Exoglucanase activities of the recombinant Clostridium thermocellum CelS, a major cellulosome component.

The recombinant CelS (rCelS), the most abundant catalytic subunit of the Clostridium thermocellum cellulosome, displayed typical exoglucanase characteristics, including (i) a preference for amorphous or crystalline cellulose over carboxymethyl cellulose, (ii) an inability to reduce the viscosity of a carboxymethyl cellulose solution, and (iii) the production of few bound reducing ends on the solid substrate. The hydrolysis products from crystalline cellulose were cellobiose and cellotriose at a ratio of 5:1. The rCelS activity on amorphous cellulose was optimal at 70 degrees C and at pH 5 to 6. Its thermostability was increased by Ca2+. Sulfhydryl reagents had only a mild adverse effect on the rCelS activity. Cellotetraose was the smallest oligosaccharide substrate for rCelS, and the hydrolysis rate increased with the substrate chain length. Many of these properties were consistent with those of the cellulosome, indicating a key role for CelS.     

Antimicrobial properties of fibrous cellulose and other polymeric materials modified with chlorhexidine]

Since fibrous cellulose and other polymeric materials are widely used as dressings, it was of interest to study the mechanism of the antimicrobial action of the products from such materials physically and chemically modified by chlorhexidine, a broad spectrum antiseptic. As vehicles and prolongation agents the following products were used: dressing gauze and cation exchange derivatives of cellulose and starch i.e. monocarboxycellulose (MCC), phosphate cellulose (PC), carboxymethyl cellulose (CMC), monocarboxyl starch (MCS) and alginic acid (AA), a natural compound. It was shown that chemical attachment of chlorhexidine provided a 2-4-fold increase in the antimicrobial effect of the preparations as compared to the use of physical sorption. The antimicrobial effect of the polymeric form of chlorhexidine based on MCC and PC was much higher than that based on MCC, MCS and AA.     

Preparation of cellulose–starch composite gel and fibrous material from a mixture of the polysaccharides in ionic liquid

This paper reports the preparation of a cellulose–starch composite gel from an ionic liquid solution. The gel was obtained by keeping the homogeneous mixture of cellulose (10% w/w) and starch (5% w/w) in 1-butyl-3-methylimidazolium chloride (BMIMCl) for several days at room temperature and characterized by elemental analysis, X-ray diffraction, and thermal gravimetric analysis. Furthermore, the production of the fibrous material composed of cellulose and starch by reconstitution from the homogeneous mixture (10% w/w each) in BMIMCl is demonstrated.     

Effect ofMyrothecium sp. cellulase on different types of cellulose and cellulosic materials

Three types of cellulase preparations were applied to different types of cellulose and cellulosic materials. The action of these types of cellulase on cellulose powder was increased with the increase of enzyme concentration. Both carboxymethyl cellulose (CMC) and sodium carboxymethyl cellulose (Na-CMC) released high amounts of reducing sugar as affected by cellulase application. Different types of paper pulp were moderately hydrolyzed, while agricultural wastes were slightly hydrolyzed. Vegetable and fruits cellulose were equally hydrolyzed but at low rate. Pretreatment of cellulose or cellulosic materials by grinding or by swelling with phosphoric acid gave rise to increased hydrolysis by the enzyme. Cellobiose was detected chromatographically as an intermediate product of hydrolysis of both cellulose and carboxymethyl cellulose with glucose.     

Purification of papain by immobilized metal affinity chromatography (IMAC) on chelating carboxymethyl cellulose

Chelating carboxymethyl cellulose was prepared in bead form by immobilizing iminodiacetic acid on carboxymethyl cellulose which was earlier crosslinked and activated by epichlorohydrin. The prepared matrix was used to purify papain by a factor of 2.6 from commercial papain, and by a factor of 4 from papaya latex by batch adsorption and immobilized metal affinity chromatography respectively. Purification factors obtained were equal in batch mode and double in column mode, to purifications obtained on Chelating Sepharose® Fast Flow. Flow rates up to 38 ml/cm2 h were easily possible on the prepared chelating carboxymethyl cellulose.     

Modification of carboxymethyl cellulose through oxidation

The periodate oxidation reaction of carboxymethyl cellulose involve the primary and secondary hydroxyl groups of the pyranose ring. The reaction is accompanied by the opening of the pyranose ring and resulting product is dialdehyde carboxymethyl cellulose along with some hydrated forms. In this process the glucosidic bond becomes weaker; the formation of carboxyl groups induces a depolymerization, thus reducing the polymerization degree and the physical and mechanical strength of the material. The reaction has been has been carried out at pH 3.5, temperature 45 °C for 0.5-4 h.     

Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.     

Formation of biocompatible nanoparticles by self-assembly of enzymatic hydrolysates of chitosan and carboxymethyl cellulose

A simple preparation method for biocompatible nanoparticles in high concentration (0.5 wt %) by self-assembly of chitosan and carboxymethyl cellulose hydrolysates was developed. Chitosan and carboxymethyl cellulose were hydrolyzed beforehand with chitosanase and cellulase respectively to make fragments having lower molecular weights. Nanoparticles were spontaneously formed only by mixing the two hydrolysate solutions. The particle size distribution was relatively narrow, about 200 nm in mean size. The mean particle size decreased from 226 nm to 165 nm with decreasing molecular weight of chitosan hydrolysate from 9.5 to 6.8 kDa. The mixing ratio of chitosan and carboxymethyl cellulose hydrolysates also affected particle size. Changes in particle size are discussed in relation to a possible mechanism of polyionic complexation. The chitosan-carboxymethyl cellulose nanoparticles were stably suspended over 1 week even under low pH (pH 3.0), high ionic strength (NaCl 1 M), or low temperature (4 degrees C) conditions.     

Carboxymethylation of cellulose using reactive extrusion

Carboxymethyl cellulose was prepared using a continuous, reduced solvent, reactive extrusion process with a short reaction time. The effects of the amounts of NaOH (30 g, 40 g and 50 g), water:ethanol ratio (100%, 70%, 50%, 30% and 10% H₂O) and their interactions on the physical, chemical and morphological properties of carboxymethyl cellulose were studied. Experiments were conducted using to a 5 × 3 blocked factorial design. X-ray diffraction analyses revealed higher degrees of crystallinity and fractions of cellulose-II crystalline structure when 100% H₂O was used as compared to that for 70%, 50%, 30% and 10% H₂O and a commercially available brand of carboxymethyl cellulose, AQUASORB A500. Statistical analysis revealed a significant interaction between the effects of NaOH and H₂O on the degrees of substitutions. The degrees of substitutions decreased with increasing amounts of NaOH and tended to increase with increasing alcohol concentrations. Liquid uptake measurements revealed that the extent of saline uptake, measured at intervals of 1 min, 5 min and 10 min, by carboxymethyl cellulose prepared with 100% H₂O, especially when 40 g and 50 g NaOH was used, was higher than that for 70%, 50%, 30% and 10% H₂O and AQUASORB A500. This may have been because of the higher crystallinity in carboxymethyl cellulose prepared with 100% H₂O. Carboxymethyl cellulose prepared with 70% H₂O and 30 g and 50 g NaOH had the highest saline absorption, using the soak method, before and after centrifugation, respectively. Scanning electron microscopy for carboxymethyl cellulose prepared with 100% and 10% H₂O, through images at 120X magnification, revealed fibers 100 μ to >800 μ in length and 0.8-3.3 μ in breadth. Some non fibrous particles, 0.8-6.7 μ in dimensions, also were observed for 100% H₂O. Images at 900× magnification revealed partially damaged fiber surfaces.     

Primary structure of human class II histocompatibility antigens (HLA-D). I. Isolation, purification and characterization of the HLA-D alpha/beta chain complex from a homozygous lymphoblastoid B cell line, H2LCL (HLA-A3,3;B7,7;Dw2, 2;DR2,2;MT1,1;DC1,1;MB1,1

The complex of alpha and beta chains of HLA-D membrane antigens has been isolated from a lymphoblastoid homozygous B cell line, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1), by an exclusively chemical two-step procedure and characterized by electrophoresis as well as isoelectric focusing in polyacrylamide gel. Cells were gained using long term cultivation in large scale, the crude membrane by differential centrifugation. The proteins of the crude membrane were then solubilized in NP-40, pH 5.0. The first purification step for HLA-D antigens consisted in an ion-exchange chromatography on carboxymethyl cellulose using the solubilization buffer. By this procedure the complex of proteins with relative molecular masses of Mr approximately 34 000 and Mr approximately 29 000 was in a high percentage not bound to the carboxymethyl cellulose. The bound fraction contained the HLA-A, -B and -C antigens and a component with Mr approximately 31 000 corresponding to the well known Ii-fraction. The bound proteins could be recovered from the column by a sodium chloride gradient. The proteins not bound to the carboxymethyl cellulose were precipitated with acetone, dissolved, dialysed against SDS buffer, pH 7.2 and then submitted to the second purification step, the Sephacryl S-300 chromatography. By this procedure the corresponding complex could be further separated from higher and lower molecular proteins. The complex was used as the starting material for the separation of alpha and beta chains. Amino-acid sequences established of the isolated chains have already been communicated.     

