Tomoyuki Nishimoto

Cloning and sequencing of trehalose synthase gene from Pimelobacter sp. R48.

The gene encoding trehalose synthase (catalyzing the conversion of maltose into alpha, alpha-trehalose by intramolecular transglucosylation) was cloned from Pimelobacter sp. R48. Sequence analysis revealed a 1719-bp synthase gene and a 573-residue amino-acid sequence. The 220 N-terminal residues were homologous to those of maltases from Saccharomyces carlsbergensis and Aedes aegypti.

CLONING AND SEQUENCING OF TREHALOSE SYNTHASE GENE FROM THERMUS AQUATICUS ATCC33923

The gene encoding trehalose synthase (catalyzing the conversion of maltose into alpha, alpha-trehalose by transglucosylation) was cloned from Thermus aquaticus ATCC33923. Sequence analysis revealed a 2892 bp synthase gene and a 963 residue amino-acid sequence. The 547 N-terminal residues were homologous to the full-length synthase from Pimelobacter sp. R48 (53.8% identity).

Purification and Characterization of a Thermostable Trehalose Synthase from Thermus aquaticus

Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80-82% at 30-40°C. The activity was inhibited by Cu(2+) , Hg(2+), Zn(2+), and Tris.

Purification and characterization of glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide in Bacillus globisporus C11.

Glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide (CTS; cyclo {→6}-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl- (1→)) from α-1,4-glucan were purified from Bacillus globisporus C11. The former was a 1,6-α-glucosyltransferase (6GT) catalyzing the α-1,6-transglucosylation of one glucosyl residue to the nonreducing end of maltooligosaccharides (MOS) to produce α-isomaltosyl-MOS from MOS. The latter was an isomaltosyl transferase (IMT) catalyzing α-1,3-, α-1,4-, and α,β-1,1-intermolecular transglycosylation of isomaltosyl residues. When IMT catalyzed α-1,3-transglycosylation, α-isomaltosyl-(1→3)-α-isomaltosyl-MOS was produced from α-isomaltosyl-MOS. In addition, IMT catalyzed cyclization, and produced CTS from α-isomaltosyl-(1→3)-α-isomaltosyl-MOS by intramolecular transglycosylation. Therefore, the mechanism of CTS synthesis from MOS by the two enzymes seemed to follow three steps: 1) MOS→α-isomaltosyl-MOS (by 6GT), 2) α-isomaltosyl-MOS→α-isomaltosyl-(1→3)-α- isomaltosyl-MOS (by IMT), and 3) α-isomaltosyl-(1→3)-α-isomaltosyl-MOS→CTS +MOS (by IMT). The molecular mass of 6GT was estimated to be 137 kDa by SDS-PAGE. The optimum pH and temperature for 6GT were pH 6.0 and 45°C, respectively. This enzyme was stable at from pH 5.5 to 10 and on being heated to 40°C for 60 min. 6GT was strongly activated and stabilized by various divalent cations. The molecular mass of IMT was estimated to be 102 kDa by SDS-PAGE. The optimum pH and temperature for IMT were pH 6.0 and 50°C, respectively. This enzyme was stable at from pH 4.5 to 9.0 and on being heated to 40°C for 60 min. Divalent cations had no effect on the stability or activity of this enzyme.

Enzymatic synthesis of kojioligosaccharides using kojibiose phosphorylase

We have attempted to synthesize kojioligosaccharides (oligosaccharides having the alpha-1,2 glycosidic linkage at the nonreducing end) using two methods. In the first, mixtures of various proportions of glucose and beta-D-glucose-1-phosphate (beta-G1P) were allowed to react in the presence of kojibiose phosphorylase (KPase). In the second, maltose was allowed to react with KPase and maltose phosphorylase (MPase) simultaneously. In the former method, kojioligosaccharides having only the alpha-1,2 glucosidic linkage were synthesized and the average degree of polymerization (D.P.) of oligosaccharides increased with decreasing proportions of glucose. In the second method, kojioligosaccharides were obtained at approximately 70% yields under optimum conditions. 4-alpha-D-Kojibiosyl-glucose, kojitriose and kojitetraose, the principal kojioligosaccharides synthesized, were not hydrolyzed by salivary amylase, artificial gastric juice, pancreatic amylase, or small intestinal enzymes.

