The non-Newtonian behavior and dynamic viscoelasticity of a series of aqueous mixtures of xanthan and galactomannan isolated from Leucaena leucocephala de WIT were measured with a rheologiometer. At a concentration of 0.2% of total gums, gelation did not occur at room temperature, but at a low temperature (0°C). A much stronger interaction was observed with mixtures containing deacetylated, deacylated, or native xanthan than with depyruvated xanthan . The maximum dynamic modulus was obtained when the ratio of xanthan to galactomannan was 2:1 . The dynamic viscoelasticity parame ters for mixtures with deacetylated and native xanthan decreased rapidly at temperatures above 20 and 15°C, respectively. It was concluded that the side chains of the galactomannan molecule prevent intermolecular interaction between xanthan and galactomannan. The results obtained support the interaction mechanism between xanthan and locust-bean gum previously proposed.

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A serine proteinase (Sep II; [EC 3.4.21.14]) from Aspergillus sojae was covalently modified by dextran-dialdehyde. The modified Sep II was purified by DEAE-Toyopearl and Sephacryl S-200 column chromatographies. The native enzyme Sep II had a molecular weight of 23, 000 and an isoelectric point of pI 5.2, whereas the modified Sep II had values of 70, 000 and pI 4.5, respectively. A marked increase in the stability at pH 11.0 was observed for the modified Sep II. The Km values for succinyl-AAVA-p-nitroanilide were 0.087 and 0.167 mM for the native and modified enzymes, respec-tively. Cross-reactivity examined by OUCHTERLONY double immunodiffusion showed a decrease in the reactivity of the modified Sep II. These results indicated that the modification of Sep II with dextran-dialdehyde might be useful for the improvement of the enzyme characteristics.

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A grape Invertase was inactivated by incubation with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) or ρ-chloromercuribenzoic acid (PCMB) . The rate of inactivation by EDC or PCMB was reduced in the presence of substrate raffinose, while the effect of maltose, which was neither a substrate nor an inhibitor, was significantly smaller than that of raffinose . The K_m value of the enzyme was not affected by the inactivation with EDC or PCMB . Many carboxyl groups were observed to be modified during the inactivation by EDC. The modification of the carboxyl groups seems to occur at both the active site and the surface of the enzyme at the same time . The complete inactivation of the enzyme by PCMB needed the modification of one sulfhydryl group per enzyme molecule . These results of chemical modification with EDC and PCMB strongly indicated that the carboxyl and sulfhydryl groups are essential for enzyme activity.

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Complex moduli, E* of 3 kinds of mochi cakes prepared with apparatus of stamping, extruding and mixer kneading were measured at frequencies from 3 to 100 Hz and temperatures from 20 to 60°C and 60 to 90°C, respectively. Measurements were carried out with an apparatus for longitudinal vibrations in a cubic specimen at the former temperature range and with another apparatus based on shear mode for concentrated liquid at the latter temperature range. Dynamic Young's modulus, E′ showed large extent of 10⁶-10⁷ Pa and did not change in the frequency range from 3 to 30 Hz, but increased slightly at 100 Hz for all the samples and for every temperature range. Dynamic rigidity, G′, however, was small (about 10³ Pa) in the latter temperature range and appeared positively dependent on frequency. The temperature dependences of E′ and dynamic loss, E″, were very large but those of G′ and G″ were smaller than in the case of E*. The loss energy ratio, ΔK/K′=π tan δ/(2+π tan δ) calcu-lated using the values of G*, showed a peak value at 30 Hz for all the samples and for every temperature range, especially in stamping sample was smaller than another ones. Thus, differences between the preparation methods may explain the different results. The temperature dependence of the apparent specific volume showed the trend Stamp <Extruder≤ Mixer which was confirmed by microscopic observation and by image analysis of the vacuole-size distribution. No difference was found between the samples in the distribution of residual tissue. Rheological evaluation at high temperature or immediately after preparation of mochi was recom-mended as the optimum.

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Rice a-amylase isozymes, AmylA and Amy3D, were expressed by Saccharomyces cerevisiae. Reaction properties of those isozymes purified by immuno-affinity chromatography were characterized to elucidate their functional roles. The pH optima of AmylA (pH 4 .2) and Amy3D (pH 5.5) correlate with the pH value of the endosperm tissue at times in rice seedling development when these isozymes are expressed. AmylA showed higher reactivities to soluble starch and starch granules than Amy3D. On the other hand, Amy3D showed much higher reactivity to maltoheptaose than AmylA. These results suggest that the individual isozymes play different functional roles during the seedling development process. A mutant enzyme, [N240Q]AmylA, which does not have an N-linked carbohydrate chain was created by site-directed mutagenesis . Thermostability of the mutant was much lower than the wild-type enzyme, which suggests that the carbohydrate chain of AmylA is important for the enzyme stability. The differences in reaction properties between the wild type enzyme and the mutant suggest that the carbohydrate chain of AmylA significantly affects the hydrolysis efficiency and the substrate recognition of AmylA for soluble starch . From the comparison of amino acid sequence of AmylA to those of other α-amylases whose 3D structures were clarified, the carbohydrate chain of A mylA is suggested to be positioned on the surface near the active cleft. Therefore, direct interactions between the carbohydrate chain and the substrate might affect the hydrolysis efficiency and substrate recognition.

