In this study, changes in rice qualities from High-Pressure (HP) treatment were investigated. Milled rice grains were presoaked in water at 25 and 55°C for 30 min, subjected to HP treatment at 400 MPa for 10 min, and soaked in water overnight. The effects of presoaking temperature and HP treatment on the physical and chemical properties of rice were evaluated. The viscosities of milled rice grains increased with the soaking process. The total sugar content increased and change in the internal structure of the rice grains occurred after HP treatment. The structural change seemed to promote the water penetration and brought about a higher degree of swelling in rice granules, which might result in a higher degree of gelatinization and higher digestibility of HP-treated cooked rice. HP treatment also brought about denaturation of water soluble proteins and an increase in some free amino acids. HP processing was thus proved to be one of the advantageous processing methods for cooked rice with better palatability.
An alkaliphilic actinomycete, Nocardiopsis sp. strain TOA-1, produced extracelluar maltotriose-producing amylases. Two amylases (AmyA-1 and AmyA-2) were purified to homogeneity by three steps of chromatography. The molecular masses of AmyA-1 and AmyA-2 were estimated to be 56 and 60 kDa, respectively. Optimal pH and temperature of both AmyA-1 and AmyA-2 were pH 9.5 and 65°C. These enzymes were stable at pH 7 and even at 13. AmyA-1 and AmyA-2 produced only maltotriose from starch, amylose, amylopectin, glycogen and γ-cyclodextrin at an early stage of reaction and small amounts of glucose and maltose were also produced upon prolonged incubation. The activities of AmyA-1 and AmyA-2 were significantly inhibited by Fe2+, Fe3+ and N-bromosuccinimide. Substrate specificities were slightly different between AmyA-1 and AmyA-2.
In order to use effectively the starch from the extracted residue of crude drugs, adsorption characteristics of cationic methylene blue (MB) and anionic methyl orange (MO) were investigated. Starches (S) were prepared from the roots of Panax ginseng C.A. Meyer (PG) and Panax notoginseng (Burk.) F.H. Chen (PN), the rhizomes of Pinellia ternate (Thunb.) Breitenbach (PT) and Alisma orientale Juzepczuk (AO), and the seeds of Coix lacrymajobi Linné var. ma-yuen Stapf (CL). The adsorption isotherm was evaluated using Akaike's information criterion (AIC) value, and the adsorption of cationic MB and anionic MO by the starches was classified into the Langmuir type and Freundlich type, respectively. The starches adsorbed more MB than MO. The amounts adsorbed were affected not by the particle size or the content of cationic minerals, but by the phosphorus content. Cationic MB was adsorbed more strongly than anionic MO on the negatively charged surface of the starches because of the presence of phosphorus. S-CL was superior in adsorption capacity for both cationic MB and anionic MO to S-PG, S-PN, S-PT, S-AO, and S-ST (potato starch) at 5 and 25°C. S-CL had a porous and stripe structure on the granular surface, and contained a small amount of phosphorus.
Carboxymethyl cellulase (CMCase) was purified from the mid-gut gland of a marine mollusc, Patinopecten yessoensis. The enzyme hydrolyzed CMC in a semi-acidic condition with maximal hydrolytic activity at pH 6.0 and 35°C. The monomeric protein was observed to have an Mr value of 43 K (SDS-polyacrylamide gel electrophoresis). Inactivation of the enzyme by EDTA was reversed by Ca2+ and Mg2+. The enzyme did not act on p-nitrophenyl β-glucoside, laminarin, or xylan. Crystalline cellulose and cellooligosaccharides such as cellotriose, cellotetraose, and cellopentaose were negligibly hydrolyzed. These results suggest that the Patinopecten 43 K-CMCase could be a 1,4-β-endoglucanase rather than a cellulase.