Production of cyclic tetrasaccharide from starch using a novel enzyme system from Bacillus globisporus C11.

Production of cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS, cyclic tetrasaccharide) from starch was attempted using 1,6-alpha-glucosyltransferase (6GT) and 1,3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus C11. The optimal conditions for production from partially hydrolyzed starch were as follows: substrate concentration, 3%; pH 6-7; temperature, 30 degrees C; 6GT, 1 unit/g-dry solid (DS); IMT, 10 units/g-DS. The production of CTS was demonstrated and 544 g of CTS hydrate crystal powders were obtained from 3500 g of partially hydrolyzed starch. Two major by-products were also isolated from the reaction mixture and identified as the branched derivatives of CTSs, 4-O-alpha-D-glucopyranosyl-CTS and 3-O-alpha-isomaltosyl-CTS.

6-α-Glucosyltransferase and 3-α-isomaltosyltransferase from Bacillus globisporus N75

Abstract A bacterial strain, Bacillus globisporus N75, produced two glycosyltransferases, 6-α-glucosyltransferase (6GT) and 3-α-isomaltosyltransferase (IMT), jointly catalyzing formation of cyclo →6)-α- d -Glc p -(1→3)-α- d -Glc p -(1→6)-α- d -Glc p -(1→3)-α- d -Glc p -(1→ (CTS) from α-1,4-glucan. The N75 enzymes produced CTS from dextrin in a 43.8% yield at the reaction temperature of 50°C, which was 10°C higher than a critical temperature of CTS-forming by the enzymes from B. globisporus C11. The optimum temperatures for 6GT and IMT reactions were 55°C and 50°C, respectively. The thermal stability of both enzymes was 45°C under the condition at pH 6.0 for 60 min. The genes for 6GT and IMT were cloned from the genomic DNA of N75. The amino acid sequences deduced from the 6GT and IMT genes showed 82% and 85% identities, respectively, to the sequences of the enzymes from C11. CTS yield was decreased by high concentrations of the substrate. It was found that the reaction yield was improved by adding cyclomaltodextrin glucanotransferase (CGTase). We demonstrated mass-production of CTS from starch by using the N75 enzymes and CGTase.

Enzymatic synthesis of novel oligosaccharides from l-sorbose, maltose, and sucrose using kojibiose phosphorylase

Glucosyl-L-sorbose, -maltose, and -sucrose were synthesized using kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 with beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor. One disaccharide and two trisaccharides thus synthesized were isolated by Toyopearl HW-40S column chromatography. The results of KPase digestion, methylation analysis, and 13C-NMR studies indicated that these oligosaccharides were alpha-D-glucopyranosyl-(1-->5)-alpha-L-sorbopyranose, alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose (4-alpha-D-kojibiosyl-glucose), and alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside, which are all novel oligosaccharides. Glucosyl-L-sorbose was partially hydrolyzed to glucose and L-sorbose by alpha-glucosidases, while glucosyl-sucrose and glucosyl-maltose were not hydrolyzed by glucoamylase, alpha-glucosidases, or CGTase.

Action of a Thermostable Trehalose Synthase from Thermus aquaticus on Sucrose

A thermostable trehalose synthase from Thermus aquaticus ATCC 33923, which catalyzes the interconversion between maltose and trehalose by intramolecular transglucosylation, converted sucrose into trehalulose (1-O-α-d-glucopyranosyl-d-fructose). The trehalulose-forming activity of the enzyme was very low compared with that of maltose and trehalose. Kinetic studies showed that sucrose competitively inhibited the interconversion activity between maltose and trehalose. Consequently, these three substrates, maltose, trehalose, and sucrose, are thought to bind the same active site of trehalose synthase.

Structural and mutational analysis of substrate recognition in kojibiose phosphorylase

Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc‐α1,4‐Glc), kojibiose (Glc‐α1,2‐Glc), trehalose (Glc‐α1,α1,‐Glc), and nigerose (Glc‐α1,3‐Glc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of α‐glucosyl oligosaccharides. Here, we determined the crystal structures of kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, Trp391, Glu392, and Thr417, recognize kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that Trp391 and Glu392, especially the latter, are required for the kojibiose activity.