Tomoki Erata

Effects of endogenous endo-β-1,4-glucanase on cellulose biosynthesis in Acetobacter xylinum ATCC23769

Endo-beta-1,4-glucanase (CMCax; EC 3.2.1.4) from Acetobacter xylinum ATCC23769 was expressed as a 6 x His-tagged fusion protein in Escherichia coli. The optimal temperature, pH, K(m) and V(max) of the purified His-tagged CMCax toward carboxymethyl cellulose were 50 degrees C, 4.5, 20 mg/ml and 37.2 microM/min, respectively. The number of recognition residues of cello-oligosaccharide by this enzyme were five (cellopentaose) or longer, and the stereochemical course of hydrolysis was of the inverting type. Addition of a small amount (1.5 mg/l) of His-tagged CMCax into a culture medium enhanced cellulose production 1.2-fold. CMCax overproduction in A. xylinum also enhanced the yield of cellulose production. Transmission electron microscopic analysis revealed that the cellulose ribbons secreted from the CMCax overproducing strain were dispersed compared with those from the wild type strain in the same manner as by carboxymethyl cellulose addition. These results could suggest that CMCax from A. xylinum influences in cellulose ribbon assembly, which is considered to be a rate-determined process in cellulose synthesis.

The production of a new water-soluble polysaccharide by Acetobacter xylinum NCI 1005 and its structural analysis by NMR spectroscopy

A new water-soluble polysaccharide (WSP) was isolated from a culture of Acetobacter xylinum NCI 1005 grown on sucrose. The structure of the WSP was analysed by nuclear magnetic resonance spectroscopy and determined to be a beta-(2-->6)-linked polyfructan, which is structurally different from the polymer synthesized from glucose instead of sucrose by the same strain. The discovery of this new polysaccharide has revealed that the bacterium is able to synthesize two different kinds of water-soluble polysaccharides.

Structural analyses of new tri- and tetrasaccharides produced from disaccharides by transglycosylation of purified Trichoderma viride beta-glucosidase.

A new β-glucosidase was partially purified from Trichoderma viride cellulase. This β-glucosidase catalyzed a transglycosylation reaction of cellobiose to give β-D-Glc-(1→6)-β-D-Glc-(1→4)-D-Glc (1, yield: 18.8%) and β-D-Glc-(1→6)-β-D-Glc-(1→6)-β-D-Glc-(1→4)-D-Glc (2, 3.7%), regioselectively. Furthermore, the enzyme regioselectively converted laminaribiose and gentiobiose into β-D-Glc-(1→6)-β-D-Glc-(1→3)-D-Glc (3, 15.3%) and β-D-Glc-(1→6)-β-D-Glc-(1→6)-D-Glc (4, 20.2%), respectively. The structures (1–4) of the products were determined by 1H and 13C NMR spectroscopies. This high regio- and stereoselectively of the β-glucosidase could be applied for oligosaccharide synthesis.

Cross-polarization/magic-angle spinning 13C nuclear magnetic resonance study of cellulose I–ethylenediamine complex

Complete assignments of the cross-polarization/magic-angle spinning (CP/MAS) 13C nuclear magnetic resonance (NMR) spectrum of the cellulose I-ethylenediamine (EDA) complex, which is the intermediate of the reaction from cellulose I to cellulose III(I), were performed. In this paper, we used the 13C-enriched cellulose that was biosynthesized by Acetobacter xylinum ATCC10245 strain from culture medium containing D-(2-13C), D-(3-13C), or D-(5-13C)glucose as a carbon source. After conversion into cellulose I-EDA complex by sufficient EDA treatment, the CP/MAS 13C NMR spectra of the 13C-enriched cellulose I-EDA complexes were measured. As a result, 13C resonance lines of each carbon of the cellulose moiety in the complex appeared as a singlet, suggesting that all glucose residues of the complex are magnetically equivalent. The difference in chemical shifts for each carbon between cellulose I-EDA and cellulose I(alpha), I(beta), and III(I), respectively, suggests that the conformation of the cellulose chains for cellulose I-EDA differs from that for cellulose I(alpha), I(beta), and III(I). In addition, fitting analysis of the 13C spectrum of Valonia cellulose I-EDA complex revealed that the complex contains one EDA molecule per two glucose residues in the cellulose chain.

