Kazumasa Shimizu

Structure of the reducing end-groups in spruce xylan

Abstract Borohydride reduction of spruce meal followed by mild hydrolysis with acid gave a large proportion of 4- O -(α- d -galactopyranosyluronic acid)- d -xylitol. When the reduction was preceded by mild treatment with alkali, galactonic acid end-groups were formed. Enzymic degradation of holocellulose from reduced spruce-meal followed by ion-exchange chromatography led to the isolation of a tetrasaccharide having a xylitol end-group. Its structure revealed that the reducing end-group in spruce xylan has the structure β- d -Xyl p -(1→3)-α- l -Rha p -(1→2)-α- d -Gal p A-(1→4)- d -Xyl, which is the same as that in hardwood xylan.

Structure of rice-straw arabinoglucuronoxylan and specificity of Streptomyces xylanase toward the xylan.

Rice-straw treated with 5% ammonia water was hydrolyzed with the beta-xylanase of Streptomyces sp. E-86, and four kinds of hetero-oligosaccharides, two arabinoxylo- and two glucuronoxylo-oligosaccharides, were isolated from the hydrolysate by charcoal column, gel filtration, ion-exchange, and aminopropyl silica column chromatographies. The structures of the oligosaccharides were identified as 3(2)-alpha-L-arabinofuranosyl-xylobiose, 3(2)-alpha-L-arabinofuranosylxylotriose, 2(3)-4-O-methyl-alpha-D-glucuronosylxylotriose, and 2(3)-alpha-D-glucuronosylxylotriose by the analysis of component sugar, partial acid hydrolysis, methylation analysis etc. Based on the structures of above oligosaccharides, the structure of rice-straw arabinoglucuronoxylan and the specificity of the xylanase toward the xylan are discussed.

Isolation from a softwood xylan of oligosaccharides containing two 4-O-methyl-d-glucuronic acid residues☆

Abstract Partial hydrolysis of a larch arabino(4- O -methylglucurono)xylan afforded two series of oligouronides composed of 4- O -methyl- d -glucuronic acid and d -xylose residues. The first series included aldouronic acids up to the aldopentaouronic acid. Methylation analysis indicated that the aldopentao- and aldotetrao-uronic acids were mixtures of isomers. One aldotetraouronic acid was isolated and identified as O -β- d -Xyl p -(1 → 4)- O -β- d -Xyl p -(1 → 4)- O -(4- O -Me-α- d -GlcA p )-(1 → 2)- d -Xyl. The two isomeric aldotriouronic acids were separated from each other. The acids of the second series, which were composed of two uronic acids and 2-4 d -xylose residues, were identified as follows: O -β- d -Xyl p -(1 → 4)- O -(4- O -Me-α- d -GlcA p )-(1 → 2)- O -β- d -Xyl p -(1 → 4)- O (4- O -Me-α- d -GlcA p )-(1 → 2)- O -β- d -Xyl p -(1 → 4)- d -Xyl, O -(4- O -Me-α- d -GlcA p )-(1 → 2)- O -β- d -Xyl p -(1 → 4)- O -(4- O -Me-α- d -GlcA p )-(1 → 2)- O -β- d -Xyl p -(1 → 4)- O -β- d -Xyl p -(1 → 4)-D-Xyl, O -(4- O -Me-α- d -GlcA p )-(1 → 2)- O -β- d -Xyl p -(1 → 4)- O -(4- O -Mec-α- d -GlcA p )-(1 → 2)- O -β- d -Xyl p -(1 → 4)-D-Xyl, and O -(4- O -Me-α- d -GlcA p )-(1 → 2)- O -β- d -Xyl p -(1 → 4)- O -(4- O -Me-α- d -GlcA p )-(1 → 2)-D-Xyl. The first three compounds were new acidic oligosaccharides. The 4- O -methyl- d -glucuronic acid in the second series was present in a larger proportion than in the first series, indicating that a large proportion of the uronic acid side-chains were located on two contiguous D-xylose residues in the backbone of the softwood xylan.

