Kiyoharu Fukuda

Effects of Cellulose Crystallinity, Hemicellulose, and Lignin on the Enzymatic Hydrolysis of Miscanthus sinensis to Monosaccharides

The effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides were investigated. A air-dried biomass was ground by ball-milling, and the powder was separated into four fractions by passage through a series of sieves with mesh sizes 250–355 μm, 150–250 μm, 63–150 μm, and <63 μm. Each fraction was hydrolyzed with commercially available cellulase and β-glucosidase. The yield of monosaccharides increased as the crystallinity of the substrate decreased. The addition of xylanase increased the yield of both pentoses and glucose. Delignification by the sodium chlorite method improved the initial rate of hydrolysis by cellulolytic enzymes significantly, resulting in a higher yield of monosaccharides as compared with that for untreated samples. When delignified M. sinensis was hydrolyzed with cellulase, β-glucosidase, and xylanase, hemicellulose was hydrolyzed completely into monosaccharides, and the conversion rate of glucan to glucose was 90.6%.

Crystal structure of a glycoside hydrolase family 6 enzyme, CcCel6C, a cellulase constitutively produced by Coprinopsis cinerea

The basidiomycete Coprinopsis cinerea produces the glycoside hydrolase family 6 enzyme CcCel6C at low and constitutive levels. CcCel6C exhibits unusual cellobiohydrolase activity; it hydrolyses carboxymethyl cellulose, which is a poor substrate for typical cellobiohydrolases. Here, we determined the crystal structures of CcCel6C unbound and in complex with p‐nitrophenyl β‐d‐cellotrioside and cellobiose. CcCel6C consists of a distorted seven‐stranded β/α barrel and has an enclosed tunnel, which is observed in other cellobiohydrolases from ascomecetes Hypocrea jecorina (HjeCel6A) and Humicola insolens (HinCel6A). In HjeCel6A and HinCel6A, ligand binding produces a conformational change that narrows this tunnel. In contrast, the tunnel remains wide in CcCel6C and the conformational change appears to be less favourable than in HjeCel6A and HinCel6A. The ligand binding cleft for subsite −3 of CcCel6C is also wide and is rather similar to that of endoglucanase. These results suggest that the open tunnel and the wide cleft are suitable for the hydrolysis of carboxymethyl cellulose.

Cloning and Transcript Analysis of Multiple Genes Encoding the Glycoside Hydrolase Family 6 Enzyme from Coprinopsis cinerea

We searched the genome database of the basidiomycete Coprinopsis cinerea (Coprinus cinereus) and found five genes encoding the glycoside hydrolase family 6 (GH6) enzyme, CcCel6A, CcCel6B, CcCel6C, CcCel6D, and CcCel6E, designated in order of increasing locus number (CC1G_01107.1, CC1G_04166.1, CC1G_08276.1, CC1G_08277.1, and CC1G_10605.1). The amino acid sequence of CcCel6A suggests a two-domain structure consisting of an N-terminal family 1 carbohydrate-binding module (CBM1) and a GH6 catalytic domain, while the other genes lack CBM1. The transcripts of CcCel6A were observed at the active growth stage in cellulose culture, whereas they were absent from glucose culture. Cellobiose strongly induced transcription of CcCel6A. On the other hand, transcripts of CcCel6B, -D, and -E were detected in both glucose and cellulose cultures, and transcription of them was induced weakly by cellobiose. The transcript level of CcCel6C was not influenced by glucose or cellobiose.

Characterization of Glycoside Hydrolase Family 6 Enzymes from Coprinopsis cinerea

Recombinants, Cel6 from Coprinopsis cinerea (CcCel6)A, -B, and -C, were expressed by Escherichia coli. These enzymes produced cellobiose from phosphoric acid-swollen cellulose. When Avicel was used as the substrate, CcCel6A showed higher activity toward the substrate than CcCel6B or -C. In reaction with carboxymethylcellulose, CcCel6B and -C hydrolyzed the substrate, whereas CcCel6A failed to react.

Effect of lime pretreatment of brown midrib sorghums.

The effect of lime pretreatment of brown midrib sorghums on enzymatic saccharification was investigated. Under most of the pretreatment conditions, the saccharification yields of bmrs were higher than those of the normal counterparts. This result suggests that bmr is useful to reduce pretreatment costs, because the amount of lime necessary for the pretreatment of biomass can reduced by using bmr mutants.

