Cultivation of cells on modified porous cellulose beads

New microcarriers for the growth of animal cells have been synthesized and studied. The preparations are porous cellulose beads, modified by diamines. Spreading and growth of L cells, MEVO and HETR cells on these beads were observed. As a result of the cultivation the number of animal cells increased 5-10-fold.     

Spatial development of the cultivation of a bone marrow stromal cell line in porous carriers

The spatial development of the cultivation of a bone marrow stromal cell line (SR-4987) in porous carriers was investigated in order to construct a three-dimensional hematopoietic culture system. Low-rate continuous agitation, 20 rpm, was an appropriate method to achieve initial adhesion of cells onto a cellulose porous beads (CPB, 100 μm pore diameter) in a spinner bottle, compared with other methods such as centrifugation and intermittent agitation. Cell growth with continuous agitation at 70 rpm after initial cell adhesion was not inferior to that at 20 rpm. A 2- and 10-fold increase in the inoculum cell concentration for CPB and another type of porous cellulose beads (Micro-cube (MC), 500 μm pore diameter) resulted in a 1.2- and 2-fold increase in final cell concentrationm, respectively. Cells attached to the MC beads and a polyester nonwoven dic (Fibra-cell (FC)) could grow and spread well on the carriers and a fibroblast-like shape was observed under scanning electron microscopy while the cells on CPB were globular. The flatness and inner surface area of these carriers may be the reason for the differences in cell morphology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preparation and Characterization of Highly Porous Direct Compression Carrier Particles with Improved Drug Loading During an Interactive Mixing Process

The aim of this study was to prepare highly porous carrier particles by emulsion solvent evaporation and compare the loading capacity of these beads with two traditional carriers, sugar beads, and microcrystalline cellulose granules during an interactive mixing process. The porous carrier particles were prepared by an emulsion solvent evaporation process using cellulose propionate as a binder, anhydrous dibasic calcium phosphate, and ion exchange resins as a fillers, and polyethylene glycol as a pore inducer. Micronized furosemide or griseofulvin powder was mixed with the same volume of each carrier in an interactive mixing process. The tableting properties, drug loading per unit volume of carrier, content uniformity of the mixtures, and dissolution of the drugs from the mixtures were measured. The results showed that highly porous microcapsules with desirable hardness equivalent to that of sugar beads and MCC granules were successfully prepared. On average the loading capacity of the new carrier was 310% that of sugar beads and 320% that of MCC granules during an interactive mixing process with very good content uniformity. The tableting properties of the microcapsules were equivalent to that of microcrystalline cellulose granules, and the dissolution of the drugs from interactive mixtures prepared with the new carrier was equivalent to that of drug suspensions. This showed that the prepared microcapsule carrier could be used to improve the loading capacity during an interactive mixing and to prepare tablets by direct compression.     

Application of bacterial cellulose pellets in enzyme immobilization

Over recent years, there has been a growing interest in the use of cellulose materials in bioprocessing technologies. Bacterial cellulose which is the pure cellulose has unique physical properties which differ from those of plant cellulose and has therefore attracted attention as a new functional material. The applications of bacterial cellulose rarely use the pellet type but it has potential in enzyme immobilization since pellet form is usually used in this field. In this research, Glucoamylase which is widely used in the food industry was immobilized on bacterial cellulose beads after testing using various activation procedures. The results showed that the epoxy method with glutaraldehyde coupling was the best method. After comparison of the different types of bacterial cellulose beads for glucoamylase immobilization, the wet bacterial cellulose beads of the smallest size (0.5–1.5 mm) were the best support. The immobilization of enzyme enhances its stability against changes in the pH value and temperature especially in the lower temperature region. The relative activity of the immobilized glucoamylase was still above 77% at pH 2.0 and it was the highest value in the literature. The relative activities were more than 68% in the lower temperature region even at 20 °C. Thus, bacterial cellulose beads are a practical potential support for the preparation of immobilized enzymes in industrial applications.     