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Xanthomonas campestris K-11151 produced a novel a-amylase in periplasm. The enzyme purified to homogeneity by several criteria showed almost the same activities on α-, β-, γ-cyclodextrins, soluble starch and amylose. Moreover, it was active on branched cyclodextrins, and pullulan but had very little activity on glycogen. This substrate specificity suggests that this enzyme has the combined activities of α-amylase, cyclodextrinase, and neopullulanase. The gene which codes for this enzyme was cloned into Escherichia coli JM109. An open reading frame of 1578 by was deduced as the structural gene. The enzyme was expressed in E. coli by the lac promoter of pUC plasmid and transported to its periplasmic space. The expressed enzyme showed the same N-terminal sequence, thermal stability, optimum temperature and substrate specificity on various substrates as those of the enzyme from Xanthomonas. The primary structure deduced from its nucleotide sequence showed low and high homology to those of X. campestris extracelluar and Bacillus megaterium α-amylases, respectively. The enzyme has been suggested to have the (α/β)₈ barrel structure common to the α- amylase family but to have the shorter 2nd and longer 4th and 6th loops than other α-amylases.

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Streptococcus mutans and Streptococcus sobrinus, which are principal etiological agents in the development of human dental caries, secrete three kinds (GTF-I, -SI and -S) and four kinds (GTF-I, -S₁, -S₂ and -S₃) of glucosyltransferases, respectively, and form the cariogenic dental plaque in the presence of dietary sucrose. To identify the most important streptococcal enzyme or gene for dental caries formation, various cariogenic properties of S. sobrinus and S. mutans strains were evaluated by biochemical and genetical approaches, respectively. The mechanisms of WIG synthesis and cellular adherence were analyzed by the reconstruction system using the purified GTFs from S. sobrinus B13N strain. Further, two GTF-deficient B13N mutants were isolated and their artificial -plaque forming and caries-inducing abilities were examined . Consequently, it turned out that the existence of GTF-I enzyme was essential for the WIG synthesis, the cellular adherence, the artificial-plaque formation, and the caries formaion by S. sobrinus. Three gtf genes (gtfB, gtfC and gtfD) derived from S. mutans GS5 strain were individually introduced into S. milleri Is57 strain, and in vitro and in vivo cariogenicities of the three S. milleri transformants and the reference strains were compared with each other. Consequently, it turned out that the gtfC transformant expressing GTF -SI enzyme possessed an ability to artificial plaque and an ability to induce caries in gnotobiotic rats . These results suggest that the GTF-I enzyme (gtfl product) of S. sobrinus and the GTF-SI enzyme (gtfC product) of S. mutans may be especially important for the development of human dental caries.

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Xyloglucans are the major component of plant cell wall matrix. Cellulose microfibrils are crosslinked by xyloglucans to form an interwoven network structure of the cell wall. The cleavage and reconnection of xyloglucan cross-links is required for rearrangement of the network structure of the cell wall, an essential process for cell growth and differentiation in plants. Endo-xyloglucan transferase (EXT) is a newly identified class of transferase that catalyzes "molecular grafting" between xyloglucan cross-links in the cell wall. Based on the amino acid sequence information of the purfie EXT protein, cDNAs encoding EXT proteins were cloned from azuki bean and four other plant species. Northern blot analyses have disclosed that the gene expression of EXT is closely correlated with cell growth in stems of azuki bean seedlings. To elucidate the regulatory mechanism of the EXT gene expression in the bean plants, we analyzed the effects of plant hormones and sucrose on the EXT-RNA levels in the stem sections. We found that auxin and gibberellin increased the EXT-mRNA levels in the sections within 2 hr. Sucrose application, which enhanced wall deposition, also increased the EXTmRNA level. These results suggest involvement of EXT in both cell expansion and cell wall deposition in plants.

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Three mould 1, 2-α-mannosidases have been characterized. They were all monomeric enzymes with similar molecular weights of about 60 kDa, showed maximum activity at pH 5, and shared a similar sensitivity for glycosidase inhibitors. Chemical modification of mould 1, 2-α-mannosidases by water soluble carbodiimide resulted in a loss of their activity, suggesting that acidic amino acids were responsible for the catalytic action. Using 1-deoxymannojirimycin (dMM), a competitive inhibitor of Penicillium 1, 2-α-mannosidase as a protecting reagent in the modification, we identified an aspartic acid residue which was involved in the interaction of the enzyme with its substrate . An α-carboxyl structure was formed via an imide intermediate in the reaction with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).

