Effect of polymorphic form on the functional properties of cellulose: A comparative study

The powder and tableting properties of cellulose II powders (MCCII) and (SDCII) were evaluated and compared with common direct compression binders. The cellulose II polymorphs offered more benefits in terms of functionality as compared with cellulose I (Avicel^® PH-102) spray dried lactose and starch. Spray dried cellulose II (SDCII) had a better disintegrant ability, but a lower compactibility than microcrystalline cellulose I (Avicel^® PH-102). However, when mixed and compressed with acetaminophen, SDCII was as compactable as cellulose I. Further, unprocessed cellulose II has a comparable compressibility to that of cellulose I. SDCII was found to be less friable, less sensitive to magnesium stearate, and possessed better acetaminophen loading capacity than unprocessed cellulose II and comparable to that of cellulose I. The cellulose II materials showed potential for use as a direct compression excipient.     

Cellulose biosynthesis: current views and evolving concepts

* AIMS: To outline the current state of knowledge and discuss the evolution of various viewpoints put forth to explain the mechanism of cellulose biosynthesis. * SCOPE: Understanding the mechanism of cellulose biosynthesis is one of the major challenges in plant biology. The simplicity in the chemical structure of cellulose belies the complexities that are associated with the synthesis and assembly of this polysaccharide. Assembly of cellulose microfibrils in most organisms is visualized as a multi-step process involving a number of proteins with the key protein being the cellulose synthase catalytic sub-unit. Although genes encoding this protein have been identified in almost all cellulose synthesizing organisms, it has been a challenge in general, and more specifically in vascular plants, to demonstrate cellulose synthase activity in vitro. The assembly of glucan chains into cellulose microfibrils of specific dimensions, viewed as a spontaneous process, necessitates the assembly of synthesizing sites unique to most groups of organisms. The steps of polymerization (requiring the specific arrangement and activity of the cellulose synthase catalytic sub-units) and crystallization (directed self-assembly of glucan chains) are certainly interlinked in the formation of cellulose microfibrils. Mutants affected in cellulose biosynthesis have been identified in vascular plants. Studies on these mutants and herbicide-treated plants suggest an interesting link between the steps of polymerization and crystallization during cellulose biosynthesis. * CONCLUSIONS: With the identification of a large number of genes encoding cellulose synthases and cellulose synthase-like proteins in vascular plants and the supposed role of a number of other proteins in cellulose biosynthesis, a complete understanding of this process will necessitate a wider variety of research tools and approaches than was thought to be required a few years back.     

细菌纤维素合酶的基本特性及作用机理

细菌纤维素是一种天然的生物质高分子聚合物。相较于植物纤维素,其具有更高的纯度和优异的力学性能。有望作为一种绿色的新型高分子材料被广泛应用。细菌纤维素合酶作为合成细菌纤维素的关键酶,其主导细菌纤维素的合成过程。因此,对其合成机理的探索有助于实现细菌纤维素大量生产和广泛应用。本文从细菌纤维素合酶的基本特性出发,综述了菌种筛选、提升产量和合酶的细胞定位等内容;围绕纤维素合酶的作用机理阐述了体外合成方法的影响因素,以及利用该方法探究各亚基相关作用的现状。以此探究细菌纤维素合酶的合成机制,并提出了当前研究中存在的问题。同时,展望了该领域未来的研究方向,以期通过对合成机理的探讨为细菌纤维素的大规模应用提供理论基础。     

Isolation and Characterization of Two Cellulose Morphology Mutants of Gluconacetobacter hansenii ATCC23769 Producing Cellulose with Lower Crystallinity

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.     