New affinity chromatography method for the separation of vanillic acid induced forms of fungal peroxidase from Trametes versicolor on porous glass beads activated with vanillin

An affinity chromatography method of separating peroxidase from the mycelium of Trametes versicolor on porous glass beads activated with vanillin is described. Two forms of peroxidase isolated by this technique were rechromatographed on CM-32 cellulose columns. Both isolated forms of enzyme were induced in the mycelium with vanillic acid.     

Preparation and kinetic behavior of immobilized whole cell biocatalysts

Actinoplanes missouriensis (for glucose isomerase), Kluyveromyces fragilis (for beta-galactosidase), and Saccharomyces cerevisiae (for invertase) cells were successfully entrapped within cellulose and cellulose di- and triacetate beads employing several carried solvent systems. Cellulose beads prepared using a melt of dimethylsulfoxide (DMSO) and N-ethylpyridinium chloride (NEPC), or cellulose diacetate using a mixture of acetone and DMSO as solvent, were found to be promising as carriers for the invertase system, cellulose triacetate beads with DMSO as solvent for yeast beta-galactosidase, and cellulose beads with a melt of DMSO and NEPC as solvent for glucose isomerase. The kinetic behavior of A. missouriensis glucose isomerase whole cell cellulose beads in a plug-flow column reactor was studied as an example system in greater detail.     

Corrigendum to “Dihydropyrimidine dehydrogenase: a flavoprotein with four iron–sulfur clusters” [Biochim. Biophys. Acta 1701 (2004) 61–74]

Lixelle is a direct hemoperfusion-type adsorption column that was developed to selectively eliminate β2-microglobulin (β2-m) from the circulating blood of patients with dialysis-related amyloidosis (DRA). The adsorbent in Lixelle comprises porous cellulose beads to which hydrophobic hexadecyl alkyl chain is covalently bound. One milliliter of wet Lixelle beads eliminates more than 1 mg of β2-m in vitro. In hemodialysis patients who were treated with Lixelle, Lixelle improved joint pain, nocturnal awakening, pinch strength, motor terminal latency, and their activity of daily living. The adsorbent adsorbs β2-m selectively but not specifically, as well as inflammatory cytokines such as interleukin-1β and IL-6 which are considered to be involved in the development of DRA. Lixelle treatments reduce the circulating levels of β2-m and inflammatory cytokines, thereby improving the symptoms of patients with DRA.     

beta(2)-Microglobulin-selective direct hemoperfusion column for the treatment of dialysis-related amyloidosis

Lixelle is a direct hemoperfusion-type adsorption column that was developed to selectively eliminate beta2-microglobulin (beta2-m) from the circulating blood of patients with dialysis-related amyloidosis (DRA). The adsorbent in Lixelle comprises porous cellulose beads to which hydrophobic hexadecyl alkyl chain is covalently bound. One milliliter of wet Lixelle beads eliminates more than 1 mg of beta2-m in vitro. In hemodialysis patients who were treated with Lixelle, Lixelle improved joint pain, nocturnal awakening, pinch strength, motor terminal latency, and their activity of daily living. The adsorbent adsorbs beta2-m selectively but not specifically, as well as inflammatory cytokines such as interleukin-1beta and IL-6 which are considered to be involved in the development of DRA. Lixelle treatments reduce the circulating levels of beta2-m and inflammatory cytokines, thereby improving the symptoms of patients with DRA.     

