A New Concept for Interpretation of the Viscoelastic Behavior of Aqueous Sodium Carboxymethyl Cellulose Systems.

Viscoelastic behaviors of aqueous systems of commercially available sodium carboxymethyl cellulose (NaCMC) samples with the degrees of substitution (DS) of approximately 0.68 and 1.3, and the weight-average molar masses (Mw) higher than 200 kg mol-1 dissolved in pure water and aqueous sodium chloride solutions were investigated over a wide concentration (c) range of NaCMC samples. The dependencies of the specific viscosity (ηsp), the average relaxation time (τw), and the reciprocal of the steady-state compliance (Je-1) on c were discussed. The relationships ηsp ∝ c3, τw ∝ c2, and Je-1 ∝ c, characteristic of the rod particle suspensions, were clearly observed in a range lower than the c where the critical gel behavior was observed. Thus, a new concept based on the rheology of rod particle suspensions was employed to interpret the viscoelastic behaviors obtained in the c range. In this context, NaCMC polymer molecules are assumed to behave as extended rod particles with length (L) and diameter (d), including effective electrostatic repulsive distances, due to the dissociation of Na+ in aqueous systems. Thus, the number density of polymer molecules is given to be ν = c/Mw, and viscoelastic parameters such as ηsp, τw, and Je-1 are calculated using the theoretical model for rod particle suspensions proposed by Doi and Edwards. This concept reasonably described not only the viscoelastic data obtained in this study but also those from other groups using NaCMC samples with different DS and Mw values.

Evidence of a Rod-like Structure for Hydroxypropyl Cellulose Samples in Aqueous Solution.

Because hydroxypropyl cellulose (HpC) is a popular polymeric material that forms a liquid crystalline phase in solutions with various kinds of solvents, including water, it is commonly thought that HpC has a typical rod-like structure in solution. In this study, the structures of commercial HpC samples in aqueous solution with average molar substitution numbers (MS) ranging from 3.6 to 3.9 and weight-average molar masses (Mw) ranging from 36 to 740 kg mol-1 were investigated in detail. We first used multiple techniques, including standard static and dynamic light scattering (SLS and DLS), neutron and X-ray scattering experiments, and viscometric measurements, to obtain clear evidence of rod-like structures quantitatively. The dependence of excess scattering intensities for HpC samples under dilute conditions on the magnitude of the scattering vector over a wide range from 8.9 × 10-3 to 3.0 × 10 nm-1 was reasonably described by the form factor of rod particles with length (L) and diameter (d). Although the determined L value was close to the contour length (lc) calculated from the Mw values in the lower Mw range, L became obviously less than lc with increasing Mw. The radius of gyration (Rg) determined via SLS measurements was proportional to L by a factor of approximately 3.5 ∼ √12 over the Mw range examined. These observations revealed that the conformation of HpC molecules changes from an elongated single chain to a certain folded structure, maintaining the shape of the rod-shaped particles. Moreover, the Mw dependencies of the intrinsic viscosities and translational diffusion coefficients of the HpC samples resulting from DLS measurements were reasonably described with a theoretical rod-like particle model, assuming that L and d are identical to those resulting from the scattering behaviors.

Rigid Rod-like Viscoelastic Behaviors of Methyl Cellulose Samples with a Wide Range of Molar Masses Dissolved in Aqueous Solutions.

The viscoelastic behaviors of aqueous solutions of commercially available methyl cellulose (MC) samples with a degree of substitution of 1.8 and a wide range of weight average molar masses (Mw) were investigated over a wide concentration (c) range at some temperatures from -10 to 25 °C. The viscoelastic parameters useful to discuss the structure and dynamics of MC-forming particles in aqueous solutions were precisely determined, such as the zero-shear viscosity (η0), the steady-state compliance (Je), the average relaxation time (τw), and the activation energy (E*) of τw. Because previously obtained scattering and intrinsic viscosity ([η]) data revealed that the MC samples possess a rigid rod-like structure in dilute aqueous solutions over the entire Mw range examined, the viscoelastic data obtained in this study were discussed in detail based on the concept of rigid rod particle suspension rheology. The obtained Je-1 was proportional to the number density of sample molecules (ν = cNAMw-1, where NA means the Avogadro's constant) over the ν range examined irrespective of Mw. The reduced relaxation time (4NAτw(3νJe [η]ηmMw)-1), where ηm means the medium viscosity, was proportional to (νL3)2, L; the average particle length depending on Mw for each sample was determined in a previous study; and the reduced specific viscosity (ηspNAL3(Mw [η])-1), where ηsp means the specific viscosity, was proportional to (νL3)3 in a range of νL3 < 3 × 102. These findings were typical characteristics of the rigid rod suspension rheology. Therefore, the MC samples behave as entangling rigid rod particles in the νL3 range from rheological points of view. A stepwise increase in E* was clearly observed in a c range higher than the [η]-1 value irrespective of Mw. This observation proposes that contact or entanglement formation between particles formed by MC molecules results in an increase in E*.