We found a microorganism, Penicillium chrysogenum 31B, that has high ability to release ferulic acid from sugar beet pulp. Approximately 85% of alkaline-extractable ferulic acid in sugar beet pulp could be released using the culture supernatant of P. chrysogenum 31B. However, the culture supernatant did not efficiently extract ferulic acid from wheat bran, peel of corn seed, or sugar-cane bagasse. A ferulic acid esterase (FAE-1) was purified from the culture filtrate of P. chrysogenum 31B. The molecular mass of the enzyme was determined to be 62 kDa by SDS-PAGE. Optimum conditions for enzyme activity were 50°C and pH 6-7. The enzyme showed activity towards methyl esters of hydroxycinnamic acids including ferulic acid, p-coumaric acid, and caffeic acid, but was not active on methyl sinapinate or 3,4-dimethoxy cinnamate. The lack of activity of FAE-1 toward these substrates appears to be due to the presence of two methoxy groups on the benzene ring. The substrate specificity of FAE-1 seemed to be similar to that of ferulic acid esterase (CinnAE) of Aspergillus niger. However, there was a difference between FAE-1 and CinnAE in respect to activity towards methyl vanillate. It is remarkable that FAE-1 hydrolyzed methyl vanillate, which, to our knowledge, is the first report of a ferulic acid esterase hydrolyzing a hydroxybenzoic acid methyl ester.
In the past two decades, there has been vast expansion in the research of fructooligosaccharides (FOS), including their chemistry, biochemistry and enzymology in higher plants. However, in spite of these considerable advances in fructan science, many other aspects of the mechanisms of fructan metabolism have not been fully understood. The bulbing and yield of dry onion cultivars vary, but they depend on the contents of the high dry matter and non-structural carbohydrates (fructooligosaccharides) which contribute to keepability. The knowledge of the mechanisms of the synthesis and the degradation of the FOS occurring during growth and storage are of great interest. Important progress has been made in the research area of onion FOS, and in addition to their role as accessible reserve carbohydrates in bulbs during sprouting, FOS participate in many physiological processes of production, protection and preservation of the bulbs. This review aims to contribute to a better understanding of the metabolism of FOS in onion bulbs, based on recent investigations. The activities of the main enzymes involved in the synthesis and the hydrolysis of the FOS during the pre-harvest (growth) and post-harvest (storage) life of the bulbs are reviewed, although present knowledge is too limited to explain clearly the mechanisms triggering the enzymes activities or the mechanisms by which FOS contribute to quality and long keepability of the bulbs.
Sembei produced from normal rice is one of the traditional Japanese rice crackers (Beika) and is generally produced by roasting the dried rice paste. The degree of swelling after roasting the rice paste is generally thought to depend on the property of rice paste characterized by forming molecular networks of starch during the drying process. So, the dispersion degrees of rice pastes prepared in various drying processes were tested in order to reveal the effects of temperature and drying time. It was suggested that the hardness of the molecular networks formed was dependent on the drying rate. In the manufacture of sembei, control of the drying rate is very important until the moisture of the rice paste reaches about 25% because the dispersion degree is alterable. When the drying rate undergoes unusual acceleration before in rice paste reaches about 25%, it could be thought that sufficient swelling of the paste does not occur in the process of roasting.
The effects of culture composition on growth and quality of Flammulina velutipes, an edible mushroom (Enokitake in Japanese) were investigated using sawdust-based medium and corn cob-based medium. The changes in pH during cultivation of the mushroom were similar in both media. An increase of soluble proteins in cultures was recorded; however it was larger in corn cob-based medium than in sawdust-based medium. Among cellulolytic enzymes such as cellulase and xylanase, activity determined in corn cob-based medium was much larger than in sawdust-based medium. In addition, the yield of fruit bodies increased by using the corn cob-based medium, and the compositions in fruit bodies cultured by each medium showed some differences. From these results, it is suggested that components of corn cob and related enzymes might be concerned in the increase of yield of fruit bodies, and that components in the fruit bodies might be influenced by culture medium.