Degradation of cellulose by nitrogen-fixing strain of Bacillus polymyxa.

During an intensive screening programme, several strains of cellulolytic bacteria were isolated. One nitrogenase-positive strain able to degrade filter paper, Avicel cellulose, carboxymethyl cellulose and cellobiose was selected for further study. On the basis of biochemical characteristics and Mol % of G+C content, the selected strain was identified as Bacillus polymyxa. The highest production of the enzymes degrading filter paper (FP-ase) and carboxymethyl cellulose (CMCase) by B. polymyxa was observed in Park's medium suplemented with Avicel cellulose. The investigated strain of bacteria produced cellulosome-like structures as was shown by transmission electron microscopy.     

Role of Sodium Carboxymethyl Cellulose and Hydroxyethyl Starch in Hematopoietic Cell Line Cultures

Sodium carboxymethyl cellulose and hydroxyethyl starch were found to support rapid growth of two hematopoietic cell lines. The polymers were not metabolized by the cells. In the presence of these compounds, lower rates of glucose utilization and lactic acid production were observed. The uptake of glucose by the cells decreased as the concentration of the polymer in the medium was increased. These results indicate that sodium carboxymethyl cellulose and hydroxyethyl starch probably protect the cells against physical stress in suspended cultures.     

Modification of microstructure and selected physicochemical properties of bacterial cellulose produced by bacterial isolate using hydrocolloid-fortified Hestrin–Schramm medium

Bacterial cellulose (BC) is a biopolymer with applications in numerous industries such as food and pharmaceutical sectors. In this study, various hydrocolloids including modified starches (oxidized starch—1404 and hydroxypropyl starch—1440), locust bean gum, xanthan gum (XG), guar gum, and carboxymethyl cellulose were added to the Hestrin-Schramm medium to improve the production performance and microstructure of BC by Gluconacetobacter entanii isolated from coconut water. After 14-day fermentation, medium supplemented with 0.1% carboxymethyl cellulose and 0.1% XG resulted in the highest BC yield with dry BC content of 9.82 and 6.06 g/L, respectively. In addition, scanning electron microscopy showed that all modified films have the characteristic three-dimensional network of cellulose nanofibers with dense structure and low porosity as well as larger fiber size compared to control. X-ray diffraction indicated that BC fortified with carboxymethyl cellulose exhibited lower crystallinity while Fourier infrared spectroscopy showed characteristic peaks of both control and modified BC films.     

Extracellular proteins secreted by the basidiomycete Schizophyllum commune in response to carbon source

The secretion of 1,4-beta-D-glucanases by the basidiomycete Schizophyllum commune in response to cellulose or cellobiose has been studied. The proteins were labeled with 35S, and the secretion of enzymes was measured by beta-glucosidase and carboxymethyl cellulase activities and by immunoprecipitation with specific antibodies. The antigen proteins used were a beta-glucosidase (Mr, 93,000), an avicelase (avicelase II; Mr, 64,000), and a carboxymethyl cellulose (carboxymethyl cellulase I; Mr 41,000). The beta-glucosidase was initially secreted as an Mr 110,000 form, which was followed later by lower-molecular-weight (88,000 to 93,000) forms. The avicelase II, which accounted for about 50% of the secreted labeled protein, had an Mr of 64,000. Secretion of the related avicelase I (Mr 61,000) followed later. The carboxymethyl cellulose I was secreted in two molecular weight forms, Mr 44,000 and 41,000. The evidence is consistent with the idea that three genes account for the secreted glucanase activities. Other species result from different glycosylation or proteolytic cleavage processing, which may occur during or after secretion. The beta-glucosidase secretion appears to be regulated differently than that of avicelase II or carboxymethyl cellulase I; the latter two were regulated coordinately under the conditions used in this work. No common immune determinants between the three antigens were observed.     

