Alkaline α-amylase from Bacillus sp. strain KSM-K38 (AmyK38) is a calcium-free enzyme that is stable against chelating and oxidative reagents. Recently, the thermostability of this enzyme was improved by replacement of its amino-terminal 11 amino acid residues with the corresponding residues of α-amylase from Bacillus sp. strain KSM-1378. In this study, to further improve the thermal stability, we compared the three-dimensional structure of AmyK38 with that of a hyper-thermostable α-amylase from Bacillus licheniformis. Using site-directed mutagenesis, we created a new possible ionic interaction in the flexible loop region from Gln167 to Gln170 of AmyK38 because the corresponding region in α-amylase from B. licheniformis has an ionic interaction between Glu167 and Lys170. Substitution of Gln167 or Tyr169 with Glu or Lys, respectively, was found to enhance the thermostability of AmyK38. Combination of both substitutions with replacement of the amino-terminal 11 residues further improved the thermostability of the enzyme.

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In this study, alginate was isolated from the residues of Hizikia fusiformis, after extraction of the fucoidan. The yield, total carbohydrate, uronic acid, ash and moisture of alginate was 2.6% (based on wet material), 85.2, 80.5, 12.1 and 2.2%, respectively. The molecular mass of alginate was calculated to be about 7.0×10⁴. The ¹H- and ¹³C-NMR and IR spectra of alginate from H. fusiformis were in agreement with those of standard alginate. From the integral of the anomeric proton signals of D-mannuronic (M) and L-guluronic (G) acid residues in the ¹H-NMR spectrum, the molar ratio of both residues was calculated to be M: G=0.53: 1. Eight alginate oligosaccharides were also isolated by gel permeation chromatography after using commercially available enzymes and were characterized by 2 D NMR techniques and ESI-MS spectrometry as Δ(4-deoxy-L-erythro-hex-4-enopyranosyluronate) GGGG, ΔGGG, ΔGG, ΔG, ΔMMMM, ΔMMM, ΔMM and ΔM.

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Isoprimeverose-producing oligoxyloglucan hydrolase (IPase; EC 3.2.1.120) is a unique β-glycosidase that cleaves xyloglucan oligosaccharides at the non-reducing end, producing isoprimeverose. Here, we describe the first reported identification and characterization of a prokaryotic IPase. We purified an IPase with a molecular mass of 105 kDa from the culture supernatant of an Actinomycetes species, Oerskovia sp. Y1, and characterized its pH and thermal stability. The enzyme was stable between pH 3.5 and 7.5, and its optimum pH was 4.5. We also found that it was stable at temperatures up to 45°C, and the optimal temperature for enzyme activity was 55°C. The K_m value for XXXG (the letters G and X refer to an unbranched Glc residue and an α-D-Xylp-(1→6)-β-D-Glcp segment, respectively) was determined to be 0.7 mM, and the specific activity was 85 U per mg protein. HPLC analysis revealed that IPase cleaves XXXG to X and XXG, then cleaves XXG to X and XG, and finally cleaves XG to X and G. Transglycosylation activity was also clearly evident; HPLC analysis revealed that the enzyme could transfer isoprimeverose to XXXG to produce XXXXG.

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Studies of the structure and function of Carbohydrate-Active enZymes (CAZymes) have made a great deal of progress over the last decade. The glycoside hydrolase (GH) family is a prominent class of CAZymes. There are more than 100 GH families, with wide variations in their 3 D structures (folds). This review focuses on the recently determined crystal structures of 4 GH families: GH42, GH57, GH54 and GH94. Possible evolutionary relationships between apparently unrelated GH families are discussed based on structural and mechanistic similarities.

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Cyclic isomaltooligosaccharides or cyclodextrans (CIs) are cyclic oligosaccharides of α-1,6 linked glucose residues. CIs are highly water-soluble and were found to strongly inhibit glucansucrase activity of mutans streptococci, so, CIs are expected to be utilized as cariostatic compounds. They are produced from dextran catalyzed by cyclic isomaltooligosaccharide glucanotransferase (CITase) and substrate dextran is produced from sucrose catalyzed by dextransucrase. CIs were found and isolated from the culture supernatant of Bacillus circulans T-3040 strain when it was cultured with dextran. The structure of CIs were determined by enzyme digestion test, ¹³C-NMR analysis, and mass spectrum analysis. In order to produce CIs for commercial scale, the high dextran producing strain Leuconostoc sp. S-51 was isolated and the B. circulans T-3040 strain was mutated to produce about 110 times as much CITase as that of wild type strain. We also successfully detected CIs in brown sugar, which suggests CIs exist in nature.

