Mishio Kawamura

Formation of a cycloinulo-oligosaccharide from inulin by an extracellular enzyme of Bacillus circulans OKUMZ 31B

A strain of Bacillus circulans OKUMZ 31B, isolated from soil, has been shown to produce an extracellular enzyme that converts inulin into cycloinulo-oligosaccharides. The main product was identified as cycloinulo-hexaose. The enzyme is arbitrarily designated as cycloinulo-oligosaccharide fructanotransferase.

The crystal structure of cycloinulohexaose produced from inulin by cycloinulo-oligosaccharide fructanotransferase

Abstract The crystal of cycloinulohexaose trihydrate, C 36 H 60 O 30 ·3H 2 O, is trigonal, space group R 3, with unit-cell dimensions a = 24.688 (17), c = 6.477 (3) A for a hexagonal cell, Z = 3. The molecule, which consists of six (2 → 1)-linked β- d -fructofuranose residues, has C 3 symmetry. The conformations of two d -fructofuranosyl moieties in an asymmetric unit are 4 T 3 with P = 348.1° and τ m = 38.9° for Fl, and 4 T 3 with P = 350.5° and τ m = 41.2° for F2. The conformations of OCH 2 CO in the 18-crown-6-ring are gauche - for O-1−C-1−C-2−O-1′ (+ 52.3°) and trans for O-1−C-1′−C-2′−O-1 (+ 163.4°).

Enzymatic synthesis of stable, odorless, and powdered furanone glucosides by sucrose phosphorylase.

Sucrose phosphorylase from Leuconostoc mesenteroides catalyzed transglucosylation from sucrose to 4-hydroxy-3(2H)-furanone derivatives. When 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone or 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (EHMF) were used as acceptors, their transfer ratios were more than 45%. In the case of glucosylation of HDMF, the major transfer product was identified as 2,5-dimethyl-3(2H)-furanone 4-O-α-D-glucopyranoside (DMF-G). In the case of glucosylation of EHMF, two major transfer products were obtained, and their structures were identified as 2-ethyl-5-methyl-3(2H)-furanone 4-O-α-D-glucopyranoside (2E5MF-G) and 5-ethyl-2-methyl-3(2H)-furanone 4-O-α-D-glucopyranoside (5E2MF-G) on the bases of spectrometric investigations. These glucosides were more stable than each aglycone. The glucosylated HDMF, DMF-G, was an odorless chemical, on the other hand, HDMF had a pineapple flavor. The glucosylated EHMF (EMF-G) were white odorless powders, though aglycone EHMF was a pale yellow syrup like a caramel with an intense sweet odor. Although DMF-G and EMF-G showed little radical-scavenging activity, hydrolyzates of these glucosides by an intestinal acetone powder from pigs had antioxidative activity as well as their aglycones. It was suggested that these glucosides improved some physical properties and may become prodrugs by glucosylation.

Purification and some properties of cycloinulo-oligosaccharide fructanotransferase from Bacillus circulans OKUMZ 31B☆

Abstract Cycloinulo-oligosaccharide fructanotransferase was purified from the cultured medium of Bacillus circulans OKUMZ 31B, to electrophoretic homogeneity, by anion-exchange column chromatography on DEAE-Toyopearl 650M, hydrophobic column chromatography on Butyl-Toyopearl 650M, gel-filtration column chromatography on Sephacryl S-2000HR and anion-exchenge column chromatography on SuperQ-Toyopearl 650M. The enzyme has a molecular weight of 132 000 and a pI of 4.1. The enzyme was most active at pH 7.5 and 40°C, and was stable at pH 6.0–9.0 and below 40°C. The enzyme catalyses the conversion of inulin into cycloinulohexaose and cycloinuloheptaose in the ratio of ca. 4:1, and a small amount of cycloinulo-octaose. The enzyme has an isoform which may be a proteolyticaly modified species of the CFTase because of its reduced molecular weight, 126 000.

Synthesis of a Series of Fructooligosaccharides with Sucrose and Cycloinulohexaose Extending over Ten Degrees of Polymerization Using Cycloinulooligosaccharide Fructanotransferase from Bacillus circulans OKUMZ 31B

Prolonged incubation with sucrose as an acceptor and cycloinulohexaose as a donor at a high concentration of cycloinulooligosaccharide fructanotransferase afforded a series of fructooligosaccharides. Their chain-length distribution extended over 10 degrees of polymerization when the donor concentration was increased to 120 mM. Increasing the acceptor concentration proved effective in improving the yield of inulin-type oligosaccharides because hydrolysis was suppressed.

Synthesis of methyl 6-O-β-inulotriosyl-α-d-glucopyranoside by intermolecular transglycosylation reaction of cycloinulo-oligosaccharide fructanotransferase

Abstract Incubation of cycloinulohexaose and methyl α- d -glucopyranoside in the presence of cycloinulo-oligosaccharide fructanotransferase gave some hetero-oligosaccharides. The main product was a tetrasaccharide whose sugar composition was methyl α- d -glucopyranoside- d -fructose in a ratio 1:3. This oligosaccharide was isolated from the reaction mixture by charcoal-column chromatography and was identified as methyl O -β- d -fructofuranosyl-(2 → 1)- O -β- d -fructofuranosyl-(2 → 1)- O -β- d -fructofuranosyl-(2 → 6)-α- d -glucopyranoside (methyl 6- O -β-inulotriosyl-α- d -glucopyranoside), by two-dimensional NMR spectroscopy.

A permethylated cyclic fructo-oligosaccharide host that can bind cations in solution

Permethylated cycloinulohexose 1 acts, like an 18-crown-6 derivative with alkali and alkaline earth cations in organic solvents, where the cation is not bound by the central 18-crown-6 moiety, but by the upper OMe-3 groups of 1.

Effects of various saccharides on cycloinulo-oligosaccharide fructanotransferase reaction: production of β-inulotriosyl-α-d-mannopyranoside and 1-O-β-inulotriosyl-α-l-sorbopyranose

The effects of various saccharides on the reaction of cycloinulo-oligosaccharide fructanotransferase with cycloinu-lohexaose were examined. In addition to beta-D-fructofuranosides and methyl alpha-D-glucopyranoside, D-mannose and L-sorbose were found to be effective acceptors in the reactions, and they enhanced the hydrolytic activity as effectively as methyl alpha-D-glucopyranoside. Hetero-tetrasaccharides were isolated as the major transfer products from both reaction mixtures. The isolates were identified by NMR spectroscopy as beta-inulotriosyl-alpha-D-mannopyranoside and 1-O-beta-inulotriosyl-alpha-L-sorbopyranose. Methyl beta-D-glucopyranoside was slightly effective and methyl alpha-D-mannopyranoside was not effective at all as the acceptor, but these saccharides strongly enhanced the hydrolytic activity. D-Glucosamine inhibited the enzyme activity.