Structural and mechanical roles of wood polymer assemblies in softwood revealed by gradual removal of polysaccharides or lignin.

A deep understanding of the inherent roles of wood polymers such as cellulose, hemicelluloses, and lignin in the hierarchical structure of wood is of key importance for advancing functional wood-based materials but is currently lacking. To address this gap, we clarified the underexplored contributions of wood polymer assemblies to the structural support and compressive properties of wood by chemically removing polysaccharides or lignin from wood blocks of a conifer Cryptomeria japonica. Compositional and structural evaluations revealed that cellulose, hemicelluloses, and lignin contributed to the dimensional stability of wood, especially that the polysaccharide network at cell corners sustained the honeycomb cell structure. Wood polymer assemblies featuring the anatomical structure of wood were also evaluated in terms of compressive properties. The modulus and strength reflected the density and anisotropy, whereas fracture behavior was well characterized by each wood polymer assembly through the classification of stress-strain curves based on principal component analysis. The difference in fracture behaviors indicated that the rigid lignin and flexible cellulose assemblies, possibly mediated by hemicelluloses, complementarily determine the unique compressive response of wood. These findings enable the adjustment of wood functionality and the selection of composite components for wood modification while inspiring the development of novel wood applications.

Structure and Conformation of Hydroxypropylmethyl Cellulose with a Wide Range of Molar Masses in Aqueous Solution─Effects of Hydroxypropyl Group Addition.

The structure of hydroxypropylmethyl cellulose (HpMC) samples with a wide range of weight average molar masses (Mw) from 23 to 5000 kg mol-1, a controlled degree of substitution (DS) of 1.9 by methyl groups, and a molar substitution number (MS) of 0.25 by hydroxypropyl groups dissolved in aqueous solution was examined using static light scattering (SLS), dynamic light scattering (DLS), small-to-wide angle neutron scattering (S-WANS) techniques, and intrinsic viscosity ([η]) measurements. The determined Mw and the radius of gyration (Rg) showed the relationships Rg ∝ Mw1.0 and [η] ∝ Mw1.7 in a range of Mw < 100 kg mol-1, similar to rigid rod molecules in solution. However, exponents in the relationships decreased gradually with increasing Mw and reached ∼0.5 in a high Mw region, which is a typical value of flexible chain molecules for both Rg and [η]. These observations suggest that the HpMC samples behave as semiflexible rods with a certain persistence length (lp). The ratios of the hydrodynamic radius via DLS measurements to Rg also supported semiflexible rod behavior. Particle form factors and the average lengths (L) resulting from SLS and S-WANS experiments are well described with rigid rod particles in the range of Mw < 100 kg mol-1 and semiflexible rods with lp ∼ 100 nm in Mw > 100 kg mol-1. Because the average contour length (lc) calculated from Mw is approximately twice as long as L in the Mw range < 100 kg mol-1, the formed HpMC particles possess a folded hairpin-like elongated rigid rod structure. However, the lc/L value increases gradually in the range Mw > 200 kg mol-1, where the formed HpMC particles behave as semiflexible rods. The formed particle structure was substantially different from that found in methyl cellulose samples with a similar DS value, which showed rod-like behavior over a wide Mw range. The addition of hydroxypropyl groups only at MS = 0.25 effectively changed the formed particle structure.

A combination of scanning electron microscopy and broad argon ion beam milling provides intact structure of secondary tissues in woody plants.

