To clarify factors causing differences in the taste of cooked rice between three rice varieties, cooking properties of cooked rice with water (white rice) and with soy sauce (Sakura-meshi) were examined for the degree of gelatinization, physical properties and sensory evaluation. The effects of preservation conditions and reheating on the taste of cooked rice were also examined. White rice showed a smaller decrease in degree of gelatinization than that of Sakura-meshi during storage at low temperature. White rice regarded to be unfavorable in taste showed a low degree of gelatinization when determined immediately after cooking, and large breaking energy as well as a large increase in breaking parameter values during storage. The firmness of the white rice ranged from 5.0 to 6.5kg as measured with a tensipresser. Sakura-meshi was observed to be softer and less sticky than white rice. Param-eters obtained by a multibite test of cooked rice with a tensipresser differed by rice variety and could be an important factor for examination of the physical properties of cooked rice. The relation between physical properties and sensory evaluation indicated that the taste of white rice immediately after cooking largely related to firmness. However, the taste of white rice which was stored at a low temperature and then reheated related largely to adhesiveness rather than firmness. Sakura-meshi showed clear differences in the physicochemical properties of the three varieties of rice compared with white rice.

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Using three rice varieties with different tastes and newly developed rice varieties as samples, the relation between the texture of cooked rice (white rice) and starch structure was examined. The amylose contents of the rice starches ranged from 6.3 to 27.3% on gel filtration. The chain length distribution of amylopectins indicated that Fr.II ranged from 17.3 to 25.3% and Fr.III/Fr.IIratios ranged from 2.7 to 3.2. Rice starch with a lower amylose content showed a lower Fr.III/Fr.II ratio. The chain length distribution of amylopectins proved that amylopectin from favorable white rice showed a small amount of Fr. I and a large amount of Fr.II as well as a low Fr.III/Fr.II ratio. The average chain length of amylopectins, as determined by modified SMITH's degradation method, ranged from 18.2 to 23.1. These values agreed well with those determined by the enzymatic method. Rice starch having a low amylose content showed a shorter average chain length. White rice significantly differed in physicochemical properties and sensory evaluation, especially in firmness and adhesiveness by rice variety. Thus, the taste of white rice appeared to be closely related to the structure of the rice starch.

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The flow behaviors (i.e., shear thinning flow, thixotropy, yield value and apparent activation energy) of tropical starch pastes were studied by a rotational viscometer using potato and corn starches as controls. Each flow curve could be described by a power-law equation: τ=K γⁿ, and viscosity index (K) and flow behavior index (n) were calculated. The n of cassava and sago pastes and arrowroot paste at 4% was similar to that of potato starch pastes. On the other hand, the n of edible canna pastes and arrowroot at 2 and 3% was similar to that of corn starch pastes. Starches, the n of which was represented by that of potato starch paste, consisted of both amylose and amylopectin with relatively large molecular weights. On the other hand, starches, the n of which was represented by that of corn starch paste, consisted of both amylose and amylopectin with relatively small molecular weights. Thixotropy was found with edible canna, cassava and sago pastes and 4% arrowroot paste between 10 and 60°C. Potato and cassava starch pastes, which consisted of both amylose and amylopectin with relatively large molecular weights, showed large thixotropy. Edible canna, arrowroot and corn starch pastes, which consisted of amylose and amylopectin with a combination of small and intermediate molecular weights, showed relatively small thixotopy. Sago starch was out of course with n and thixotropy. Yield values were found with 4% arrowroot starch paste at 10 and 20°C and corn starch pastes at 3 and 4% from 10 to 60°C. The apparent activation energy ranged between 8.5 and 17 kJ mol^-1 for the six varieties of starches.

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When the crude extract from developing kidney bean (Phaseolus vulgaris L.) seeds was incubated soluble starch, β-limit dextrin or maltose, hydrolyzing activities were detected. The activity staining of gels of native polyacrylamide gel electrophoresis (i.e., zymograms) at pH 5.6 showed three bands on soluble starch and two on β-limit dextrin. Two active bands on both substrates were to heat treatment (at 70°C), while the other band from soluble starch had no activity. These results suggest that the heat-stable active bands correspond to α-amylases and the other to β-amylasea branching enzyme. Western blot analysis revealed that some bands bound to antiserum against amylase purified from germinating seeds. The zymogram at pH 7.0 indicated additional active from those observed at pH 5.6 on amylose. The mobilities of these two newly detected bands coincident with branching enzymes 1 and 2 purified from developing kidney bean seeds, suggesting the active band observed on soluble starch but not on β-limit dextrin at pH 5.6 was β-amylase. When the heat-treated crude extract was incubated with fl-limit dextrin at pH 5.6, glucose, maltose, other oligosaccharides were produced as evidenced by thin-layer chromatography, similar to α-amylase purified from germinating seeds. These data indicate that α-amylase exists in developing seeds.