Adsorption and Desorption of Cellulase Components during Enzymatic Hydrolysis of Steamed Shirakamba (Betula platyphylla Skatchev) Wood

Abstract The adsorption behavior of Trichoderma viride cellulases on steamed shirakamba ( Betula platyphylla Skatchev) wood was studied. Substrate/enzyme (S/E) ratio, hydrolysis time and saccharification extent as well as the presence or absence of lignin in the substrate were taken into consideration as factors affecting the enzyme adsorption. Filter paperase (FPase) components were adsorbed more selectively than other cellulase components on the substrate. The presence of lignin in the steaamed hardwood tended to slow down the enzyme adsorption, but it did not appear to restrict the extent of hydrolysis of the carbohydrate moiety. The changes in the composition of the enzyme preparation during the course of hydrolysis were analyze by fast protein liquid chromatography (FPLC). The irreversible adsorption of specific cellulase components was not observed in the prolonged hydrolysis of steamed shirakamba wood containing abundant lignin. The removal of lignin from steamed hardwood prior to hydrolysis is not always necessary for the recycling of cellulolytic enzymes.

Screening of fungal β-xylanases for production of acidic xylooligosaccharides using in situ reduced 4-O-methylglucuronoxylan as substrate

Fungal beta xylanases were screened for production of acidic xylooligomers from 4-O-methylglucuronoxylan. In situ reduced hardwood xylan was used as substrate because the products of neutral- and acidic-branched xylooligomers help define substrate specificity of the enzymes. Borohydride reduction in situ transformed 30% of 4-O-methyl-alpha-D-glucopyranosyluronic acid residues into 4-O-methyl-alpha-D-glucopyranosyl residues and reduced C-1 end groups in the xylan backbones. A total of ten beta-xylanase fractions from four fungi were partially purified by chromatography by anion exchange and molecular sieving, and graded qualitatively for enzymatic hydrolysis of the substrate. The yield of acidic xylooligomers was highly affected by whether alpha-glucuronidases were present in the beta-xylanase fractions. Some fractions gave free 4-O-methyl-alpha-D-glucopyranosyluronic acid, but none of the enzyme fractions could release free 4-O-methyl-alpha-D-glucose. Among the beta-xylanase fractions studied, xylanase II of Trichoderma viride was the best producer of aldotetraouronic acid [2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylotriose]. The results obtained suggested that there was a difference in the steric hindrance of the branch point on fungal beta-xylanases between 4-O-methyl-alpha-D-glucopyranosyl and 4-O-alpha-D-glucopyranosyluronic acid residues.

β-Elimination of 2-O-(4-O-methyl-α-d-glucopyranosyluronic acid)-d-xylose with methylsulphinyl carbanion and hydrolysis of the hex-4-enopyranosiduronic linkage

Abstract On treatment with m sodium methylsulphinylmethanide at 25°, 2- O -(4- O -methyl-α- d -glucopyranosyluronic acid)- d -xylose ( 1 ) was rapidly degraded by β-elimination, to form 2- O -(4-deoxy-β- l - threo -hex-4-enopyranosyluronic acid)- d -xylose ( 2 ). The kinetics of hydrolysis of 1 and 2 in 0.5 m sulphuric acid have been studied. Compound 2 was hydrolysed 70 times faster than 1 . Compared with the rate coefficients of other related compounds, 2 was hydrolysed at approximately the same rate as 2- O -(4- O -methyl-α- d -glucopyranosyl)- d -xylose, 3.5 times more slowly than xylobiose, and twice as fast as the xylosidic bond in O -(4- O -methyl-α- d -glucopyranosyluronic acid)-(1→2)- O -β- d -xylopyranosyl-(1→4)- d -xylose.

β-elimination of aldobiouronic acids during methylation by the Hakomori method

Abstract On methanolysis and subsequent saponification, the aldobiouronic acids 2- O -(4- O -methyl-α- d -glucopyranosyluronic acid)- d -xylose ( 1 ) and 4- O -(α- d -galactopyranosyluronic acid)- d -xylose ( 2 ) gave four ( 3–6 ) and two ( 7 and 8 ) methyl glycosides, respectively. These glycosides were well separated by ion-exchange chromatography and their structures were established by methylation analysis, mass spectrometry of their permethylated derivatives, and 13 C-n.m.r. spectroscopy. On methylation by the method of Hakomori, compounds 3–6 , having 4- O -substituted uronic acid residues, afforded β-elimination products ( 15–18 ) in addition to the permethylated derivatives ( 9–12 ). On the other hand, compounds 7 and 8 , having HO-4′ unsubstituted, gave the products of direct methylation ( 13 and 14 ), which gave, on re-methylation, permethylated, unsaturated aldobiouronic acids ( 19 and 20 ).