Effect of board density on bending properties and dimensional stabilities of MDF-reinforced corrugated particleboard

We investigated the bending properties of composite boards produced by reinforcing both sides of corrugated particleboard with medium-density fiberboard (MDF). Thickness swelling and linear expansion (LE) were measured to assess the dimensional stabilities of the composite board. Although the apparent density of the composite board was 0.48 g/cm3, its strength was found to be equivalent to that of 18-type particleboard as described in JIS A 5908. The board’s parallel/perpendicular anisotropy in strength was 0.9. The modulus of rupture (MOR) of the composite board increased with board density only up to a certain density, beyond which the MOR was constant. On the other hand, the thickness swelling of both corrugated particleboard and the composite board was smaller than that of flat-type particleboard, satisfying the JIS A 5908 standard of 12%. Linear expansion (soaking in water of ordinary temperature for 24 h) of corrugated particleboard was 0.7%–0.9% in the parallel direction and 2.1%–3.1% in the perpendicular direction; hence, anisotropy in linear expansion existed in the corrugated particleboard. The linear expansion of the composite board was 0.6%–0.9% in the parallel direction and 1.8%–2.5% in the perpendicular direction. Although the LE of the composite board was lower than that of corrugated particleboard, it is necessary to improve the LE of composite board for practical use.

Heterologous expression, crystallization and preliminary X-ray characterization of CcCel6C, a glycoside hydrolase family 6 enzyme from the basidiomycete Coprinopsis cinerea

CcCel6C is a gene that encodes a glycoside hydrolase family 6 (GH6) enzyme in the Coprinopsis cinerea genome. In the evolutionary tree of GH6 enzymes, the encoded enzyme was closely related to Cel6B from Humicola insolens, previously called endoglucanase VI, while its amino-acid sequence revealed a region corresponding to the C-terminal active-site-enclosing loop typical of cellobiohydrolase II. Here, the crystallization of CcCel6C produced in Escherichia coli is reported. The square prismatic crystal belonged to the triclinic space group P1, with unit-cell parameters a = 44.04, b = 45.11, c = 48.90 A, alpha = 77.81, beta = 87.34, gamma = 68.79 degrees. Diffraction data were collected to 1.6 A resolution.

Improvement of dimensional stability and weatherability of composite board made from water-vapor-exploded wood elements by liquefied wood resin impregnation.

High-density and high-resin-content boards were produced by phenolic resin impregnation into board materials prepared by the water-vapor-explosion process (WVE) to develop high-durability wood composite boards for exterior use. Wet-dry cyclic tests and accelerated weathering tests were conducted, and the fundamental properties were determined to examine the effect of resin impregnation on board qualities. Bending and internal bond strength of resin-impregnated boards (I-board) satisfied the criterion for 18-type particleboard described in JIS A 5908. Thickness swelling (TS) after 24-h water immersion was approximately 2%. Resin impregnation improved the dimensional stability of the boards. In wet—dry cyclic testing, TS of I-board was the same as that of plywood. The retention ratio of modulus of rupture of I-board was large; thus, I-board had high bond durability. Color change of I-board was less than that of ordinary particleboard after a 500-h accelerated weathering test. I-Board had lower surface roughness than boards produced by a spray application method (S-board) and higher water repellency, although the difference in resin contents of the face layer was small. Thus, it is suggested that the surface properties and weatherability of I-board were improved by impregnation of phenolic resin. High-density and resin-impregnated boards made from the WVE elements are expected to withstand actual exterior use.

PROGRESS OF WOOD DECAY AND VERIFICATION OF PREDICTION MODEL UNDER ISOTHERMAL CONDITIONS

*������� 20 ���� 21 �����������[1][2] [3]��������������������������������� �1� ������� ������ ������ ������� � � � Research Specialist, Dept. of Environmental Engineering, Building Research Institute, Dr. Eng. �2� ������� ���������� ��� ���� Prof., Division of Ecosciences., Tokyo Univ. of Agriculture and Technology, Dr. Agr. �3� ������ ����� ��������� ����� � � Director, Dept. of Environmental Engineering, Building Research Institute, Dr. Eng. ��� ����� ��������� ������ Central laboratory, Japan Testing Center for Construction Materials