Fumiaki Nakatsubo

Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium

The lignin-degrading basidiomycete Phanerochaete chrysosporium synthesizes veratryl alcohol (3,4-dimethoxybenzyl alcohol) via phenylalanine, 3,4-dimethoxycinnamyl alcohol and veratrylglycerol. Study of the conversion of 3,4-dimethoxycinnamyl alcohol to veratrylglycerol and veratryl alcohol showed is to be (a) catalyzed by a secondary metabolic system, (b) markedly suppressed by culture agitation, and (c) strongly inhibited by l-glutamate. The amount of veratryl alcohol synthesized de novo was positively correlated with the O2 concentration after primary growth. Other work has shown that the cinnamyl alcohol terminal residue in a lignin substructure model compound is degraded via arylglycerol and benzyl alcohol structures in ligninolytic cultures of P. chrysosporium, and that the ligninolytic system exhibits traits (a)-(c) above. Ligninolytic activity is also strongly and positively correlated with O2 concentration. The results here suggest, therefore, that the actual biosynthetic secondary metabolic product is 3,4-dimethoxycinnamyl alcohol, but that this is degraded by the ligninolytic system to veratryl alcohol via veratrylglycerol. Veratryl alcohol is only slowly metabolized by the fungus, and accumulates.

Property enhancement of optically transparent bionanofiber composites by acetylation

The authors studied acetylation of bacterial cellulose (BC) nanofibers to widen the applications of BC nanocomposites in optoelectronic devices. The slight acetylation of BC nanofibers significantly reduces the hygroscopicity of BC nanocomposites, while maintaining their high optical transparency and thermal stability. Furthermore, the degradation in optical transparency at elevated temperature (200°C) was significantly reduced by acetylation treatment. Therefore, the acetylation of bionanofibers has an extraordinary potential as treatment for property enhancement of bionanofiber composites.

Synthesis of Guaiacylglycerol-β-guaiacyl Ether

Guaiacylglycerol-ß-guaiacyl ether (i), the model compound of arylglycerol-ß-aryl ether structure in lignin was synthesized in high yield through five reaction steps from vanillin. The key step of this synthetic method was the condensation reaction between ethyl 2-methoxyphenoxy acetate (4) and benzyl vanillin (5). At this step, lithium diisopropyl amide was used as the basea and /?-hydroxy ester (6) was obtained in 95 % yield as an oily substance consisted of two isomers, from which only erythro isomer was obtained as crystal in 51 % yield. The residual oily substance was converted to its carbamate (7) and crystallized in 70% yield. The crystalline /Miydroxy ester (6) and the carbamate (7) were then converted to the final compound (i) by the lithium aluminum hydride reduction and subsequent hydrogenation with Pd-C. The overall yield of the guaiacylglycerol-/?guaiacyl ether (i) from benzyl vanillin (5) was about 72%.

Stoichiometric studies of tannin-protein co-precipitation

Co-precipitation of a series of galloylglucoses (hydrolysable tannins) with bovine serum albumin (BSA) was studied stoichiometrically by analysing both galloylglucoses and BSA in the precipitates using HPLC. BSA-precipitating ability increased mainly with an increase in the number of galloyl groups in a galloylglucose molecule but was also affected by the position of the galloyl group (penta- > tetra- > 2,3,6-tri- > 2,3,4-tri- >> di- >> monogalloylglucose). The precipitated BSA increased linearly with an increase in the number of galloyl groups bound to a BSA molecule. BSA-precipitating abilities of the galloylglucoses were closely related to their relative affinities for BSA. These results suggest a two-stage mechanism: initial complexation of galloylglucose with BSA and subsequent precipitation, as a mechanism of the co-precipitation.

Metabolism of a phenylcoumaran substructure lignin model compound in ligninolytic cultures of Phanerochaete chrysosporium

The degradation of the phenylcoumaran substructure model compound methyl dehydrodiconiferyl alcohol by the white-rot wood decay fungus Phanerochaete chrysosporium was investigated using culture conditions optimized for lignin oxidation. Initial attack was in the cinnamyl alcohol side chain, which was oxidized to a glycerol structure. This was subsequently converted by loss of the two terminal carbon atoms, Cβ′ and Cγ′, to yield a Cα′-aldehyde structure, which was further oxidized to the Cα′-acid compound. The next detected intermediate, a phenylcoumarone, was produced by double bond formation between Cα and Cβ, and oxidation of the Cγ-alcohol to an aldehyde group. Further oxidation of Cγ to an acid yielded the next intermediate. The final identified degradation product was veratric acid. No products from the 5-substituted aromatic ring, and no phenolic products, were found. The initial glycerol-containing intermediate was a mixture of the threo and erythro forms, and no optical activity could be found, suggesting that its formation might have involved nonstereospecific Cα′-Cβ′ epoxidation followed by non-enzymatic hydrolysis of the epoxide.

