Mitsuo Takai

Stabilization of low denaturation temperature collagen from fish by physical cross-linking methods.

Collagen matrices were prepared from atelo salmon collagen (SC). SC has a lower denaturation temperature (19 degrees C) than mammalian collagen. SC matrices were successfully stabilized by ultraviolet irradiation and dehydrothermal treatment, and their optimum conditions were determined. By sponging at 37 degrees C, partial denaturation of the collagen molecules resulted in shrinkage of the matrices.

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.

ESR studies of radicals generated by ultrasonic irradiation of lignin solution. An application of the spin trapping method

Abstract An electron spin resonance (ESR) method combined with a spin trapping reagent was successfully applied to trap and characterize unstable radicals which were generated by ultrasonic irradiation of dimethylsulfoxide (DMSO) solution of hardwood, Fagus crenata lignin. It was found, consequently, that a secondary carbon radical, ∼CH· was trapped as the nitroxide spin adduct when the DMSO solution was subjected to ultrasonic irradiation in the presence of a spin trapping reagent: 2, 4, 6-tri-tert-butylnitrosobenzene (BNB) at 50 °C for 60 minutes under vacuum. This means that the alkyl phenyl ether bonds known as lignin linkage bonds were homolytically scissoned by the ultrasonic irradiation, although the phenoxy radical, Ph-O· as the counter radical of the secondary carbon radical was not trapped by the BNB spin trap. Further, the data showed that the primary carbon radical, ∼OCH2· is trapped by the BNB to form the corresponding spin adduct, indicating that the hydrogen abstraction from the ortho methoxy group in the syringyl and/or guaiacyl moiety is induced by the secondary carbon radical when irradiated. Based on these findings it was concluded that the Ph-O· radicals produced by the homolytic rupture of the alkyl phenyl ether bonds were not trapped by the BNB spin trap. This suggests that large steric hindrances operate between the syringyl with two methoxy moieties and/or guaiacyl with a methoxy moiety at the ortho position, and the BNB molecule bearing two bulky ortho tert-butyl groups.

A method for isolation of milled-wood lignin involving solvent swelling prior to enzyme treatment

SummaryA new lignin isolation method has been developed. Wood and pulp were subjected to ball milling, swelled in an organic solvent, and then treated with a cellulase. The enzyme digestion time could be shortened to 1 day with this method. The lignin obtained has been named Swelled Enzyme Lignin (SEL). Swelling and enzyme digestion conditions and their effects on lignins were investigated. The SELs from wood could be directly washed with water, while those from pulp had to be washed with aqueous acetic acid because they were water soluble. The purification of crude SELs was accomplished by extracting them with dioxane-water, and then precipitating and washing with ethyl ether. Lignin yields were 24–67% based on the total amount of lignin present. The characteristics of the SELs were further investigated by gel-permeation chromatography (GPC), infrared and 13C nuclear magnetic resonance (NMR) spectroscopy.

Homolytic scission of interunitary bonds in lignin induced by ultrasonic irradiation of MWL dissolved in dimethylsulfoxide.

Summary An electron spin resonance (ESR) method combined with a spin trapping technique was applied to trap and characterize unstable radicals which were generated by ultrasonic irradiation of the dimethylsulfoxide (DMSO) solution of a softwood, Yezo Spruce (Picea jezoensis carr.) lignin. It was found that an unstable secondary carbon radical, ~CH • in the solution was trapped as the stable nitroxide spin adduct when the DMSO solution was subjected to ultrasonic irradiation in the presence of a spin trapping reagent: 2,4,6-tri-tert-butylnitrosobenzene (BNB) at 50°C for 30 min. This means that the alkyl phenyl ether bonds, ~CH-O-phenyl, known as interunitary bonds in lignins were homolytically cleaved by the ultrasonic irradiation, although the phenoxy radical Ph-O •, called guaiacoxy radical, i.e. the counter radical of the secondary carbon radical, was not trapped by the BNB spin trap. This suggests that the trapping of the guaiacoxy radical, having a methoxy group in an ortho-position, by the BNB molecule, carrying two bulky butyl groups in the ortho-positions, is sterically hindered.

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.

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.