The Cellulases Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act Synergistically To Solubilize Native Cotton Cellulose but Not To Decrease Its Molecular Size

Degradation of cotton cellulose by Trichoderma reesei endoglucanase I (EGI) and cellobiohydrolase II (CBHII) was investigated by analyzing the insoluble cellulose fragments remaining after enzymatic hydrolysis. Changes in the molecular-size distribution of cellulose after attack by EGI, alone and in combination with CBHII, were determined by size exclusion chromatography of the tricarbanilate derivatives. Cotton cellulose incubated with EGI exhibited a single major peak, which with time shifted to progressively lower degrees of polymerization (DP; number of glucosyl residues per cellulose chain). In the later stages of degradation (8 days), this peak was eventually centered over a DP of 200 to 300 and was accompanied by a second peak (DP, (apprx=)15); a final weight loss of 34% was observed. Although CBHII solubilized approximately 40% of bacterial microcrystalline cellulose, the cellobiohydrolase did not depolymerize or significantly hydrolyze native cotton cellulose. Furthermore, molecular-size distributions of cellulose incubated with EGI together with CBHII did not differ from those attacked solely by EGI. However, a synergistic effect was observed in the reducing-sugar production by the cellulase mixture. From these results we conclude that EGI of T. reesei degrades cotton cellulose by selectively cleaving through the microfibrils at the amorphous sites, whereas CBHII releases soluble sugars from the EGI-degraded cotton cellulose and from the more crystalline bacterial microcrystalline cellulose.     

A spectroscopic assessment of cellulose and the molecular mechanisms of cellulose biosynthesis in the ascidian Ciona intestinalis

Tunicates are the only animal group known to synthesize cellulose. The current hypothesis is that a horizontally acquired cellulose synthase gene of bacterial origin might have contributed to the establishment of this unique trait. Cellulose biosynthesis in tunicates thus provides an opportunity to understand how a foreign gene was assimilated into a new genome to establish a new trait. Because little is known of the molecular mechanisms underlying cellulose biosynthesis, we set up a practical assessment of cellulose in the ascidian tunicate Ciona intestinalis. We first demonstrated and characterized cellulose in the tunic of adult specimens by chemical purification and by subsequent scanning electron microscopic observation and X-ray diffractometry. Next, we showed that Fourier transform infrared spectroscopic microscopy (FTIR microscopy) can be used to assess cellulose in the small tunic of individual larval specimens without chemical purification. Using FTIR microscopy together with a blastomere isolation technique, we demonstrated that cellulose biosynthesis occurred cell-autonomously in the animal hemisphere of an embryo where the future epidermis, the known site of cellulose biosynthesis, will arise. We combined FTIR microscopy with morpholino antisense oligonucleotide-mediated gene knockdown technology to generate a reverse genetic system to identify genes involved in cellulose biosynthesis. FTIR microscopy was thus able, in combination with current research resources, to contribute to cellular and molecular investigations of animal cellulose biosynthesis.     

Facile approach for the dispersion of regenerated cellulose in aqueous system in the form of nanoparticles

This study reports a facile method to disperse cellulose in deionized water, wherein a critical condition of regenerated cellulose is discovered, where it completely disperses up to a maximum of 5 g L(-1) concentration in deionized water with the help of ultrasonication. The dispersed cellulose is characterized by TEM and DLS, the latter among which shows 200 nm hydrodynamic radii of cellulose nanoparticles dispersed in deionized water. FTIR analysis of dispersed cellulose reveals that dispersed cellulose losses its crystallinity during regeneration and dispersion step employed in this study. The dispersed cellulose reported in this study is able to form free-standing, transparent films, which were characterized by SEM, XRD, TGA, EDX, and FTIR spectroscopy and show resistance against dissolution in water. Additionally, the dispersed cellulose is able to undergo at least three times faster enzymatic hydrolysis in comparison to pristine microcrystalline cellulose under similar reaction conditions. The dispersed cellulose reported here could be a better material for reinforcement, preparation of hydrogels, and drug delivery applications under physiological environment.     