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From tea leaves (Camellia sinensis var. sinensis cvs. Shuixian and Maoxie) to be processed to oolong tea, we have isolated and identified alcoholic aroma precursors of geraniol, linalool, etc., which are known to contribute to the floral aroma of oolong tea and black tea, as β-primeverosides (6-O-β-Dxylopyranosyl-β-D-glucopyranosides), guided by an enzymatic hydrolysis followed by GC analysis. Aroma precursors of linalool oxides III and IV were found to be exceptionally present as 6-O-β-D-apiofuranosyl-β-D-glucopyranosides. We have also purified β-primeverosidases from fresh leaves of cv. Yabukita for Japanese green tea, cv. Shuixian for oolong tea and a cultivar of C. s. var. assamica. The enzymatic characteristics were very similar to each other (molecular weight, 60.2-60.5 kDa; optimum temperature, 45°C; stable temp., 40-45°C; optimum pH, 4; pH stability, pH 3-5). The enzyme was confirmed to effectively hydrolyze the aroma precursors, β-primeverosides as well as 6-O-β-Dapiofuranosyl-β-D-glucopyranoside, into disaccharides and each aglycon (alcoholic aroma) without further hydrolysis. We were also interested in the molecular mechanism of the floral aroma emission in flowering. We applied the same experimental procedures as used for the tea to the study of Gardenia jasminoides, Jasminum sumback, and Rosa damascene to find that disaccharide glycosides such as β-primeverosides, β-vicianosides, etc. are also important alcoholic aroma precursors in flowers. In Gardenia jasminoides, glycosidic activities in floral buds reach a maximum at flower opening stage. Purification of the enzyme concerned with the floral aroma formation is now in progress. The enzyme has been characterized as a glycosidase which hydrolyzes the aroma precursors, the disaccharide glycosides, into aglycons and disaccharides.

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Erythritol (1, 2, 3, 4-butanetetrol, molecular weight: 122.12) is a tetra-carbon sugar alcohol contained in fermented foods, animal bodies and plants, and humans have consumed it naturally from ancient times. Erythritol possesses such characteristics as 75% of the sweetness level of sucrose, low hygroscopicity, high endothermic reaction, and easy crystallization. Physiological aspects include low energy value (0.2 kcal/g), low laxative effect and no affect to blood glucose level or secretion of insulin. The National Food Research Institute of the Ministry of Agriculture, Forestry and Fisheries in Japan and Nikken Chemicals Co., Ltd. have done research and development concerning erythritol production by fermentation, and have discovered micro-organisms which can produce erythritol at a high yield and not generate much foam. As the consequence, we succeeded in applying manufacturing technology for highly efficient production of erythritol from glucose by fermentation. Nikken launched the commercial production of erythritol from 1990 and plans to introduce industrial uses in the future.

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Xylanase C (XynC) from Aspergillus kawachii is stable between pH 1-9 and the optimum pH is 2.0. In order to obtain the structural basis of its extreme acid stability and low pH optimum, we conducted X-ray cystallography of the enzyme. Good crystals were grown by using sodium sulfate as a precipitant, and the space group of the crystal was P4₃2₁2 with unit cell dimensions a=b=62 .1 Å, c=113.3 Å. The structure of the enzyme was solved by molecular replacement using the coordinates of Trichoderma reesei XYN I. The final R-factor was 0.194 for data between 6.0 and 2.0 A resolution with F>3σ(F). A structural feature around Asp37 was clearly observed, which is thought to be characteristic for xylanases with a low pH optimum. A mutational analysis confirmed that the residue is important for the low pH optimum of the enzyme. The “Ser/Thr surface” of the enzyme was covered by acidic residues, and thought to be a cause of its extreme acid stability.

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Alkaliphilic Bacillus sp. strain 41M-1 was isolated from soil. The strain secreted a xylanase (xylanase J) that had an alkaline pH optimum. The gene encoding xylanase J was cloned and sequenced. The putative xylanase J gene contained an open reading frame of 1062 by and encoded a 27-amino-acid (aa) leader peptide followed by a 327-aa mature enzyme. The deduced amino acid sequence of xylanase J was compared with those of other bacterial xylanases. The potential catalytic domain belonging to family G was located at the N-terminus of xylanase J. A linker-like sequence occurred between the catalytic domain and an additional functionally-unknown domain at the Cterminus. A mutational analysis of xylanase J indicated that G1u93 and G1u183 should act as catalytic residues, and that Trpl8, Trp86, Tyr84 and Tyr95 might play important roles in substrate binding. Furthermore, the substitution of Asp20 by Asn or Trp144 by Phe caused an acidophilic shift in the optimum pH of xylanase J. A deletion derivative of xylanase J lacking the C-terminal region retained xylanase activity, suggesting that the C-terminal region was not essential for catalytic activity. Wild type xylanase J bound to insoluble xylan specifically, while the C-terminal truncated enzyme completely lost binding ability. On the other hand, the fusion protein between glutathione S-transferase and the C-terminal region of xylanase J showed xylan-binding activity. These results suggested that the C-terminal region of xylanase J is a novel xylan-binding domain. Wild-type xylanase J hydrolyzed insoluble xylan more efficiently than the C-terminal truncated enzyme. Thus, the xylan-binding domain enhances hydrolyzing activity toward the insoluble xylan of the catalytic domain.