Solid 7Li-NMR and in situ XRD studies of the insertion reaction of lithium with tin oxide and tin-based amorphous composite oxide

The lithium insertion reactions with tin (II) oxide (SnO) and tin-based composite oxide (abbreviated as TBCO) are studied by solid 7Li-NMR Knight shift, T1 and T1ρ relaxation rate, TEM and in situ XRD methods. By the insertion reaction for SnO, the lithium oxide and β-tin are produced first at Li/Sn = 2; at Li/Sn = 3 to 6 the products are not simple and a mixture of LiSn2, LiSn, Li5Sn2 and Li7Sn2 alloys is detected during the insertion. For the TBCO, which is revealed as amorphous, mainly constituted by randomly distributed very short-range (order of 10-9 m) regions by TEM observation, it is found that electrochemically inserted lithium forms Li2O and produces metallic tin (Sn) in the first step (Li/Sn 2). During the second step, the Li/Sn ratio of formed lithium-tin alloy is kept at almost 4. By the analyses of 7Li NMR Knight shifts, line shape and in situ XRD, the lithium-inserted TBCOs are characterized as almost amorphous and mixtures of highly ionic components.

Transglycosylation of cellobiose by partially purified Trichoderma viride cellulase

A commercial cellulase from Trichoderma viride was fractionated into three fractions, F1, F2, and F3, in order to investigate transglycosylation activities. Among these fractions, F3, which demonstrated highly hydrolytic activity toward p-nitrophenyl beta-D-glucopyranoside and Avicel, most effectively catalyzed the transglycosylation of cellobiose and converted cellobiose into beta-Glc-(1-->6)-beta-glc-(1-->4)-Glc and beta-Glc-(1-->6)-beta-Glc-(1-->6)-beta-Glc(1-->4)-Glc. The F3 fraction contained the enzyme to catalyze beta-glucosyl transfer toward only the C-6 position of the sugar acceptor, and thus it is expected to be of use for syntheses of functional oligosaccharides.

CPMAS 13C NMR and X-ray studies of cellooligosaccharide acetates as a model for cellulose triacetate

A series of crystalline oligomers from α-D-cellobiose octaacetate through α-D-cellohexaose eicosaacetate were prepared by homogeneous acetylation of the corresponding cellooligosaccharides and characterized by cross-polarization and magic angle sample spinning (CPMAS) carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy and X-ray analysis to obtain the structural models of cellulose triacetate (CTA) in the solid state. Progressing toward the hexamer, the NMR spectral features of the oligomers, in comparison with two allomorphs of CTA I and CTA II, gradually approached those of CTA I. Specifically, chemical shifts of both the hexamer and pentamer were in agreement with those of CTA I. In addition, X-ray diffraction patterns of the oligomers established that the crystalline pentamer and hexamer had a CTA I lattice despite recrystallization from ethylacetate-n-hexane. Therefore, we conclude that the pentamer and hexamer are useful models for the CTA I structure.

Effect of far-infrared light irradiation on water as observed by X-ray diffraction measurements

X-ray diffraction measurements were made on water irradiated with far-infrared (FIR) light. It was found that the X-ray diffraction intensity at a 2θ angle of 30° increased by about 2 times with the irradiation. The increase in the X-ray diffraction intensity was interpreted in terms of the destruction of water clusters.

Evidence of multisite exchange in AgI–AgPO3 glasses: 109Ag NMR hole-burning spectra

Abstract A narrow 109 Ag NMR spectrum of superionic conductor glass containing AgI exhibits strong broadening at low temperature or at low concentration of AgI. This broadening of the spectrum is attributed to the inhomogeneity of the local structure around Ag + ions in the glass. The temperature and composition dependence of the spectra can be well explained by a multisite exchange (MSE) model. The existence of the local inhomogeneity and the exchange of Ag + ions among them can be detected by site-selective hole-burning NMR technique. In this paper, the first measurement of the NMR hole-burning spectra of a superionic conductor glass of AgI–AgPO 3 is reported. A clear hole is observed below −20 °C, which diffuses to the higher-frequency region corresponding to AgI-rich sites, where the exchange rate is faster. This technique is very useful to investigate site exchange of ions in disordered systems.

Isolation of a thermotolerant bacterium producing medium-chain-length polyhydroxyalkanoate

Aims: The aim of this study was to isolate a thermotolerant micro‐organism that produces polyhydroxyalkanoates (PHAs) composed of medium‐chain‐length (mcl) HA units from a biodiesel fuel (BDF) by‐product as a carbon source.