Amylomaltase (EC 126.96.36.199) from Thermus aquaticus catalyzes an intramolecular transglycosylation of α-1,4 glucan and produces cycloamylose, which is a cyclic α-1,4 glucan with a degree of polymerization of 22 and higher. The amylomaltase has weak but significant hydrolytic activity together with its major transglycosylation activity, which consequently decreases the yield of cycloamylose. To diminish the hydrolytic activity of this enzyme, random mutations are introduced into the gene coding for this enzyme. In the random mutagensesis experiment, it is suggested that tyrosine 54 (Y54), far away from the catalytic site, was involved in hydrolytic activity. In order to investigate the function of Y54, we have performed saturating mutagenesis at Y54 within the amylomaltase and examined the properties of the mutated enzymes. The reaction specificities of the mutated enzymes were surprisingly changed by only one amino acid replacement at Y54. Y54G mutated enzyme had higher cyclization activity in addition to the lower hydrolytic activity. These mutated enzymes also provided useful information to gain further understanding for the activity and the specificity of this enzyme.
Glucodextranase (GDase) hydrolyzes α-1,6-glucosidic linkages of dextran from the non-reducing end to produce β-D-glucose. GDase is classified under GH15, whose major member is glucoamylase (GA) that hydrolyzes α-1,4-glucosidic linkages of starch. We have cloned a GDase gene from the Gram-positive bacterium Arthrobacter globiformis I42 and determined the crystal structure at 2.42-Å resolution. The structure of GDase is composed of four domains N, A, B and C. Domain N consists of 17 antiparallel β-strands and domain A forms an (α/α)6 barrel structure, which is conserved between GAs. Furthermore, the complex structure with acarbose was also determined at 2.42-Å resolution. The structure of GDase complexed with acarbose revealed that the positions and orientations of the residues at subsites -1 and +1 are nearly identical for GDase and GA; however, Glu380 and Trp582 located at subsite +2, which form the entrance of the catalytic pocket, and the position of the open space and constriction of GDase are different from those of GAs. On the other hand, domains B and C are not found in GAs. The primary structure of domain C is homologous with the surface layer homology (SLH) of pullulanases from Gram-positive bacteria, and the three-dimensional structure of domain C resembles the carbohydrate-binding domain of some glycohydrolases. The hydrophobicity of domain B is higher than that of the other three domains. These findings suggest that domains B and C serve as cell wall anchors and contribute to the effective degradation of dextran at the cell surface.
Construction of various rare sugar oligosaccharides by glycosidase-catalyzed transglycosylation reaction may require α-glycosidases that possess unique glycon specificity. In order to obtain such α-glycosidase, we carried out two studies to: 1) investigate unknown glycon specificities of several α-glycosidases using various types of rare sugar containing glycosides as substrates, and 2) change the glycon specificities of the α-glucosidase from Geobacillus stearothermophilus by site-specific mutagenesis. Through the former studies, several α-glycosidases were found to possess hydrolytic activities towards specific glycon monodeoxy analogs of p-nitrophenyl (pNP) α-D-glycopyranosides. Using Aspergillus niger α-glucosidase that showed activity towards 2-deoxy glucoside and jack bean α-mannosidase that showed activity towards 6-deoxy mannoside (α-D-rhamnoside), the glycon 2-deoxy derivative of isomaltoside (ethyl 2-deoxy-α-D-arabino-hexopyranosyl-1,6-β-D-thioglucopyranoside) and α-D-rhamnodisaccharide derivative (ethyl α-D-rhamnopyranosyl-1,2-α-D-thiorhamnopyranoside) were prepared by their transglycosylation reaction in good yields. For the latter studies, fifteen mutant enzymes of Geobacillus stearothermophilus α-glucosidase were prepared and their hydrolytic activities towards the maltose, eight diastereomers of pNP α-D-aldohexopyranoside, and possible monodeoxy- and mono-O-methyl analogs of pNP α-D-gluco, -manno and -galactopyranosides were elucidated. For these mutant enzymes, there were differences between the specificities for pNP α-D-glucopyranoside and those for maltose, while significant changes were not confirmed in the specificity for other pNP α-D-aldohexopyranosides or the partially modified analogs of pNP α-D-glycopyranosides.