Bacterial cellulose-chitosan composite hydrogel beads for enzyme immobilization

In this work, we report the preparation of bacterial cellulose (BC)-chitosan composite hydrogel beads by co-dissolution of BC and chitosan in 1-ethyl-3-methylimidazolium acetate and subsequent reconstitution with distilled water. The BC-chitosan hydrogel beads were used as enzyme supports for immobilizing Candida rugosa lipase by physical adsorption and covalent cross-linking. BC-chitosan hydrogel beads immobilized lipase more efficiently than microcrystalline cellulose (MCC)-chitosan hydrogel beads. The amount of protein adsorbed onto BCchitosan beads was 3.9 times higher than that adsorbed onto MCC-chitosan beads, and the catalytic activity of lipase was 1.9 times higher on the BC-chitosan beads. The lipase showed the highest thermal and operational stability when covalently cross-linked on BC-chitosan hydrogel beads. The half-life time of the lipase cross-linked on BC-chitosan bead at 60°C was 22.7 times higher than that of free lipase. Owing to their inherent biocompatibility and biodegradability, the BC-chitosan composite hydrogel beads described here could be used to immobilize proteins for various biomedical, environmental, and biocatalytic applications.     

Properties of cellulase immobilized on agarose gel with spacer

Cellulase produced by fungus Trichoderma viride was immobilized on agarose beads (Sepharose 4B) activated by cyanogen bromide and also on activated agarose beads that contained spacer arm (activated CH-Sepharose 4B and Affi-Gel 15). The CMCase activity retained by immobilized cellulase on activated Sepharose containing the spacer tended to be higher than that immobilized without spacer, although the extent of protein immobilization was lower. Also, the higher substrate specificity for cellulase immobilized on beads with spacer was obtained for cellobiose, acid-swollen cellulose, or cellulose powder. The hydrolysis product from their substrates was mainly glucose.     

Evaluation and comparison of tailor-made stationary phases based on spherical silica-based beads for capillary electrochromatography via peptide separation analysis

Small cyclic peptides have been employed to elucidate the performance of novel sorbents as stationary phases in capillary electrochromatography (CEC). In this paper chain length dependencies for ordinary liquid chromatographic sorbents are reported together with findings acquired on beads specifically designed to suit CEC. The latter, tailor-made, spherical, porous silica exhibits a distinguished surface modification to meet the criteria anticipated to enhance performance profiles in CEC. With well-characterised peptides resembling the analytes, probing of the CEC system in a systematic manner (predominantly via the organic modifier content of the background electrolyte (BE)) reveals insight into the complex interplay occurring in such analytical systems at the molecular and sub-molecular level in particular upon various modes of interaction.     

Examination of immobilized fungal cells by phase-contrast and scanning electron microscopy

Conidia of Penicillium urticae were immobilized in kappa-carrageenan beads and then shaken, in a growth-supporting medium to yield an in situ grown population of mycelia. The physical stability of these beads and the degree of mycelial growth inside the beads were significantly affected by the concentrations of kappa-carrageenan and locust bean gum (LBG) in the bead matrix and by the porous or nonporous nature of the interior. Thus 16-h-old porous and nonporous beads, prepared from 1.25% kappa-carrageenan, 0.5% LBG, and conidia, possessed a very dense mycelial mass at the surface. Only the porous beads possessed a moderately dense mycelial mass at the centre. The conidia at the centre of nonporous beads either failed to germinate or formed very small germ tubes. When washed, 36-h-old porous beads were repeatedly (i.e., 48 h) transferred into nitrogen-free medium, the density of mycelia at the centre increased to equal that at the surface after three transfers or 8 days. Mycelia at the surface exhibited signs of physical damage, while those in the centre did not. The addition of 100 micrograms/mL of cycloheximide to these replacement cultures was reflected by the distortion of interior mycelia.     

