Yoshiki Horikawa

Nanotube and Three-Way Nanotube Formation with Nonionic Amphiphilic Block Peptides

Amphiphilic block polypeptides having a helical hydrophobic block with a uniform chain length and a hydrophilic nonionic block were newly synthesized and self-assembled into homogeneous nanotubes with ca. 60 nm diameter and ca. 200 nm length. The tubular assembly was shown to be elongated by heating over micrometer length without changing the diameter. Notably, a distinctive three-way nanotube was obtained just by mixing two kinds of amphiphilic polypeptides with the same helical hydrophobic block but different chain lengths of the hydrophilic block. The morphology of the molecular assemblies was shown to be tunable from a curved sheet-shaped assembly to a long or short nanotubular assembly and a three-way nanotubular assembly by suitable molecular design of the hydrophobic block, selection of the chain length of the hydrophilic block, mixing two-type block peptides, and processing such as heating.

Chemometric Analysis with Near-Infrared Spectroscopy for Chemically Pretreated Erianthus toward Efficient Bioethanol Production

In this paper, we report the combination of a near-infrared (NIR) spectroscopic method with multivariate analysis in order to develop a calibration model of the saccharification ratio of chemically pretreated Erianthus. The regression models clearly depend on the NIR spectral regions, and the information of CH and aromatic framework vibrations contributed most effectively to the alkaline dataset. From interpretations of the regression coefficient, lignin and cellulose were negatively and positively correlated with the saccharification ratio, respectively, and this result was supported by the data from wet chemical analysis. A more complex dataset was obtained from varied chemical pretreatments; here, the saccharification ratio was either small or had no linear correlation with each structural monocomponent. These results enabled the successful construction of the PLS regression model. NIR spectroscopy can be a rapid screening method for the saccharification ratio, and furthermore, can provide information of the key factors influencing the realization of more efficient enzymatic accessibility.

Near-Infrared Chemometric Approach to Exhaustive Analysis of Rice Straw Pretreated for Bioethanol Conversion

We report a simple analytical procedure combining near-infrared (NIR) spectroscopy with multivariate analysis to detect the saccharification efficiency of pretreated rice straw. Three types of sample preparation methods were tested to develop a powerful calibration model, with the disk sample used as the standard protocol. From the spectra dataset of NaOH-treated biomass, we obtained a good calibration for the saccharification ratio and some major structural components by partial least-squares regression. Adding dataset from hot water and dilute sulfuric acid pretreatments to NaOH sample dataset, an acceptable calibration model to predict the saccharification ratio as well as the glucose, xylose, and lignin contents was generated. NIR has a great potential for rapid screening of saccharification efficiency of pretreated biomass, which would allows us to control the quality of processing toward better bioethanol production.

Extraction of cellulose-synthesizing activity of Gluconacetobacter xylinus by alkylmaltoside.

This study reinvestigated the synthesis of cellulose in vitro with a well-known cellulose-producing bacterium, Gluconacetobacter xylinus. Alkylmaltoside detergents, which are more frequently used in recent structural biological researches, are uniquely used in this study to solubilize cellulose-synthesizing activity from the cell membrane of G. xylinus. Activity comparable to that previously reported is obtained, while the synthesized cellulose is crystallized into a non-native polymorph of cellulose (cellulose II) as well as the previous studies. In spite of this failure to recover the native activity to synthesize cellulose I microfibril in vitro, the product is a polymer with a degree of polymerization greater than 45 as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was thus concluded that the established protocol can solubilize cellulose-synthesizing activity of G. xylinus with polymerizing activity.

The crystalline phase of cellulose changes under developmental control in a marine chordate

The native form of cellulose is a fibrillar composite of two crystalline phases, the triclinic Iα and monoclinic Iβ allomorphs. Allomorph ratios are species-specific, and this gives rise to natural structural variations in cellulose crystals. However, the mechanisms contributing to crystal formation remain unknown. We show that the two crystalline phases of cellulose are tailored to distinct structures during different developmental stages of the tunicate chordate Oikopleura dioica. Larval cellulose consisting of Iα allomorph constitutes the body cuticle fin, whereas adult cellulose consisting of Iβ allomorph frames a mucous filter-feeding device, the “house.” Both structures are secreted from the epidermis in accordance with the mutually exclusive expression patterns of two distinct putative cellulose synthase genes. We discuss a possible linkage between structural variations of the crystalline phases of cellulose and the underlying evolutionary genetics of cellulose biosynthesis.

Enzymatic Polymerization Catalyzed by Immobilized Endoglucanase on Gold

Enzymatic polymerization was carried out on gold by immobilized genetically engineered endoglucanase II (EGII) from Trichoderma viride , and the polymerization behavior and the produced cellulose were analyzed in comparison with those by free enzymes. Mutant EGIIs were EGII(core2) and EGII(core2H), which consist of two sequential catalytic core domains with one His-tag (His6) on N-terminal and with totally two His-tags on both terminals, respectively. His-tagged EGIIs were immobilized via Ni chelators of nitrilotriacetic acid (NTA) introduced on gold surface. From SPR measurements, the affinity of EGII(core2H) to the surface was higher than that of EGII(core2), and the molecular occupation area of EGII(core2H) was larger than that of EGII(core2), indicating that EGII(core2H) was immobilized with utilizing two His-tags introduced on both terminals. The hydrolytic activity of the immobilized EGII(core2H) using cellohexaose as substrate was slightly lower than that of free EGII(core2H) when they were compared at the same amount in the hydrolytic system. Enzymatic polymerization catalyzed by both immobilized EGII(core2) and EGII(core2H) proceeded with producing highly crystalline cellulose in comparison with free enzyme. Immobilization of the endoglucanase is thus effective to obtain crystalline cellulose due to the high density of the catalytic domain on gold.