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We have been investigating the mechanism of cellulose degradation by various cellulases . Cellulases were classified into three groups such as R-glucosidase, endo-R-1, 4-glucanase and exo-β-1, 4-glucanase, and the differences of reactivities on Avicel and CMC as substrates discriminated endo-β-1, 4-glucanase from exo-β-1, 4-glucanase. However, results which deviate from the above classification were obtained when we investigated the reactivities of some cellulase components on other cellulosic substrates, such as bacterial cellulose, with a different type of structure . From these results, we propose that bacterial celluloses with different crystalline structures are convenient substrates for characterizing the mode of action of cellulases produced from various microorganisms.

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The β-glucosidase (EC 3.2.1.21, βGA) preparation purified from Aspergillus niger (Novozym 188DCN0003) was confirmed to be a monomer protein of Mw 137, 000. With this preparation, binding of glucono-l:5-lactone (GLN) to βGA was studied using fluorescence-spectrophotometric and stopped flow kinetic experiments on the basis of Trp-residue (s), and an inhibition kinetic experiment for the cellobiose substrate. The dissociation constant (K_d) and inhibitor constant (K_i) (competitive inhibition) for the GLN-βGA complex were evaluated to be 10μM and 15μM, respectively. The subsite structure of βGA was analyzed with the steady-state kinetic method. It was confirmed that the active site was composed of six subsites, affinity at subsite 2 (A₂) was the largest and subsites 4-6 had negative affinity. We tried to interpret the binding of GLN on the basis of the subsite structure, where subsite 1 carried an indispensable role in the productive binding mode. Determining the kinetic parameters for several β-glucosides as substrates, it was shown that the molar activity (k₀) and Michaelis constant (K_m) were very characteristic of the substituent species in aglycone phenyl-residue. Furthermore, the value of k₀/K_m, which reflects the productive-binding mode, strongly depends on substituent constant n (degree of hydrophobicity), suggesting that subsite 2, in which the aglycone residue is bound, is intimately related to the "hydrophobic-driven" mechanism for ES-complex formation. Further, the effect of acetonitrile (0-32.3%) on the hydrolytic reaction was examined for the cellobiose substrate. The results support the hydrophobic-driven mechanism.

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D-enzyme (EC 2.4.1.25) is believed to be involved in starch metabolism, but the function in vivo is not known. In order to investigate the role of D-enzyme, several approaches have been undertaken. A biochemical analysis of the purified potato D-enzyme suggested that high molecular weight starch (amylose and amylopectin) can serve as donor and acceptor, and very long α-1, 4-glucans or even highly branched glucans can be transferred by the enzyme. Transgenic potato plants with dramatically reduced D-enzyme activity were obtained by introducing sense and antisense D-enzyme cDNA sequences with the appropriate promoter sequence. The tubers from these plants sprouted later and the growth of sprouts was slower than the wild type. However, no significant difference was found in starch produced in tubers, either in its quantity or quality. From these results, the possible role of Denzyme in starch metabolism is discussed.