Formulation and roentgenographic studies of naproxen-pectin-based matrix tablets for colon drug delivery

A study has been carried out to assess the potential use of pectin in combination with two added hydrocolloids, i.e., hydroxy-propyl-methyl cellulose and hydroxyethyl cellulose in varied concentrations and coated with ethyl cellulose and cellulose acetate phthalate. The results of in vitro drug release showed that the matrix tablets prepared with pectin, hydroxy ethyl cellulose (20 percent) when coated with ethyl cellulose and cellulose acetate phthalate were found to be 63.0 percent, 8.4 percent, and 4.5 percent, respectively, in after eight hours during drug release study period. These results were confirmed with the results of roentgenographic studies in nine healthy human volunteers to find the shape and integrity of the dosage form. The X-ray photographs revealed that the enteric-coated tablet was visible only up to 5.5 hours and at the end of eighth hour, the photograph has not shown any presence of tablet indicating the loss of shape and size by the microflora present in the colon region. So, the results of in vitro and roentgenographic studies revealed that pectin, hydroxy ethyl cellulose (20 percent) base coated with ethyl cellulose and cellulose acetate phthalate was found to be a promising carrier for naproxen to colon.     

Reorientation of cellulose nanowhiskers in agarose hydrogels under tensile loading

Agarose hydrogels filled with cellulose nanowhiskers were strained in uniaxial stretching under different humidity conditions. The orientation of the cellulose whiskers was examined before and after testing with an X-ray laboratory source and monitored in situ during loading by synchrotron X-ray diffraction. The aim of this approach was to determine the process parameters for reorienting the cellulose nanowhiskers toward a preferential direction. Results show that a controlled drying of the hydrogel is essential to establish interactions between the matrix and the cellulose nanowhiskers which allow for a stress transfer during stretching and thereby promote their alignment. Rewetting of the sample after reorientation of the cellulose nanowhiskers circumvents a critical increase of stress. This improves the extensibility of the hydrogel and is accompanied by a further moderate alignment of the cellulose nanowhiskers. Following this protocol, cellulose nanowhiskers with an initial random distribution can be reoriented toward a preferential direction, creating anisotropic nanocomposites.     

Chemoreception for cellulose

Rats that had been trained to avoid a suspension containing1% purified cellulose, subsequently avoided suspensions containingas little as 0.1% cellulose, but did not avoid suspensions containingcom, wheat or rice starch. Subsequent experiments examined thebasis for this unusual form of chemoreception. Viscosity measurementsindicated that cellulose and starch have similar effects ofviscosity; the suspending agent used in these experiments, xanthangum, masked much of the textural effects of cellulose or starchin water. Therefore, it does not seem likely that rats sensecellulose via its textural effects. On the other hand, ratsthat had been trained to avoid cellulose suspensions, also avoidedaqueous extracts of cellulose that had been filtered to removecellulose particles. This result suggests that a water-solubleimpurity contributes to cellulose chemoreception. This water-solubleimpurity could be washed off cellulose with water, but returnedafter the washed cellulose was dried. It is likely that cellulosereacts with the atmosphere to produce small amounts of water-solublecompounds that rats can sense     

Resistin and RELM-alpha gene expression in white adipose tissue of lactating mice

An ORF2 gene located upstream of the cellulose synthase (bcs) operon of Acetobacter xylinum BPR2001 was disrupted and a mutant (M2-2) was constructed. In static cultivation, the parent strain produced a tough, colorless, and insoluble cellulose pellicle, whereas M2-2 culture produced a thin, yellow, and fragile pellicle. The results of X-ray diffraction and 13C solid-state NMR indicated that the product of M2-2 is a mixture of cellulose I, cellulose II, and amorphous cellulose. The cellulose I to cellulose II ratio of the mixture was evaluated from the signal areas of C6 to be about 1:2. Electron microscopy revealed that the product of M2-2 included ribbon-like cellulose and irregularly shaped particles attached to the ribbons. On the other hand, the mutant complemented with plasmid pSA-ORF2/k containing the ORF2 gene and BPR2001 produced only cellulose I. These results indicate that the ORF2 gene is involved in the production and crystallization of cellulose I microfibrils by this microorganism.     