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A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an α-1,6-linkage [ICG5; cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS-PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50°C, and stable from pH 4.5 to 9.0 at up to 35°C. The addition of 1 mM Ca²⁺ enhanced the thermal stability of the enzyme up to 40°C. It acted on maltooligosaccharides that have degrees of polymerization (DP) of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular α-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular α-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme. The gene for IGTase was cloned from the genome of B. circulans AM7. The IGTase gene, designated igtY, consisted of 2985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The four conserved regions common in the α-amylase family enzymes were found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme (IgtZ) was purified. The enzyme acted on maltooligosaccharides that have a DP of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme, which has a novel amino-acid sequence, should be assigned to α-amylase. It is notable that both IGTase and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.

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Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the α-1,4-linkage and its subsequent transfer to the α-1,6-position. A novel BE encoded by an uncharacterized ORF (TK1436) was identified in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). TK1436 orthologs are distributed in archaea of Thermococcales, cyanobacteria, some actinobacteria and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion exchange chromatography, eluting slower than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain α-glucans, indicating that the reaction product contained many α-1,6-branching points. TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70°C, and thermostability up to 90°C as determined by the iodine-staining assay. These properties were the same when a protein devoid of the C-terminal HhH motifs (TK1436ΔH protein) was used. The average molecular weight of branched glucan after reaction with TK1436ΔH protein was over 100 times larger than that of the starting substrate. These results indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family.

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Amylose is a functional biomaterial and is expected to be used for various industries. However at present, manufacturing of amylose is not done, since the purification of amylose from starch is very difficult. It has been known that amylose can be produced in vitro by using α-glucan phosphorylases. In order to obtain α-glucan phosphorylase suitable for manufacturing amylose, we isolated an α-glucan phosphorylase gene from Thermus aquaticus and expressed it in Escherichia coli. We also obtained thermostable α-glucan phosphorylase by introducing amino acid replacement onto potato enzyme. α-Glucan phosphorylase is suitable for the synthesis of amylose; the only problem is that it requires an expensive substrate, glucose 1-phosphate. We have avoided this problem by using α-glucan phosphorylase either with sucrose phosphorylase or cellobiose phosphorylase, where inexpensive raw material, sucrose or cellobiose, can be used instead. In these combined enzymatic systems, α-glucan phosphorylase is a key enzyme. This paper summarizes our work on engineering practical α-glucan phosphorylase for industrial processes and its use in the enzymatic synthesis of essentially linear amylose and other glucose polymers.

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We cloned and sequenced the gene encoding exo-β-glucosaminidase (GlcNase) from Amycolatopsis orientalis, and found that the gene has an open reading frame of 1032 residues with a calculated molecular mass of 110,557. The GlcNase has been classified as a member of family GH-2. Sequence alignments identified a group of GlcNase-related protein sequences forming a distinct subclass of family GH-2. When mono-N-acetylated chitotetraose [(GlcN)₃-GlcNAc] was hydrolyzed by the enzyme, the GlcN unit was produced from the nonreducing end together with the transglycosylation products. ¹H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The rate of hydrolysis of the disaccharide, GlcN-GlcNAc, was somewhat lower than that of (GlcN)₂, suggesting that the N-acetyl group of the sugar residue located at (+1) site partly interferes with the catalytic reaction. Based on the time-course of the enzymatic hydrolysis of the completely deacetylated chitotetraose [(GlcN)₄], we obtained the values of binding free energy changes of +7.0, -2.9, -1.8, -0.9, -1.0 and -0.5 kcal/mol corresponding, respectively, to subsites (-2) (-1) (+1) (+2) (+3) (+4). Synergism resulting from mixing the A. orientalis GlcNase with Streptomyces sp. N174 endochitosanase was also observed when chitosan polysaccharide was used as the substrate. To identify the catalytic residue, mutations were introduced into the putative catalytic residues resulting in five mutated enzymes (D469A, D469E, E541D, E541Q and S468N/D469E) which were successfully produced. The four single mutants were devoid of enzymatic activity, indicating that Asp469 and Glu541 are essential for catalysis as predicted from sequence alignment of enzymes belonging to GH-2 family.