Characterization of fibrous compound of Golgi vesicles in tapetal cells ofDelphinium

Summary It was shown that Golgi structures abundantly appearing in tapetal cells ofDelphinium Ajacis L. developing anthers, prior to meiocytes meiosis, show a fine fibrous material within their vesicles. At the time of the formation of tapetal cell wall this fibrous component, released by an exocytotic process, is incorporated into the cell wall. The membrane of dictyosomes derived vesicles participates in the development of plasma membrane. Fibrous material appears to be morphologically similar to the fibrils of tapetal cell wall; this cell wall gives a positive reaction for cellulose and pectins, as visible in the light microscope. Moreover, the fibrous and pectinase resistant compound of dictyosomes derived vesicles and the fibrils of cell wall disappear partly after cellulase digestion which proves their cellulosic character. On the other hand pectinase treatment as well as ruthenium red staining suggest associated with cellulose pectins within Golgi vesicles.     

Properties of a genetically reconstructed Prevotella ruminicola endoglucanase.

A pUC19-derived plasmid was constructed that coded for a hybrid cellulase with the Thermomonospora fusca E2 cellulose-binding domain at its C terminus joined to the Prevotella ruminicola 40.5-kDa carboxymethyl cellulase (CMCase). The hybrid enzyme was purified and characterized enzymatically. It bound tightly to cellulose, and its specific activities on carboxymethyl cellulose, amorphous cellulose, and ball-milled cellulose were 1.5, 10, and 8 times that of the 40.5-kDa CMCase, respectively. Furthermore, the modified enzyme gave synergism with an exocellulase in the degradation of filter paper, while the 40.5-kDa CMCase did not.     

Cloning of a Gene Cluster from Cellvibrio mixtus which Codes for Cellulase, Chitinase, Amylase, and Pectinase

The soil isolate Cellvibrio mixtus UQM2294 degraded a variety of polysaccharides including microcrystalline cellulose. Among 6,000 cosmid clones carrying C. mixtus DNA, constructed in Escherichia coli with pHC79, 50 expressed the ability to degrade one or more of the following substrates: carboxymethyl cellulose, chitin, pectin (polygalacturonic acid), cellobiose, and starch. These degradative genes are encoded in a single 94.1-kilobase segment of the C. mixtus genome; a preliminary order of the genes is starch hydrolysis, esculin hydrolysis, cellobiose utilization, chitin hydrolysis, carboxymethyl cellulose hydrolysis, and polygalacturonic acid hydrolysis. A restriction endonuclease cleavage map was constructed, and the genes for starch, carboxymethyl cellulose, cellobiose, chitin, and pectin hydrolysis were subcloned.     

Properties of a genetically reconstructed Prevotella ruminicola endoglucanase.

A pUC19-derived plasmid was constructed that coded for a hybrid cellulase with the Thermomonospora fusca E2 cellulose-binding domain at its C terminus joined to the Prevotella ruminicola 40.5-kDa carboxymethyl cellulase (CMCase). The hybrid enzyme was purified and characterized enzymatically. It bound tightly to cellulose, and its specific activities on carboxymethyl cellulose, amorphous cellulose, and ball-milled cellulose were 1.5, 10, and 8 times that of the 40.5-kDa CMCase, respectively. Furthermore, the modified enzyme gave synergism with an exocellulase in the degradation of filter paper, while the 40.5-kDa CMCase did not.     

Modification of cellulose films by adsorption of CMC and chitosan for controlled attachment of biomolecules

The adsorption of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) on cellulose supports were investigated. The dynamics and extent of related adsorption processes were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Amine groups were installed on the cellulose substrate by adsorption of chitosan from aqueous solution, which allowed for hIgG to physisorb from acid media and produced a functionalized substrate with high surface density (10 mg/m(2)). hIgG adsorption from neutral and alkaline conditions was found to yield lower adsorbed amounts. The installation of the carboxyl groups on cellulose substrate via carboxymethylated cellulose (CMC) adsorption from aqueous solution enhanced the physisorption of hIgG at acidic (adsorbed amount of 5.6 mg/m(2)) and neutral conditions. hIgG adsorption from alkaline conditions reduced the surface density. BSA was used to examine protein attachment on cellulose after modification with chitosan or carboxymethyl cellulose. At the isoelectric point of BSA (pI 5), both of the surface modifications enhanced the adsorption of this protein when compared to that on unmodified cellulose (a 2-fold increase from 1.7 to 3.5 mg/m(2)). At pH 4, the electrostatic interactions favored the adsorption of BSA on the CMC-modified cellulose, revealing the affinity of the system and the possibility of tailoring biomolecule binding by choice of the surface modifier and pH of the medium.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight