Takao Uchiyama

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°).

Purification and properties of Arthrobacter ureafaciens inulase II

Abstract 1. 1. Arthrobacter ureafaciens inulase II, which converts inulin to di- d -fructofuranose 1,2′:2,3′ dianhydride (difructose anhydride III) and a small amount of oligosaccharides, was purified about 150-fold in the 43% yield from the cultured liquid of the bacteria by means of ammonium sulfate fractionation, followed by acetone fractionation and Sephadex G-100 gel filtration. A polarimetric measurement was carried out for the estimation of enzymatic activities. 2. 2. The purified enzyme was found to be homogeneous by polyacrylamide disc gel electrophoretic studies in support of an intramolecular transfructosidation reaction of the enzymatic reaction. The enzyme was optimally reactive at pH 6.0 and at 50 °C, and was stable within a broad pH range (pH 4 to 11) and below 50 °C. The activity was strongly inhibited by HgCl2.

Formation of di-d-fructofuranose 1,2′:2,3′ dianhydride from inulin by an extracellular inulase of Arthrobacter ureafaciens

Abstract 1. 1.|A strain of Arthrobacter ureafaciens isolated from soil was shown to produce an extracellular enzyme which degrades inulin to a difructose anhydride and a small amount of oligosaccharides. The products resulting from enzymic hydrolysis are different from those produced by the common inulase (β-2,1-fructan fructanohydrolase, EC 3.2.1.7) of the β-fructofuranosidase type. 2. 2.|Characterization and identification of the difructose anhydride indicated that it corresponds to di- d -fructofuranose 1,2′:2,3′ dianhydride, difructose anhydride III, described by Jackson and McDonald in 1931 ( Bur. Stand. J. Res. , 6 (1931) 709).

Crystal structure of di-β-d-fructofuranose 2′, 1:2,3′-dianhydride

Abstract The crystals of di-β- d -fructofuranose 2′,1:2,3′-dianhydride are monoclinic, space group P 2 1 , with unit-cell dimensions a = 12.8557(14), b = 7.7266(7), c = 7.0322(9)A, β = 97.395(10)°, z = 2. The structure was solved by the direct method, and refined to an R value of 0.046 and an R w value of 0.048 for 2123 observed reflections. The conformations of the furanose rings are 2 E with P = 302.5° and τ m = 39.4° for d -fructose 1, and 3 T 2 with P = 145.7° and τ m = 35.8° for d -fructose 2. The fused, 1,4-dioxane ring has a chair conformation with Cremer-Pople puckering parameters Q = 0.501 A and θ = 8.1°.

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.

Measurement of chiral amino acid discrimination by cyclic oligosaccharides: a direct FAB mass spectrometric approach

The novel cyclic oligosaccharides, permethylated cyclofructans MECF, 1b and 2b, discriminate enantiomers of chiral amino acid ester hydrochlorides.

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.