognition domain and the disappear of two critical amino acids (Tyr54 and Ser190). Then, we also report the substrate specificity of GPT for the oligosaccharides using variety of fluorescence labeled high mannose type oligosaccharides. As a point to be noticed, GPT can transfer the G1cNAc-phosphate to the G1cMan9GlcNAc2 that still remains glucose.

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cDNA encoding Schizosaccharomyces pombe a-glucosidase was cloned, and expressed in Saccharomyces cerevisiae. The deduced amino acid sequence categorized under the α-glucosidase family II showed a high homology to those of a-glucosidase from molds, plants and mammals. By site direct mutagenesis, Asp481, G1u484, and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COON) of the Asp647 residue was for the first time pointed out to be the candidate of proton donor in the a-glucosidase of family II. The carboxylate group (-COO-) of the Asp481 residue was assumed to be the secondary carboxylate group, which stabilize the oxocarbenium ion through electrostatic interaction, and the Asp481 was considered to be modified by the chemical modification with conduritol B epoxide. The role of the G1u484 residue, which was the third residue, was presumed to be to fix the reaction intermediate of substrates.

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Two intracellular enzymes, cellobiose phosphorylase (CBP) and cellodextrin phosphorylase (CDP) are involved in the phosphorolytic pathway in cellulose degradation. Those enzymes are considered to be useful in syntheses of oligosaccharides because the reactions are reversible. CBP from Cellvibrio gilvus and Clostridium thermocellum YM4, and CDP from C. thermocellum YM4 were cloned and over-expressed in Escherichia coli. All the three enzymes showed ordered bi bi mechanism. However the orders of the substrate binding of the CBPs were different. It was found that CBP from C. gilvus strictly recognized the hydroxyl groups at positions β-1, 3, and 4 of the acceptor molecule in the reverse reaction. On the other hand, the recognition of the hydroxyl groups at positions 2 and 6 was not so strict. Three branched β-1, 4-glucosyl trisaccharides were synthesized by using the reverse reaction of C. gilvus CBP. A new substrate inhibition pattern, competitive substrate inhibition, was also found in the reverse reaction of CBP using glucose as the acceptor. Specific colorimetric quantification of cellobiose was designed by using the reaction of CBP. Cellobiose was produced from sucrose at 90% yield by a combined action of three enzymes including CBP.

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Levansucrase acts on sucrose usually to synthesize a fructan which contains a sucrose unit at one terminus of the molecule and has branches formed by β-2, 1-fructofuranosyl linkage. The transfer of fructosyl residue to an acceptor by the enzyme is carried out from not only sucrose but also oli gosaccharides which contain a sucrose unit such as raffinose or fructosyl maltoside, fructosyl cellobioside, fructosyl lactoside, etc., which can be synthesized from sucrose in the presence of an acceptor sugar (maltose, cellobiose, lactose, etc.) by levansucrase. Soybean contains such oligosaccharides other than sucrose. We isolated levan from fermented soybean (Natto) and analyzed the molecular weight and terminus of the molecule. It was revealed that the molecular weight was about 1.1 × 10⁴ by HPLC using molecular markers of pullulans, and the levan isolated contained a stachyose unit at the terminal of the molecule. It was thought that the enzyme might have a donor binding site (with a high affinity toward sucrose) and an acceptor binding site in the molecule. Because synthesized trisaccharide contained only raffinose and the terminal unit of levan was raffinose in the case of levan synthesized from sucrose in the presence of melibiose, although the levan synthesized from sucrose alone contained kestose (mainly the 1-kestose and 6-kestose, ca .1:1) as the terminal unit of the levan.

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For these 20 years our research groups have investigated the reaction mechanism of glycoenzymes as follows; (1) glucoamylases from Rhizopus niveus and delemar, (2) CGTase from Bacillus stearothermophilus, (3) BLA, (4) BSA, (5) TAA, (6) xylose isomerase from Streptomyces phaeochromogenus, (7) β-glucosidase from Aspergillus niger, (8) β-galactosidase from yeast, (9) β-glucanase from yeast, (10) β-glucocerebrosidase from animal (wagyu). A lot of experimental results have been accumulated in this research era. However, all the reactions were carried out in aqueous solutions, where the reaction system is three dimensions. This is an important point in this discussion. As described in textbooks, for example The Cell, the rate in a reaction system of two dimensions is much larger (around 15 fold) than that of three dimensions. Thus we are very interested in the reaction system of two dimensions, which reaction field in a biological system is the membrane. As a consequence, research on the enzymes and proteins in membrans will be critically important for biological science and technology. Here, based on the research into glycoenzymes, we intended to develop a new research field, that is, cellular macromolecules.

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