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Endo-β-N-acetylglucosaminidases hydrolyze N,N´-diacetylchitobiose linkages of asparagine-linked oligosaccharides. They can also cleave the linkage with suitable agents having hydroxyl groups and transfer the released oligosaccharides to the agents. Thus endo-β-N-acetylglucosaminidases are very useful for synthesis of neoglycoconjugates having asparagine-linked oligosaccharides. On the other hand, the structures of asparagine-linked oligosaccharides are divided into three groups, high-mannose type, hybrid type and complex type. We discovered a novel endo-β-N-acetylglucosaminidase (Endo), named Endo HS. Endo HS can specifically hydrolyze bi-, tri- and tetraantennary complex-type oligosaccharides from glycoproteins. We have investigated the transglycosylation reaction by Endo HS. Endo HS transferred the biantennary complex type oligosaccharide from human transferrin to p-nitrophenyl (PNP)-β-D-Glc and PNP-β-D-Gal. Endo HS was strictly distinct from other enzymes in transferring oligosaccharide to the Gal moiety. The amount of the transglycosylation product increased depending on the concentration of the acceptors. Endo HS also transferred the oligosaccharide to PNP-α-D-Glc, PNP-α-D-Gal, PNP-β-D-Man, PNP-β-D-Xyl, PNP-β-D-GlcNAc and PNP-glycerol. The amount of the transglycosylation product successively increased and became constant and then barely decreased. No apparent difference in the K_m value for human transferrin as an oligosaccharide donor was observed using different acceptors such as PNP-β-D-Glc and PNP-glycerol. Endo HS also transferred the triantennary complex-type oligosaccharide from calf fetuin and the bi-, tri- and tetraantennary complex-type oligosaccharides from human α₁-acid glycoprotein to PNP-β-D-Glc. In addition to glycoproteins, Endo HS transferred biantennary complex-type oligosaccharide from glycopeptides. Furthermore, Endo HS transferred bi- and triantennary complex-type oligosaccharides from glycoasparagines to various monosaccharides, oligosaccharides, sugar alcohols and glycosides. The addition of polar organic solvents was also effective for the transglycosylation efficiency. The results demonstrate that Endo HS is a useful tool for synthesis of neoglycoconjugates having a wide variety of complex-type asparagine-linked oligosaccharides from glycoproteins.

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A method is presented for synthesizing thermo-, acid-stable glucooligosaccharides (TASOG) from sucrose (2.5-4 M) using a dextransucrase prepared from Leuconostoc mesenteroides B-512FMCM. The degree of polymerization (DP) of oligosaccharides synthesized was from 2 to 11. TASOG resisted hydrolysis of its glycosidic linkages at 140°C and pH 6.0 for 1 h. It was stable at pHs ranging from 2 to 4 at 120°C. A method for synthesizing fructo-oligosaccharides (TASOF) with high concentrations of sucrose (1-3 M) by using levansucrase prepared from L. mesenteroides B-1355C was also developed. The DP of oligosaccharides synthesized according to the present method ranged from 2 to over 15. The TASOF was also stable at pHs ranging from 2 to 4 under 120°C. The percentage of TASOF in the reaction digest was 95.7% (excluding monosaccharides; 4.3% was levan). Both oligosaccharides effectively inhibited the formation of insoluble glucan, and the growth and acid production of Streptococcus sobrinus. TASOG and TASOF potentially can be used as sweeteners for food and beverages where thermo- and acid-stable properties are required and as potential inhibitors of dental caries.

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Oxidative polymerization of phenolic glucosides catalyzed by horseradish peroxidase (HRP) was reviewed. Hydroquinone β-glucoside (arbutin, β-Arb) and hydroquinone α-glucoside (α-Arb) were polymerized similarly responding to the addition of H₂O₂. A calculated model reproduced successfully most of the experimental results: An increase of the polymer with a decrease of the monomer and a temporal accumulation of the dimer. The polymers had linkages at the 3´- and 5´-positions of hydroquinone moieties and abundant polymerization degrees of 7-17. Dimers emitted fluorescence and absorbed a wider UV region. Polymers carrying heterogeneous side residues were synthesized through two successive enzyme reactions, glycosylation and subsequent polymerization. A novel glycoside was also synthesized by the coupling of β-Arb and 2,5-dihydroxybenzoic acid followed by spontaneous intramolecluar esterification.

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