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A unicellular green alga identified as Lobosphaera sp. was selected as a sourse of trehalase. The alga grew well heterotrophically to produce intracellular trehalase. The enzyme was highly purified and characterized. The molecular mass of the native enzyme and that of subunit were estimated to be 400 kDa and 180-200 kDa, respectively. The enzyme was most active at pH 5.5 and at 65t and stable between pH 4-9 and below 65°C. The enzyme specifically hydrolyzed α, α'-trehalose to produce α- and β-anomers of n-glucose in an almost equal ratio . The enzyme catalyzed the condensation not only of n-glucose but also of 2-deoxy-D-glucose, yielding α, α'-trehalose and 2, 2'-dideoxy-α, α'-trehalose, respectively. Simultaneous incubation of the two monosaccharides resulted in the formation of 2- deoxy-α, α'-trehalose. The enzyme synthesized dideoxy-trehalose (yield, 16%) more than trehalose (5%). Monodeoxy-trehalose was produced most effectively in the reaction of D-glucose and 2-deoxyn- glucose in a weight ratio of 2 : 5 to give a yield of 6%.

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Two kinds of debranching enzymes for the degradation of glycogen and amylopectin are known. One kind includes isoamylase and pullulanase, which produce (1→4) -α-glucan chains only by hydrolysis of the branch points; the other kind includes oligo-1, 4→1, 4-glucantransferase and amylo-1, 6 glucosidase which release branched residues as glucose by two-step reactions, first by the transferase and second by the amylo-1, 6-glucosidase . The yeast debranching enzyme was first characterized as isoamylase, and later transferase/amylo-1, 6-glucosidase was also found in yeast. The question then arose as to whether one or both enzymes were present in yeast cells . First, we attempted to discriminate which debranching action was operating in yeast by using branched cyclodextrin. Only glucose appeared as the sole reducing sugar from the branched cyclodextrins (G₃-cG₇, G₅-cG₇) by the action of crude enzyme preparation. This result indicates that the only yeast debranching enzyme is transferase/ amylo-1, 6-glucosidase. Second, the action of the purified debranching enzyme (transferase/ amylo-1, 6-glucosidase) was characterized by the use of various branched cyclodextrins, malto-oligosaccharides, and high molecular weight a-glucans as substrates . The enzyme (system) yielded glucose as the sole reducing sugar from branched cyclodextrins and α-glucans, but was inactive for G₂-cG, . We conclude from these results that ma .ltosyl and maltotriosyl transfers occurred from one branched sugar to another or to linear dextrin by the transferase to form glucosyl branched saccharide, and the glucosyl stub is hydrolyzed by the amylo-1, 6-glucosidase to release glucose.

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CGTase was purified and crystallized. With this preparation, action mechanisms of CGTase were investigated by use of various kinds of substrates. It was elucidated by use of reducing end-labelled maltosaccharides that cyclization proceeds from the non-reducing end of α-1, 4 glucan. Surfactants which have straight carbon chains as a hydrophobic moiety were extremely effective in α-CD formation. On the other hand, surfactants which have a more bulky hydrophobic moiety than straight carbon chains were extremely effective for β-CD formation. Hydrolyzing activity was effectively depressed by the addition of SDS. From these results, the authors infer that cyclization proceeds on 6_5-helices and 7_6-helics to form α- and β-CD from the non-reducing end of α-1, 4 glucan and also that the action pattern of CGTase depends not only on the specificity of the enzyme itself, but also on the conformation of the substrate. Branched oligosaccharides were formed with the action of CGTase on the mixture of G₁-α-CD and 14C -labelled glucose . By the use of various kinds of enzymes, the structure of each branched oligosaccharide was determined. By the combination of the results described above, the authors proposed an enzyme model of the CGTase active site. The mixture of glucose and (G₁) ₂-α-CD (a mixture of three positional isomers) was reacted with CGTase, and the main product formed by the coupling action of CGTase was branched G₉ (BB₉). Thirty-seven percent (from (G₁)₂-α-CD) of completely nonreactable (G₁) ₂-α-CD was obtained . This nonreactable CD coincided with authentic AD type of diglucosyl-α-CD, and these results show that the author's proposed enzyme model might be reasonable.

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We have previously proposed that α-amylases and their related enzymes including neopullulanase are to be called an α-amylase family owing to their common functional and structural characteristics. In these α-amylase family enzymes, neopullulanase has a very interesting enzymatic characteristic, that is, in its active center, neopullulanase acts on both α- (1→4) - and α- (1→6) -glucosidic linkages of polysaccharides, and even catalyzes both hydrolysis and transglycosylation reactions. Hence, using this enzyme, we expected to demonstrate a hypothesis that the enzymes belonging to α-amylase family can be altered each other by protein engineering. However, the 3-D structure of neopullulanase is still unknown. Here, the 3-D structure is predicted on the basis of the X-ray crystal structure of Taka-amylase A by homology modeling. Moreover, the enzyme-substrate structures which are difficult to be elucidated by experimental technique, are predicted by docking-study and molecular dynamics calculation. Based on these informations, we successfully altered the substrate specificity toward α-(1→6) -branched oligosaccharides and pullulan by manipulating the volume of side chain of 11e358, which was located in the active cleft. It is shown that I358W mutant was rather similar to a-amylase type enzyme that can not act on α- (1→6) -linkages. We also successfully altered the reaction speci-ficity toward transglycosylation by manipulating the hydrophobic environment near the entrance route of the water molecule which was probably used in hydrolysis process. It is shown that all the mutants which were substituted by more hydrophobic amino acids had high activities for transglycosylation.

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