Biosorption of Zn(II) on the different Ca-alginate beads from aqueous solution

The performance of a new biosorbent system, consisting of a fungal biomass immobilized within an orange peel cellulose absorbent matrix, for the removal of Zn(2+) heavy metal ions from an aqueous solution was tested. The amount of Zn(II) ion sorption by the beads was as follows; orange peel cellulose with Phanerochaete chrysosporium immobilized Ca-alginate beads (OPCFCA) (168.61 mg/g) > orange peel cellulose immobilized Ca-alginate beads (OPCCA) (147.06 mg/g) > P. chrysosporium (F) (125.0 mg/g) > orange peel cellulose (OPC) (108.70 mg/g) > plain Ca-alginate bead (PCA) (98.26 mg/g). The Zn(2+) concentration was 100 to 1000 mg/L. The widely used Langmuir and Freundlich isotherm models were utilized to describe the biosorption equilibrium process. The isotherm parameters were estimated using linear and non-linear regression analysis. The Box-Behnken model was found to be in close agreement with the experimental values, as indicated by the correlation coefficient value of 0.9999.     

Biofilm of Pseudomonas C12B on glass support as catalytic agent for continuous SDS removal

The purpose of this work was to investigate the biodegradation of Sodium dodecylsulphate, a common surfactant used in commercial detergent formulations, by immobilized cells of the surfactant-degrading bacterium Pseudomonas C12B. Cells were immobilized by adsorption on porous glass beads with either unmodified or silanized surface. Data showed a direct relation between the SDS concentration in the medium and formation of the biofilm on glass beads. Bioreactors with Pseudomonas C12B cells immobilized on both types of porous glass beads were prepared. Both types showed equivalent efficiency to remove SDS. This biocatalyst was also effective to remove anionic surfactants from commercial dishwashing liquid (Jar) and shampoo (Clear) under continuous operation.     

Evaluation of activation methods with cellulose beads for immunosorbent purification of immunoglobulins

Attempts were made to evaluate the chemical properties of cross-linked cellulose beads in order to utilize them as a support material for the large scale purification of specific immunoglobulins via immunosorbent chromatography with goat anti-human IgG serving as the model affinity ligand. Since these cellulose beads have sufficient mechanical strength to sustain a high flow rate of viscous fluids, they are ideal for rapid purification of large fluid volumes. The beads were activated with cyanogen bromide, tosyl chloride, cyanuric chloride or oxidation reagents such as chromium trioxide, sodium periodate and dimethylsulfoxide-carbodiimide before the antibodies were immobilized under mild conditions. The inert hydroxyl groups were thus converted into more active cyanate ester, tosylate, reactive acyl-like chlorines, and carbonyl groups which readily react with amino groups of antibodies. Antibodies were immobilized on the activated cellulose beads under mild conditions with an average yield of 42.3%. Every immobilization method had disadvantages. The binding activity of the immobilized antibody depended on its concentration. Very high binding efficiency was achieved when the concentration was less than 0.2 mg/ml; however, the efficiency was only about 5% when the concentration was greater than 2 mg/ml. The binding activity of immobilized antibodies was affected by the steric factors imposed by the support material but not affected by the immobilization methods. Although some non-specific interaction between plasma components and the cellulose bead immunosorbent occurred, specific immunoglobulin could be purified from plasma in a single step.     

A standardized spectrophotometric assay of endoglycanase activities using dyed, amorphous polysaccharides

This is a new technique to assay virtually any endoglycanase activity where enough polysaccharide material is available to allow for production of the amorphous, dyed beads used as substrates. It allows for a direct comparison of endoglycanase activities between laboratories since dyed beads from at least the most common polysaccharides such as cellulose, xylan, mannan, and chitin are now under development and will soon be commercially available; cellulose beads already are. It is a very sensitive technique and enzyme activities can be measured using a nonsophisticated spectrophotometer.     