Novel chromogenic substrates for endo-β-galactosidase were designed on the basis of the structural features of keratan sulfate. Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ-pNP (2), which consists of two repeating units of N-acetyllactosamine, was enzymatically synthesized by consecutive additions of GlcNAc and Gal residues to p-nitrophenyl β-N-acetyllactosaminide. In a similar manner, GlcNAcβ1,3Galβ1,4GlcNAcβ-pNP (1), GlcNAcβ1,3Galβ1,4Glcβ-pNP (3), Galβ1,4GlcNAcβ1,3Galβ1,4Glcβ-pNP (4), Galβ1,3GlcNAcβ1,3Galβ1,4Glcβ-pNP (5), and Galβ1,6GlcNAcβ1,3Galβ1,4Glcβ-pNP (6) were synthesized as analogs of 2. Endo-β-galactosidases released GlcNAcβ-pNP or Glcβ-pNP in an endo-manner from each substrate. A colorimetric assay for endo-β-galactosidase was developed using the synthetic substrates on the basis of the determination of p-nitrophenol liberated from GlcNAcβ-pNP or Glcβ-pNP formed by the enzyme through a coupled reaction involving β-N-acetylglucosaminidase or β-D-glucosidase. Kinetic analysis by this method showed that the value of Vmax/Km of 2 for Escherichia freundii endo-β-galactosidase was almost equal to that for keratan sulfate, indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo-β-galactosidase assay. In addition, the hydrolytic action of the enzyme toward 2 has shown to be remarkably promoted by the presence of 2-acetamide group adjacent to p-nitrophenyl group in comparison with 4. In addition, enzymatic synthesis of GlcNAc-terminated poly-N-acetyllactosamine β-glycosides GlcNAcβ1,3 (Galβ1,4GlcNAcβ1,3)n Galβ1,3GlcNAcβ-pNP (n=1-5) has been demonstrated using a transglycosylation reaction of E. freundii endo-β-galactosidase. The enzyme catalyzed a transglycosylation reaction on 1, which served both as a donor and an acceptor, and converted 1 into p-nitrophenyl β-glycosides GlcNAcβ1,3(Galβ1,4GlcNAcβ1,3)nGalβ1,4GlcNAcβ-pNP (9, n=1; 10, n=2; 11, n=3; 12, n=4; 13, n=5). The efficiency of production of poly-N-acetyllactosamines by E. freundii endo-β-galactosidase was significantly enhanced by the addition of BSA and by a low temperature condition.
Recently, we isolated and cloned two xyloglucan-specific endoglucanases (XEGs) from Geotrichum sp. M 128 and Gram-positive bacterium sp. KM 21, and a xyloglucan-specific exoglucanase, oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH) from Geotrichum sp. M 128, all of which belong to glycoside hydrolase family 74. Geotrichum and KM 21 XEGs have endoglucanase activity toward xyloglucan but not cellulose, while Geotrichum OXG-RCBH is a unique exoglucanase that releases two Glc residue segments from the reducing end of the xyloglucan main chain. Further analysis of the substrate specificities of Geotrichum XEG and OXG-RCBH using various oligosaccharides revealed that they recognize specific xylose branching structures. Although both Geotrichum XEG and OXG-RCBH have at least four subsites (-2 to +2), specific recognition of xylose residues occurs at the +1 and +2 sites in the former and at the -1 site in the latter. Moreover, xylose branching at the reducing end (site +2) eliminates OXG-RCBH activity. This enzymatic activity is very different from those of known glycosidases. Recently, OXG-RCBH was assigned a new EC number (EC 188.8.131.52). To elucidate the molecular mechanism of OXG-RCBH, its three-dimensional structure was analyzed. The X-ray crystal structure of OXG-RCBH revealed a unique feature of this enzyme: OXG-RCBH consists of two similar domains, both of which are folded into seven-bladed β-propeller structures. The cleft between the two domains can accommodate the oligosaccharide substrate and thus constitutes a putative catalytic core. Mutation of either Asp 35 or Asp 465, located in the cleft, to Asn resulted in a protein with no detectable catalytic activity, indicating the critical role of these amino acids in catalysis.