The secondary tissues of woody plants consist of fragile cells and rigid cell walls. However, the structures are easily damaged during mechanical cross-sectioning for electron microscopy analysis. Broad argon ion beam (BIB) milling is commonly employed for scanning electron microscopy (SEM) of hard materials to generate a large and distortion-free cross-section. However, BIB milling has rarely been used in plant science. In the present study, SEM combined with BIB milling was validated as an accurate tool for structural observation of secondary woody tissues of two samples, living pine (Pinus densiflora) and high-density oak wood (Quercus phillyraeoides), and compared with classical microtome cross-sectioning. The BIB milling method does not require epoxy resin embedding because of prior chemical fixation and critical point drying of the sample, thus producing a three-dimensional image. The results showed that xylem structures were well-preserved in their natural state in the BIB-milled cross-section compared with the microtome cross-section. The observations using SEM combined with BIB milling were useful for wide-area imaging of both hard and soft plant tissues, which are difficult to observe with transmitted electron microscopy because it is difficult to obtain sections of such tissues, particularly those of fragile reaction woods.

Artificially lignified cell wall catalyzed by peroxidase selectively localized on a network of microfibrils from cultured cells.

MAIN CONCLUSION: An artificial lignified cell wall was synthesized in three steps: (1) isolation of microfibrillar network; (2) localization of peroxidase through immunoreaction; and (3) polymerization of DHP to lignify the cell wall. Artificial woody cell wall synthesis was performed following the three steps along with the actual formation in nature using cellulose microfibrils extracted from callus derived from Cryptomeria japonica. First, we constructed a polysaccharide network on a transmission electron microscopy (TEM) grid. The preparation method was optimized by chemical treatment, followed by mechanical fibrillation to create a microfibrillated network. Morphology was examined by TEM, and chemical characterization was by Fourier transform infrared (FTIR) spectroscopy. Second, we optimized the process to place peroxidase on the microfibrils via an immunoreaction technique. Using a xyloglucan antibody, we could ensure that gold particles attached to the secondary antibodies were widely and uniformly localized along with the microfibril network. Third, we applied the peroxidase attached to secondary antibodies and started to polymerize the lignin on the grid by simultaneously adding coniferyl alcohol and hydrogen peroxide. After 30 min of artificial lignification, TEM observation showed that lignin-like substances were deposited on the polysaccharide network. In addition, FTIR spectra revealed that the bands specific for lignin had increased, demonstrating the successful artificial formation of woody cell walls. This approach may be useful for studying woody cell wall formation and for producing made-to-order biomaterials.

Reconsideration of the conformation of methyl cellulose and hydroxypropyl methyl cellulose ethers in aqueous solution.

The structure and conformation of methyl cellulose (MC) and hydroxypropyl methyl cellulose (HpMC) ether samples dissolved in dilute aqueous (D2O) solutions at a temperature of 25 °C were reconsidered in detail based on the experimental results obtained using small- and wide-angle neutron scattering (S-WANS) techniques in a range of scattering vectors (q) from 0.05 to 100 nm-1. MC samples exhibited an average degree of substitution (DS) by methyl groups per glucose unit of DS = 1.8 and the weight average molar mass of M w = 37 × 103 and 79 × 103 g mol-1. On the other hand, HpMC samples possessed the average molar substitution number (MS) by hydroxypropyl groups per glucose unit of MS = 0.25, DS = 1.9, and M w = 50 × 103 and 71 × 103 g mol-1. The concentration-reduced scattering intensity data gathered into a curve for the solutions of identical sample species clearly demonstrated the relationship I(q)c -1 ∝ q -1 in a q range from 0.05 to 2.0 nm-1, and small interference peaks were found at q ∼ 7 and 17 nm-1 for all examined sample solutions. These observations strongly revealed that form factors for both the MC and HpMC samples were perfectly described with that for long, rigid rod particles with average diameters of 0.8 and 0.9 nm, respectively, and with an inner structure with characteristic mean spacing distances of ca. 0.9 and 0.37 nm, respectively, regardless of the chemically modified conditions and molar masses. A rationally speculated structure model for the MC and HpMC samples dissolved in aqueous solution was proposed.

Prediction of Lignin Contents from Infrared Spectroscopy: Chemical Digestion and Lignin/Biomass Ratios of Cryptomeria japonica.