Binding nature and denaturation of protein during interaction with galloylglucose

Analysis of insoluble complexes between tetragalloylglucose and proteins following a series of successive washes with buffer indicated (1) heterogeneity of binding between galloylglucose and protein and (2) irreversible denaturation of protein during interaction with galloylglucose. Relatively large amounts of tetragalloylglucose were removed by initial washes, indicating weak, low affinity binding, whereas smaller amounts removed by subsequent washes suggest bonds with a higher affinity. Although the maximum number of bindings sites, calculated per 10,000 M(r) of protein, was similar for BSA, myoglobin and lysozyme, the proportion of these sites that appeared to have high affinity, varied from 8 to 29%. The low proportion of strongly binding sites in lysozyme explains its relatively low tannin-complexing ability. Solubility decrease in protein during successive washing and decrease in the beta-glucosidase activity indicate that irreversible denaturation of protein occurs, which progresses with an increase in the incubation time with galloylglucose and galloylglucose/protein molar ratio in the mixture. Relative affinity of galloylglucose is directly related to the ability to cause irreversible denaturation.

Quantitative determination of tannin and protein in the precipitates by high-performance liquid chromatography

Abstract High performance liquid chromatography was applied for the determination of both tannin and protein in tannin-protein coprecipitates and the relative affinities of a series of galloylglucoses for bovine serum albumin were determined. Relative affinity is affected mainly by the number of the galloyl groups in the galloylglucose molecule with penta > tetra- > tri- ⪢ di- ⪢ monogalloylglucose. The position of the galloyl group in the glucose core also affects the relative affinity but the effect is smaller than that of the number with 2,3,6- > 2,3,4-tri- O -galloylglucose and 4,6- > 2,3-di- O -galloylglucose.

Synthesis of 1,2-Diarylpropane-1,3-Diols and Determination of their Configurations

Schlüsselwörter (Sachgebiete) ij2-Diarylpropan-i33-diol Lithium diisopropylamid Threound ErythroIsomere Phenylboronat Synthesis of i,2-Diarylpropane-i33-Diols and Determination of their Configurations Summary i-(4-Hydroxy-3i5-dirnethoxyphenyl)-2-(4-hydroxy-3-methoxyphenyl)-propane-ii3-diol (i), one of the main structural units in hardwood lignin was synthesized. The key step of this synthetic method is the condensation reaction between methyl benzylhomovanillate (3) and benzyl syringaldehyde (4). At this step, lithium diisopropyl amide was used as base, and threo and erythro isomers of ß-hydroxy ester (5) whose ratio was about 3: were obtained as crystal, respectively. These ß-hydroxy esters (5) were converted to the final compounds (i) by hydrogenation with Pd-C and hydrogen, and subsequent acetylation and reduction with lithium aluminum hydride. The configurations of these i,2-diarylpropane-i,3-diols were established by NMR analysis of their phenyl boronates. The coupling constants between aand ^-protons were 10 cps (threo) and 4.3 cps (erythro), respectively, and the values supported these configurations.

Effects of the Hydroxylation Patterns and Degrees of Polymerization of Condensed Tannins on their Metal-Chelating Capacity

We studied the effects of the hydroxylation patterns and the degrees of polymerization of condensed tannins on their metal-chelating capacity. These effects were evaluated by the relative stability of aluminium complexes with proanthocyanidin. The relative stability of aluminium complexes was determined qualitatively in methanol solution by the competition with the metallochromic reagent (chrome azurol S). A comparison of these capacities indicates the following results: 1) phenolic hydroxy groups in tannins are essential sites for the chelation with metal. 2) the sites of tannins interacting with metal ions are mainly o-dihydroxyphenyl groups in B-ring. 3) increasing degrees of polymerization of tannins tends to increase the relative stability of condensed tannin-aluminium chelate.

Vibrational property changes of spruce wood by impregnation with water-soluble extractives of pernambuco (Guilandina echinata Spreng.)

Sitka spruce (Picea sitchensis Carr.) was treated with water-soluble extractive components of pernambuco (Guilandina echinata Spreng. syn Caesalpinia echinata Lam.) by two methods: impregnation under evacuation using an aspirator and repetitive surface application using a brush. The influence of these treatments on the vibrational properties were examined. The loss tangent (tan δ) of the impregnated specimen decreased, up to nearly a half of its original value, with increasing weight gain. It is suggested that the decrease in tan δ results from impregnation of the extractive components into the amorphous region of cell walls, forming secondary bonds between matrix substances. The surface application of the extractive components, on the other hand, hardly brought about the desirable change in vibrational properties.