Akira Kai

Analysis of the biosynthetic process of cellulose and curdlan using 13C-labeled glucoses

Abstract In order to elucidate the biosynthetic process of cellulose and curdlan, 13C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing d -(1-13C)glucose, d -(2-13C)glucose, d -(4-13C)glucose, or d -(6-13C)glucose as the carbon source, and their structures were determined by 13C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.

Influence of substituent of direct dye having bisphenylenebis(azo) skeletal structure on structure of nascent cellulose produced by Acetobacter xylinum [I]: different influence of Direct Red 28, Blue 1 and 15 on nascent structure

The difference of influence of a certain kind of direct dye on the structure of nascent microbial cellulose was examined, with Direct Red 28 have a biphenylenebis(azo) skeletal structure; Direct Blue 1 having two hydroxyl, two methoxy and two sulfonate groups more than Direct Red 28; and Direct Blue 15 whose sulfonate groups position are different compared to Direct Blue 1. It became clear that the product in the presence of a direct dye (in particular, Direct Red 28) has the structure in which the dye molecule is included between the monolayer in the cellulose sheets corresponding to the (110) plane of microbial cellulose. On the other hand, the structure of the product in the presence of Direct Blue 1 and 15 contains conceived cellulose II structure which occurred due to be removal of dye during the rinsing process as a result of larger hydrophilicity than its affinity toward cellulose. Solid state 13C NMR and deuteration-IR measurements showed that the product in the presence of direct dye is in a noncrystalline state, although X-ray measurements indicated that they are in a crystalline state. These results support the inclusion of a dye between the (110) planes. Solid state 13C NMR and deuteration-IR reveal that the crystal structure of cellulose regenerated from the product in the presence of Direct Red 28 is similar to cellulose IVI, while that from each Direct Blue 1 and 15 product is cellulose II. The difference of the influence of the former and the latter on the nascent cellulose seemed to be caused mainly by the number of sulfonate groups, although the influence of hydroxyl and methoxy groups is not clear at present.

Biosynthesis of curdlan from culture media containing 13C-labeled glucose as the carbon source

13C-Labeled curdlans were biosynthesized by Agrobacterium sp. (ATCC 31749) from culture media containing D-(1-13C)glucose, D-(6-13C)glucose, or D-(2-13C)glucose as the carbon source, and their structures were analyzed by 13C NMR spectroscopy. The labeling was mainly found in the original position, that is, C-1, C-6, or C-2, indicating direct polymerization of introduced glucose. In addition, C-3 in curdlan obtained from D-(1-13C)glucose, C-1 in curdlan obtained from D-(6-13C)glucose, and C-1 and C-3 in curdlan obtained from D-(2-13)glucose were labeled. From analysis of this labeling, the biosynthesis of curdlan was interpreted as involving five routes: (1) direct synthesis from glucose; (2) rearrangement (1-13C-->3-13C); and (3) isomerization (6-13C-->1-13C) of cleaved trioses by the Embden-Meyerhof pathway, followed by neogenesis of glucose and formation of curdlan; (4) from fructose 6-phosphate formed in the pentose cycle (2-13C-->1-13C, 3-13C); and (5) neogenesis of glucose from fragments produced in various pathways of glycolysis. The 13C-labeling at C-6 and C-2 in the starting glucoses is well preserved in the C-6 carbon and the C-1 to C-3 carbons, respectively, in the curdlan produced.

C.p./m.a.s. 13C n.m.r. study on microbial cellulose-fluorescent brightener complexes

Abstract The structures of microbial cellulose-fluorescent brightener complexes, produced from the Acetobacter culture in the presence of a fluorescent brightener, and the cellulose samples regenerated from them have been examined by cross-polarization/magic-angle spinning (c.p./m.a.s.) 13C n.m.r. spectroscopy. C4 and C6 resonance lines for the cellulose components of the complexes appear at about 84.0 and 63.0 ppm, respectively, with the disappearance of their downfield crystalline components. Since the chemical shifts of the lines are in good accord with those of the non-crystalline component of native cellulose, it is concluded that the cellulose component of the complex is in the non-crystalline state. 13C spin-lattice relaxation time (T1C) measurements have also confirmed that the T1C values of the dried complexes are of the same order as those for the non-crystalline component of native cellulose. In contrast, the T1C values of the non-dried complex are much shorter than those of the dried complexes, indicating a much enhanced molecular mobility in the non-dried complex. On the other hand, when the non-dried complex is subjected to dye extraction, C4 and C6 resonance lines assignable to the crystalline component can be observed in the c.p./m.a.s. spectrum. Moreover, selective measurements of the spectrum of the crystalline component have revealed that cellulose Iβ is preferentially grown from the non-dried complex by dye extraction. In the case of the dried complexes, only very small amounts of cellulose I crystals are regenerated, possibly as a result of the formation of tight hydrogen bonds in the complex.