A simple and reliable method for the detection of sialylation in complex/hybrid-type carbohydrate chains of glycoproteins by mixed lectins

A practical and convenient method for discriminating between the presence and the absence of sialic acid in carbohydrate chains of glycoproteins was devised using paramagnetic beads and two lectins, Sambucus sieboldiana lectin (SSA) and Ricinus communis agglutinin (RCA120). The glycoproteins of transferrin or thyroglobulin were firstly captured to paramagnetic beads that were previously coated with corresponding antibody, and then the lectins of RCA120-biotin and SSA-FITC were bound to the glycoproteins on the paramagnetic beads. Finally, the fluorescence intensity of the beads was measured to determine the ratios of lectins RCA120-biotin/Cy5-streptavidin to SSA-FITC. The mixed lectins method showed bigger difference of the ratios between the presence and the absence of sialic acid, indicating higher discrimination efficiency than the ordinary non-mixed lectins method. Furthermore, statistical analysis by two-side t-test indicated that the mixed lectins method was more highly reliable than the ordinary non-mixed lectins method in discriminating between the presence and the absence of sialic acid. The reaction with the two lectins can be performed in a single tube and readily automated taking advantage of the use of paramagnetic beads.     

Selective assay for endotoxin using poly(ε-lysine)-immobilized Cellufine and Limulus amoebocyte lysate (LAL)

We developed a selective endotoxin (lipopolysaccharide; LPS) assay using poly(ε-lysine)-immobilized cellulose beads (PL-Cellufine) and Limulus amoebocyte lysate (LAL). First, LPS was selectively adsorbed on the beads in a solution containing various LAL-inhibiting or LAL-enhancing compounds (e.g., amino acids, enzymes) and the LPS adsorbed on the beads was separated from the compounds by centrifugation. Second, the LPS adsorbed on the beads directly reacted with the LAL reagent, and the LPS concentration was determined by a turbidimetric time assay. The accuracy of the adsorption method with PL-Cellufine was high compared with that of a common solution method. Apparent recovery of LPS from compound solution was 88-120%.     

Immunocapillarymigration with enzyme-labeled antibodies: Rapid quantification of C-reactive protein in human plasma

Various procedures to improve the sensitivity and precision of antigen quantitation by immunocapillarymigration are investigated. The best results are obtained when using porous strips of cellulose acetate with covalently attached antibodies and when enzyme-labeled antibodies are used to expose the antigen-covered areas of the strips. Such a system has a sensitivity of 0.15 mg/liter and a precision of 7%. It allows a rapid quantitation of human C-reactive protein without the use of laboratory instrumentation.     

Preparation and activity of carbonic anhydrase immobilized on porous silica beads and graphite rods

Techniques for the immobilization of bovine carbonic anhydrase (BCA) on porous silica beads and graphite are presented. Surface coverage on porous silica beads was found to be 1.5 x 10(-5) mmol BCA/m(2), and on graphite it was 1.7 x 10(-3) mmol BCA/m(2) nominal surface area. Greater than 97% (silica support) and 85% (graphite support) enzyme activity was maintained upon storage of the immobilized enzyme for 50 days in pH 8 buffer at 4 degrees C. After 500 days storage, the porous silica bead immobilized enzyme exhibited over 70% activity. Operational stability of the enzyme on silica at 23 degrees C and pH 8 was found to be 50% after 30 days. Catalytic activity expressed as an apparent second-order rate constant K'(Enz) for the hydrolysis of p-nitrophenyl acetate (p-NPA) catalyzed by BCA immobilized on silica beads and graphite at pH 8 and 25 degrees C is 2.6 x 10(2) and 5.6 x 10(2) M(-1)s(-1) respectively. The corresponding K(ENZ) value for the free enzyme is 9.1 x 10(2) M(-1)s(-1). Activity of the immobilized enzyme was found to vary with pH in such a manner that the active site pK, on the porous silica bead support is 6.75, and on graphite it is 7.41. Possible reasons for a microenvironmental influence on carbonic anhydrase pK(a), are discussed. Comparison with literature data shows that the enzyme surface coverage on silica beads reported here is superior to previously reported data on silica beads and polyacrylamide gels and is comparable to an organic matrix support. Shifts in BCA-active site pK(a) values with support material, a lack of pH dependent activity studies in the literature, and differing criteria for reporting enzyme activity complicate literature comparisons of activity; however, immobilized BCA reported here generally exhibits comparable or greater activity than previous reports for immobilized BCA.