Oligosaccharides are linked to the protein surface and play roles in a number of biological events. Therefore, much attention is being paid to research to investigate the function of the oligosaccharides. In order to investigate the function of oligosaccharides, many synthetic approaches have been examined by synthesizing N-linked oligosaccharides. In this paper, we introduce recent synthetic developments focusing on the synthesis of N-linked oligosaccharides.
Mode of substrate-binding of chitosanases from Streptomyces sp. N174 (N174 chitosanase) and Bacillus circulans MH-K1 (MH-K1 chitosanase) was examined by site-directed mutagenesis and physicochemical techniques, including thermal unfolding, fluorescence spectroscopy, and X-ray crystallography. Asp57 located at the central portion of the binding cleft of N174 chitosanase was mutated to asparagine and alanine (D57N and D57A), and the relative activities of the mutated enzymes were 72 and 0.5% of that of the wild type, respectively. Thermal unfolding experiments in the presence of (GlcN)n clearly indicated the importance of Asp 57 for substrate binding. Kinetic analysis of (GlcN)6 degradation catalyzed by N174 chitosanase suggested that Asp57 is most likely to participate in the substrate binding at subsite -2 through hydrogen bonding as well as electrostatic interaction. On the other hand, for MH-K1 chitosanase, we focused our attention on Tyr148 and Lys218, which are located at the bottom of the binding cleft and at the flexible loop forming the edge of the binding cleft, respectively. These residues were mutated to serine (Y148S) and proline (K218P), respectively, and the enzymatic activities of Y148S and K218P were found to decrease to 12.5 and 0.16% of the wild type. When (GlcN)3 binding ability to the chitosanase was evaluated from the change in tryptophan fluorescence intensity, the binding abilities of Y148S and K218P were found to be reduced from that of the wild type by 1.0 and 3.7 kcal/mol of binding free energy, respectively. The crystal structure of K218P revealed that the main chain and side chain structures of the loop comprising Lys218 are affected by the mutation. Thus, we concluded that the flexible loop comprising Lys218 plays an important role in substrate binding, and that the role of Tyr148 is less important but significant, probably due to stacking interaction.
Vibrio proteolyticus chitobiose (GlcNAc-β1,4-GlcNAc) phosphorylase (ChBP) catalyzes the reversible phosphorolysis of chitobiose into α-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. ChBP and its homologues, cellobiose phosphorylase (CBP) and cellodextrin phosphorylase (CDP), were classified under the glycosyltransferase (GT) class, GT-36, on the finding that they have no hydrolytic activity. As the first known structures of a GT-36 enzyme, we determined the crystal structures of ChBP including the ternary complex with GlcNAc and SO4. They are also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveal a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a β-sandwich domain and an (α/α)6 barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65). The proposed reaction mechanism of ChBP also shows similarity with those for inverting hydrolytic enzymes with the exception of the molecules attacking the C1 atom. The similarities of overall structures and catalytic mechanisms between ChBP and GH enzymes led to the reclassification of family GT-36 into a novel GH family, namely GH-94. The substrate complex structures of ChBP also provide many structural insights into its oligosaccharide synthesis reaction such as substrate specificity.
One hundred ten genes for human glycosyltransferases had been cloned and analyzed at the beginning of April, 2001 after the first mammalian glycosyltransferase gene was cloned in 1986. The term glycogene includes the genes for glycosyltransferases, sulfotransferases adding sulfate to carbohydrates and sugar-nucleotide transporters, etc. In April 2001, we started the Glycogene Project (GG project), which was a comprehensive study on human glycogenes. One hundred five novel glycogenes were identified as candidates with the aid of bioinformatic technology. All of them were cloned and expressed as recombinant enzymes, and their substrate specificities were then examined using various acceptors. Thirty-eight glycogens among the 105 candidates were determined to be glycosyltransferases, sulfotransferases and sugar-nucleotide transporters. One hundred sixty-five glycogenes were subcloned into a Gateway entry vector, and prepared as a human glycogene library. These cloned glycogens can be easily expressed as recombinant enzymes in various expression systems.