A method for the high-throughput analysis of the relative lignin contents of Cryptomeria japonica samples over a wide concentration range (3-73%), independent of the type of chemical pretreatment, was developed by using Fourier transform infrared spectroscopy. First, the assignments of the infrared absorbance related to lignin were reviewed. Then, various chemical treatments, including alkaline, acid, and hydrothermal processes, and a sodium chlorite oxidation treatment, were performed to prepare samples containing a wide range of different lignin contents. Principal component analysis indicated high variability among the chemical treatments in terms of the corresponding lignin contents as well as the resulting changes in the chemical structure of hemicellulose; this conclusion was supported by the loading vectors. The intensity of the key band of lignin at 1508 cm-1 was calculated using the absorbance at 2900 cm-1 as a reference; a reliable calibration curve with an R2 of 0.968 was obtained independent of the chemical treatment performed. This simple and rapid method for determining the lignin content is expected to be widely applicable for optimizing bioethanol production, as well as monitoring biomass degradation processes.

Transport Properties of Commercial Cellulose Nanocrystals in Aqueous Suspension Prepared from Chemical Pulp via Sulfuric Acid Hydrolysis.

A cellulose nanocrystal (CNC) sample prepared from chemical pulp via sulfuric acid hydrolysis procedures has been supplied by InnoTech Alberta Inc. in the shape of white dry powder as a prototype product. Some transport coefficients were precisely investigated for the CNC sample in aqueous suspensions at the room temperature of 25 °C such as the average rotational and translational diffusion coefficients (D r and D t) and viscoelastic relaxation times (τv) at dilute conditions. The determined values, D r ≈ 2.3 × 103 s-1 and D t ≈ 1.0 × 10-11 m2 s-1, using depolarized and usual dynamic light scattering (DLS) techniques, respectively, proposed the consistent length and width of L ≈ 170 nm and W ≈ 7.6 nm via a theoretical model for monodisperse rigid rods dispersed in pure water. The viscoelastic behavior for aqueous CNC suspensions containing spherical probe particles was examined using DLS rheological techniques. The obtained value of τv = 1.0 × 10-4 s fairly agrees with that of (6D r)-1 ≈ 7.4 × 10-5 s. Because the theoretical model for monodisperse rods denotes the relationship τv = (6D r)-1, this observation strongly confirms that the CNC sample behaves as approximately monodisperse rigid rodlike particles. Transmission electron microscopy (TEM) images clearly demonstrated a bimodal distribution in rod length with major and small minor peaks at ca. 150 and 240 nm, respectively. Then, the reason for the observed disagreement between the L values resulted from the transport coefficients and the major peak in TEM images is the presence of the small minor component with L ≈ 240 nm. Consequently, individual nanosize rodlike crystalline particles in the CNC sample well disperse without forming large aggregations because of strong interactions and behave as isolated individual rods in dilute aqueous suspensions.

Crystal Orientation of Poly(l-Lactic Acid) Induced by Magnetic Alignment of a Nucleating Agent.

The orientation of poly(l-lactic acid) (PLLA) crystals was controlled through crystal growth from a magnetically oriented nucleating agent, phenylphosphonic acid zinc (PPAZn). The one-dimensional magnetically oriented microcrystal array of PPAZn microcrystals revealed the relationship between the magnetization and crystallographic axes in the PPAZn crystal. The PPAZn microcrystals were homogeneously dispersed in PLLA via melt mixing, which decreased the molecular weight of the PLLA component due to degradation. The PPAZn microcrystals in the molten PLLA were uniaxially aligned under an 8-T static or rotating magnetic field. The wide-angle X-ray diffraction and small-angle X-ray scattering patterns of the PPAZn/PLLA composite films crystallized under each magnetic field showed that the PLLA lamellae grew from the surface of the PPAZn microcrystals, which were uniaxially oriented along the easy- or hard-magnetization axis, with the c-axis of PLLA parallel to the bc-plane of PPAZn. It was also suggested that the greater nucleating effect of PPAZn on PLLA was derived not from geometrical matching, but from factors such as favorable interactions and/or the plate-like shape of the microcrystal.

Influence of drying of chara cellulose on length/length distribution of microfibrils after acid hydrolysis.