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Cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 and its mutant enzymes are cocrystallized with acarbose at pH 4.5. A triclinic unit cell contains two independent molecules, MOLL and MOL2, related by pseudo two-fold symmetry . In both MOLL and MOL2 of wild-type . CGTase, acarbose occupies subsites 2 to 2' of the enzyme and the catalytic triad Asp229, Glu257 and Asp328 are in hydrogen-bonding contact with B and C residues which are connected by a scissile bond (residue A is at the non-reducing end of acarbose). The corresponding catalytic residues in a mutant enzyme, F183L/F259L, constituting subsite 2' form hydrogen bonds with C and D residues in MOLL and only with the D residue in MOL2. In addition, the cleavage sites of 3-ketobutylidene-2-chloro-4-nitrophenyl f3-maltopentaoside (3KB-G5CNP) are observed to be shifted in the direction of the reducing end in the catalytic reaction of the mutant. This mutant shows a 10, 000-fold increase of IC50 of acarbose on starch degrading activity as compared to that of wild-type CGTase. From these findings, we discuss the effects of the stacking interaction of Phe183 and Phe259 on substrate binding, cyclization reaction and the acarbose-inhibition mechanism of CGTase . The replacement of Tyr100 with leucine causes a drastic reduction in catalytic activity as well as an increase in IC50 of more than 170, 000-fold. Interestingly, in MOL1 and MOL2 of Y100L, acarbose forms fewer hydrogen bonds with the active site than that bound to wild-type CGTase and the O₄ atom of each scissile bond moves in the direction of Tyr195, away from G1u257. As a result, Phe183 and Phe259 can no longer stack on the D residue. Since the significant changes in the protein backbone conformation between Y100L-acarbose and wild-type acarbose have not been found, the drastic reduction of catalytic activity of the mutant Y100L is ascribed to decreased interaction between enzymes and substrates. This again points to an essential role for the residue constituting subsite 1 in the catalysis of CGTase.

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Thermostable α-glucosidase with a broad substrate specificity from Bacillus sp. SAM1606 can hydrolyze various 1-O-α-D-glucopyranosides includingg maltose, isomaltose and sucrose, and is the only known α-glucosidase that can efficiently hydrolyze a, α-trehalose. The SAM1606 α-glucosidase has several short conserved regions (CR) which are detected in the α-amylase family' enzymes. The putative catalytic residues conserved among the α-amylase family enzymes are also identified in the CR of the SAM1606 enzyme; suggesting that the enzyme can be categorized into the family. The enzyme exhibits a very high sequence similarity to two oligo-1, 6-glucosidases (016G) of Bacilli that cannot act on trehalose. To identify the critical residues which determine the broad substrate specificity of the SAM1606 enzyme by site-specific mutagenesis, we selected five residues to be mutagenized in the SAM1606 a-glucosidase by comparison of the CR sequences of these three glucosidases. These 5 residues have been specifically replaced by in vitro mutagenesis with the residues as in Bacillus 016G. The twelve mutant enzymes with one through five substitutions were expressed and kinetically characterized. None of the single and multiple mutations caused a significant reduction in Vmax for all of the substrates tested; all mutant retained Vmax values more than 20% of those of the wild-type enzyme. There was no significant variation in Km detected with maltose, sucrose or isomaltose upon each mutation. For trehalose, however, G1y273Pro as well as all multiple mutations containing the G1y273Pro caused appreciable increases in the Km value for this substrate, indicating that the loss in affinity for trehalose is critically governed by G1y273Pro, whose effect is specifically enhanced by Thr342Asn. From recent X-ray crystallographic studies of the porcine pancreatic α-amylase (PPA) complexed with acarbose, G1y273 which corresponds to I1e235 in PPA appears to be in close contact with the bound inhibitors.

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The domain structure of Aspergillus niger glucoamylase was studied by calorimetry and steady-state kinetics. Two forms of the enzyme were used; G1 is the whole protein with two main domains of the enzyme (the catalytic domain and the starch-binding domain), and G2 is an isozyme that lacks the starch-binding domain of Gi. Adiabatic differential scanning calorimetry (DSC) revealed that the catalytic domain and starch-binding domain were independent of each other concerning thermally induced unfolding, and that the former domain unfolded irreversibly and the latter reversibly . DSC and isothermal titration calorimetry also showed that there was no detectable interaction for the binding of ligands to those domains. Steady-state kinetic studies showed that the starch-binding domain did not change the kinetic parameters for the hydrolysis of p-nitrophenyl glucoside or the binding modes of inhibitors. These results lead us to the following conclusion; the essential role of the starch-binding domain seems simply to increase the local concentration of starch around the enzyme molecule .

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Soybean β-amylase was mutated by site-directed mutagenesis at residues (His93, Cys95, Asp101, Glu186, Cys208, Cys343, Glu345, Asp348, Glu380 and Leu383) conserved in highly similar regions of the α-amylase family found in plants and bacteria. The substitutions of Asp101, Glu186 and Glu380 completely eliminated the activity without inducing any significant change in the binding affinity for α-cyclodextrin (α-CD). These data and X-ray crystallographic works confirm Glu186 and Glu380 as catalytic residues of the enzyme. On the other hand, substitutions of Leu383 led to remarkable decreases in the k_cat/K_m values, and those mutants also showed marked reductions in their binding affinity to α-CD. Leu383, therefore, may be important for both substrate penetration and subsequent retention at the active site. Based on the foregoing, we propose an action mechanism of soybean β-amylase involving the interaction of three essential amino acid residues (Asp101, Glu186 and Glu380) in concert with leu383.

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