Biosynthesis of Cellulose From Culture Media Containing 13C-Labeled Glucose as a Carbon Source

Abstract 13C-Labeled celluloses were biosynthesized by Acetobacter xylinum (IFO 13693) from culture media containing D-[1-13C]glucose, D-[6-13C]glucose, or D-[2-13C]glu-cose as a carbon source with or without addition of ethanol, and their structures were analyzed by 13C NMR spectroscopy. The labeling was mainly found in the original position, that is C-1, C-6 or C-2, in cellulose obtained from D-[1-13C]glucose, D-[6-13C]-glucose or D-[2-13C]glucose, respectively, indicating direct polymerization of introduced glucoses, especially with addition of ethanol in culture medium. Furthermore, C-1 carbons in cellulose obtained from D-[6-13C]glucose, and C-1, C-3 and C-5 carbons in cellulose obtained from D-[2-13C]glucose were labeled. From the analysis of labeling, the mechanism of biosynthesis of cellulose was explained by (1) direct synthesis from glucose, (2) isomerization and rearrangement of trioses formed in the Embden-Meyerhof pathway, Entner-Doudoroff pathway, or pentose cycle, followed by neogenesis of ...

Influence of substitution of direct dye having biphenylenebis(azo) skeletal structure on nascent cellulose produced by acetobacter xylinum [II]

The influence of Direct Blue 14 and 53 dyes, both having biphenylenebis(azo) skeletal structure but different sulfonate groups substitution on the structure of the nascent microbial cellulose was examined. The product obtained from the Acetobacter culture in the presence of each dye is a characteristic dye–cellulose complex, and the dye molecule is included between the cellulose sheets in the complex corresponding to the (110) plane of microbial cellulose. Due to the inclusion of dyes between the cellulose sheets through hydrogen bonding or van der Waals forces, the hydrogen bonding between cellulose chains of microbial cellulose is hindered. The different position of sulfonate groups has no major influence on the two products except on the uniplanar orientation of the product. Celluloses regenerated from both products are cellulose II, but their fine structures are different from each other.

Biosynthesis of hetero-polysaccharides by Pestalotiopsis microspora from various monosaccharides as carbon source

Abstract Pestalotiopsis microspora was cultured in media containing various monosaccharides as a carbon source. It was found that P. microspora metabolizes various monosaccharides and the composition of polysaccharides depends strongly on the monosaccharides used for carbon source. When D -glucose, D -mannose or D -galactose were used as the carbon source, hetero-polysaccharides of similar compositions containing a considerable amount of D -mannose units beside D -glucose units were formed. With D -xylose or N-acetyl- D -glucosamine as the carbon source, hetero-polysaccharides containing large amounts of D -mannose and D -galactose units beside D -glucose units were produced. Cultures with L -arabinose as the carbon source produced almost pure glucan, while with L -rhamnose, the yield and the composition of hetero-polysaccharide is the same as that obtained from the culture with D -glucose as the carbon source. The mechanism of biosynthesis of these hetero-polysaccharides is briefly discussed.

Control of the crystal structure of microbial cellulose during nascent stage

The structure of the product, from an Acetobacter culture in the presence of Fluorescent Brightener, Direct Red 28, and Direct Blue 1, 14, 15 and 53, characterized by an X-ray diffractormeter is a crystalline complex. On the other hand, solid-state 13C-NMR spectroscopy reveals that the product is noncrystalline. However, the X-ray result of the product sample suggests that the dye molecule is included in the form of a monolayer between the cellulose sheets in the complex corresponding to the (11¯0) plane of microbial cellulose. But the celluloses regenerated from the Fluorescent Brightener product, the Direct Red 28 product, and the rest of the dye products are celluloses I, IV, and II, respectively. More specifically, the 13C-NMR spectra revealed that the crystal types of cellulose from the Fluorescent Brightener and Direct Red 28 products are Iβ and IVI, respectively. Thus, the crystal structure of the product and the regenerated cellulose depends mainly on the position and number of the sulfonate groups in a direct dye and the interactions of the dye with the noncrystalline microbial cellulose in the nascent stage. The conformation and arrangement of the nascent cellulose chain changes when a direct dye adheres to it.

Biosynthesis of 13C-labeled branched polysaccharides by pestalotiopsis from 13C-labeled glucoses and the mechanism of formation

Abstract Biosynthesis of branched glucan by Pestalotiopsis from media containing D-(1- 13 C)glucose, D-(2- 13 C)glucose, D-(4- 13 C)glucose, D-(6- 13 C)glucose or a mixture of D-(1- 13 C)glucose and D-(2- 13 C)glucose was carried out to elucidate biosynthetic mechanism of branched polysaccharides. 13 C NMR spectra of the labeled polysaccharides were determined and assigned. Analysis of 13 C NMR spectra of glucitol acetates obtained from hydrolysates of the labeled branched polysaccharides indicated that transfer of labeling from C-1 to C-3 and C-6 carbons, from C-2 to C-1, C-3 and C-5 carbons, and from C-6 to C-1 carbon. From the results the percentages of routes via which the polysaccharide is biosynthesized are estimated. They show that the biosynthesis of the polysaccharide via the Embden-Meyerhof pathway and that from lipids and proteins are more active, and the pentose cycle is less active, than in the biosynthesis of cellulose and curdlan. As for the results, labeling at C-6 carbon in the branched polysaccharide cultured from D-(6- 13 C)glucose was low, compared to that of cellulose and curdlan.