Chara is a genus of freshwater alga that is evolutionarily observed at the aquatic-terrestrial boundary, whose cellulose microfibrils are similar to those of terrestrial plants regarding the crystallinity and biosynthesis of cellulose. Oven-dried and never-dried celluloses samples were prepared from chara. Terrestrial plant cellulose samples were used as references. The lengths and length distributions of oven-dried and never-dried chara cellulose microfibrils after acid hydrolysis with or without pretreatment by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, which was used for efficient fibrillation of acid-hydrolyzed products, were observed by transmission electron microscopy. All terrestrial plant celluloses and oven-dried chara cellulose had short nanocrystal-like morphologies of 100-300 nm in length after acid hydrolysis. In contrast, the never-dried chara cellulose had much longer microfibrils of ∼970 nm in length after acid hydrolysis. These results indicated that disordered regions present periodically along the cellulose microfibrils, which cause the formation of cellulose nanocrystals after acid hydrolysis, are not present in inherent chara cellulose microfibrils in water, but are formed artificially under drying or dehydration conditions.

Changes in the degree of polymerization of wood celluloses during dilute acid hydrolysis and TEMPO-mediated oxidation: Formation mechanism of disordered regions along each cellulose microfibril.

Most commercially available plant celluloses, such as kraft pulps and cotton celluloses, have so-called leveling-off degrees of polymerization (LODPs) when subjected to dilute acid hydrolysis. The formation of LODPs is hypothesized to be caused by disordered regions that are present periodically along each cellulose microfibril in plant celluloses. Here, we prepared never-dried wood cellulose, and wood celluloses at different drying stages, and subjected them to dilute acid hydrolysis. The viscosity average degrees of polymerization (DPv) of the wood celluloses decreased in DPv with increasing dilute acid-hydrolysis times. However, the DPv values after 4h of acid hydrolysis differed from those of softwood bleached kraft pulp (SBKP), which showed typical patterns of LODPs. Thus, the disordered regions corresponding to LODPs that were observed for SBKP are probably not synthesized in the native wood. Instead, such disordered regions are formed secondarily or artificially during the isolation/purification and/or drying processes of plant cellulose fibers. The results of the dilute acid hydrolysis and the 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation of SBKP and softwood unbleached soda-anthraquinone pulp showed that the structures of disordered regions can be controlled via the preparation and drying conditions of wood cellulose used as starting materials.

Site-directed mutagenesis of bacterial cellulose synthase highlights sulfur-arene interaction as key to catalysis.

Cellulose is one of the most abundant biological polymers on Earth, and is synthesized by the cellulose synthase complex in cell membranes. Although many cellulose synthase genes have been identified over the past 25 years, functional studies of cellulose synthase using recombinant proteins have rarely been conducted. In this study, we conducted a functional analysis of cellulose synthase with site-directed mutagenesis, by using recombinant cellulose synthase reconstituted in living Escherichia coli cells that we recently constructed (cellulose-synthesizing E. coli, CESEC). We demonstrated that inactivating mutations at an important amino acid residue reduced cellulose production. In this study, an interesting loss-of-function mutation occurred on Cys308, whose main chain carbonyl plays an important role for locating the cellulose terminus. Mutating this cysteine to serine, thus changing sulfur to oxygen in the side chain, abolished cellulose production in addition to other apparent detrimental mutations. This unexpected result highlights that the thiol side-chain of this cysteine plays an active role in catalysis, and additional mutation experiments indicated that the sulfur-arene interaction around Cys308 is a key in cellulose-synthesizing activity. Data obtained by CESEC shed light on the function of cellulose synthase in living cells, and will deepen our understanding of the mechanism of cellulose synthase.

Functional reconstitution of cellulose synthase in Escherichia coli.

Cellulose is a high molecular weight polysaccharide of ß1 → 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials.

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.

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.

Preparation of fibrous cellulose by enzymatic polymerization using cross-linked mutant endoglucanase II.

A cross-linked mutant endoglucanase II was prepared for enzymatic polymerization to cellulose. The cross-linked enzyme is composed of three mutant enzymes showing polymerization activity. A characteristic feature of the polymerization with this cross-linked enzyme is formation of cellulose fibrils in contrast to plate-like crystals obtained by using a free enzyme.

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.