Effect of thermochemical pretreatment on lignin alteration and cell wall microstructural degradation in Eucalyptus globulus: comparison of acid, alkali, and water pretreatments

Pretreatment is an essential step to effectively hydrolyze lignocellulosic polysaccharides. In this paper, we investigated the degree of decompositions of lignin and cell wall structure using dilute acid, alkali, and water pretreatments to assess both chemical and ultrastructural alterations during pretreatment. The thioacidolysis method showed that β-O-4 linkages in lignin were mostly cleaved after all pretreatments, in which the highest decrease of β-O-4 units was for NaOH pretreatment, followed by hot water and H2SO4 pretreatments. The amounts of lignin degradation compounds, including vanillin and syringaldehyde, in the supernatant water also differed between the three pretreatments. Field-emission scanning electron microscopy revealed clear differences among the pretreatments in decomposing the ultrastructure of the inner surface of the fiber cell walls. Small pores were formed due to degradation of a part of the warty layer of the innermost surface in H2SO4 pretreatment. The warty layer was more degraded in hot water pretreatment and thus the cellulose microfibrils of the secondary walls were exposed. NaOH pretreatment showed that the warty layer was almost completely decomposed. The comparative study of different pretreatments using chemical methods and microscopic observations led to a better understanding of decomposition of wood cell walls by thermochemical pretreatment.

ANATOMICAL AND MECHANICAL CHARACTERISTICS OF LEAF-SHEATH FIBROVASCULAR BUNDLES IN PALMS

This study presents anatomical characteristics, mechanical properties, microfibril angles (MFAs) and Klason lignin contents of leaf-sheath fibrovascular bundles from 14 palm genera (18 species). Observed by light microscopy, all fibrovascular bundles consisted equally of thick-walled sclerenchyma fibers and vascular tissue, while the shape and localization of vascular tissues on the transverse sections varied among species. It was possible to group these fibrovascular bundles into 3 types based on the vascular tissue’s differences: type A – rounded in the central region; type B – angular in the marginal region; and type C – aliform in the central region. These three anatomical types of fibrovascular bundles showed some correlation with a current phylogenetic classification of palm species. Through mechanical tests, this research confirmed the correlation between diameter and mechanical properties of the fibrovascular bundles of palms; tensile strength and Young’s modulus showed a decreasing trend with increasing diameter. We clarified that this trend was due to a marked increase in the proportion of transverse sectional area comprised by vascular tissue with increasing diameter of fibrovascular bundles. The MFAs of fibrovascular bundles ranged from 10.3o to 47.1o, which were generally larger than those of non-woody plants, conifers, and broad-leaved trees. The Klason lignin contents of palm species were also high, ranging from 18.3% to 37.8%, with a mean value of 29.6%. These large MFAs and high lignin contents could lead to the long-term plastic deformation and relatively low tensile strength of palm fibrovascular bundles.

Cell wall characterization of windmill palm (Trachycarpus Fortunei) fibers and its functional implications

The fiber bundles from the lignified leaf sheath of windmill palm (Trachycarpus fortunei) are widely used as natural fibers for various products, and exhibit excellent durability. In this study, the cell wall of windmill palm fibers was characterized using transmission electron microscopy, high resolution field emission scanning electron microscopy, and polarized light microscopy, and chemical analysis to measure lignin content. It was found that (1) the secondary wall was composed of just two layers, outer (equivalent to S1, 0.65 ± 0.12 μm) and inner (equivalent to S2, 1.28 ± 0.30 μm) ones, with a high ratio of S1 to the whole cell wall thickness; (2) the microfibrils of the S1 are orientated in an S-helix (MFA, 127.0° ± 2.0), and those of the S2 in a Z-helix (MFA, 43.7° ± 2.2); and (3) the Klason lignin content of fiber bundles was very high (nearly 40%). It is suggested that these structural and chemical features of windmill palm fibers are involved in their mechanical properties such as high flexibility and elasticity, and also related to their high durability.

Assessment of endoglucanase activity by analyzing the degree of cellulose polymerization and high-throughput analysis by near-infrared spectroscopy

We developed a novel and practical assessment technique for endoglucanase (EG) activity by measuring the degree of polymerization (DP) of cellulose from Eucalyptus globulus. This evaluation method demonstrated that EG II from Trichoderma reesei had higher endoglucanase activity than EG I, which has not been detected in conventional experiments using carboxymethyl cellulose as a model substrate. In addition, a high-throughput protocol for DP measurement was developed by using near-infrared spectroscopy combined with a multivariate analysis. Interpreting the regression coefficient, a reciprocal correlation was observed between the relative crystallinity of the cellulosic residue after enzymatic hydrolysis and the DP.