Anaerobic breakdown of uroporpophysrins I and II to bile pigments by extracts of Clostridium tetanomorphum

Abstract The five conserved tryptophan residues in the cellulose binding domain of xylanase A from Pseudomonas fluorescens subsp. cellulosa were replaced with alanine and phenylalanine. The mutated domains were fused to mature alkaline phosphatase, and the capacity of the hybrid proteins to bind cellulose was assessed. Alanine substitution of the tryptophan residues, in general, resulted in a significant decrease in the capacity of the cellulose binding domains to bind cellulose. Mutant domains containing phenylalanine substitution retained some affinity for cellulose. The C-terminal proximal tryptophan did not play an important role in ligand binding, while Trp¹³, Trp³⁴ and Trp³⁸ were essential for the cellulose binding domain to retain cellulose binding capacity. Data presented in this study suggest major differences in the mechanism of cellulose attachment between Pseudomonas and Cellulomonas cellulose binding domains.     

Role of contact in bacterial degradation of cellulose

Abstract Bacterial cells can adhere to cellulose fibres, but it is not known if cell-to-fibre contact is necessary for cellulose degradation. This problem was explored using aerobic cellulolytic bacteria, including known species and new isolates from soil. These were tested on plates containing Avicel, Solka floc, CF11 cellulose, carboxymethyl cellulose, or phosphoric acid-treated cellulose. Cellulose degradation was measured both by formation of clearing zones and by growth when cellulose was the only carbon source. The bacteria tested were either inoculated directly on the cellulose-containing agar, or separated from it by a pure agar layer or by membrane filters (not containing cellulose). Even when separated from the cellulose-containing agar all strains grew well. Clearing zones, best seen in phosphoric acid-treated cellulose, were larger under colonies separated from cellulose by an agar layer than under those in direct contact with cellulose. Such zones could also appear under filters. Our results show that bacterial degradation of cellulose does not depend on cell-to-fibre contact and suggest that when cellulose is at a greater distance from the cell, the removal of end products reduces catabolite repression of cellulose formation.     

Crystallographic aspects of sub-elementary cellulose fibrils occurring in the wall of rose cells culturedin vitro

Summary Quantities of disencrusted sub-elementary cellulose fibrils from the cell wall of rose cells culturedin vitro were prepared. Following an X-ray and electron diffraction analysis, these fibrils gave a cellulose diffraction pattern which presented only two strong equatorial diffraction spacings at 0.409 and 0.572 nm indicating that the fibrils have a crystalline structure resembling that of cellulose IV_I. This observation is best explained in terms of a lateral disorganization of the cellulose chains within the fibrils. This disorganization cannot be eliminated and is connected with the small width of the fibrils which contain from 12 to 25 cellulose chains only. In these fibrils, most of the cellulose chains are superficial and not locked with neighboring chains in a tight hydrogen bond system as in thicker cellulose microfibrils.     

Function of a low molecular weight peptide from Trichoderma pseudokoningii S38 during cellulose biodegradation

The biochemical mechanism for cellulose decomposition by a low molecular weight peptide, named short fiber generating factor (SFGF), derived from the culture supernatant of a cellulolytic fungus Trichoderma pseudokoningii S-38, was determined. Sufficient information obtained by biochemical and biophysical studies and combined with observation with a scanning electron microscope provided further evidence for the earlier studies that the SFGF had a high capacity for chelating and reducing ferric ions, and could produce free radical by reduction of Fe(3+) to Fe(2+) in the presence of oxygen molecule. These studies suggested that the effect of SFGF on cellulose is directly related to an oxidative reaction and is different from the hydrolysis of cellulose by cellulases. The alcoholic hydroxyl groups in cellulose can be oxidized by SFGF, which leads to destruction of the hydrogen bond network in cellulose and cleavage of glycosidic linkages. Both effects led to the de-polymerization of cellulose and the formation of short fibers, and increase of reducing groups in residual cellulose, then the cellulose substrates became more susceptible for hydrolysis by cellulases.     

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane.