Alginate Beads (alginate + bead)

Distribution by Scientific Domains


Selected Abstracts


Intraportal Transplantation of Allogenic Pancreatic Islets Encapsulated in Barium Alginate Beads in Diabetic Rats

ARTIFICIAL ORGANS, Issue 11 2003
Stephan Schneider
Abstract:, The survival of microencapsulated islets transplanted into the unmodified peritoneal cavity is limited, even if capsular overgrowth is restricted to a minimum, due to an insufficient oxygen supply to the islets. Therefore, research efforts should focus on finding or creating a transplantation site, which permits a closer contact between the encapsulated islets and the blood. For this reason, the liver could be an interesting candidate. The aim of the present study was to test the hypothesis that the intraportal transplantation of allogenic islets encapsulated in small-sized barium alginate beads is safe and succeeds to induce normoglycemia in diabetic rats. The intraportal transplantation of 1,500 islets encapsulated in barium alginate beads leads within 10 h and up to 24 h to blood sugar concentrations below 40 mg/dL, most likely due to an acute cell lysis of the graft. Afterwards, the reappearance of the diabetic state could be detected in these animals. Most likely these findings are induced by a sudden hypoxia to the islets. We believe that the occlusion of small- and medium-sized portal venules by the alginate beads is responsible for this effect. Therefore, in forthcoming studies, barium alginate beads, with a diameter below 350 µm, stabilized with medical approved additives should be used. [source]


Immobilized Metal Affinity Chromatography without Chelating Ligands: Purification of Soybean Trypsin Inhibitor on Zinc Alginate Beads

BIOTECHNOLOGY PROGRESS, Issue 1 2002
Munishwar N. Gupta
Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL -1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. [source]


Tunable transport of glucose through ionically-crosslinked alginate gels: Effect of alginate and calcium concentration

JOURNAL OF APPLIED POLYMER SCIENCE, Issue 5 2008
Mari-Kate E. McEntee
Abstract Alginate beads have numerous biomedical applications, ranging from cell encapsulation to drug release. The present study focuses on the controlled release of glucose from calcium-alginate beads. The effects of alginate concentrations (1,6 wt %) and calcium chloride concentrations (0.1,1.0M) on glucose release from beads were examined. It was found that the time required for complete glucose release from beads could be tuned from 15 min to over 2 h, simply by varying alginate and calcium chloride concentrations in beads. For calcium-alginate beads with sodium alginate concentrations of 1,4 wt %, higher sodium alginate concentrations lead to more prolonged release of glucose and thus a smaller value of a rate constant k, a parameter shown to be proportional to the diffusion coefficient of glucose in the alginate gel. For beads with sodium alginate concentrations of 4,6 wt %, there was no statistically significant difference in k values, indicating a lower limit for glucose release from calcium-alginate beads. Similarly, higher calcium chloride concentrations appear to extend glucose release, however, no conclusive trend can be drawn from the data. In a 50 : 50 mixture of calcium-alginate beads of two different alginate concentrations (1 and 4 wt %), glucose release showed a two-step profile over the time range of 20,50 min, indicating that the pattern and time of glucose release from beads can be tuned by making combinations of beads with varying alginate and/or calcium chloride concentrations. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source]


Release behavior of freeze-dried alginate beads containing poly(N -isopropylacrylamide) copolymers

JOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2008
Jae-Hyung Choi
Abstract Beads composed of alginate, poly(N -isopropylacrylamide) (PNIPAM), the copolymers of N -isopropylacrylamide and methacrylic acid (P(NIPAM- co -MAA)), and the copolymers of N -isopropylacrylamide, methacrylic acid, and octadecyl acrylate (P(NIPAM- co -MAA- co -ODA)), were prepared by dropping the polymer solutions into CaCl2 solution. The beads were freeze-dried and the release of blue dextran entrapped in the beads was observed in distilled water with time and pH. The degree of release was in the order of alginate bead < alginate/PNIPAM bead , alginate/P(NIPAM- co -MAA) bead < alginate/P(NIPAM- co -MAA- co -ODA) bead. On the other hand, swelling ratios reached steady state within 20 min, and the values were 200,800 depending on the bead composition. The degree of swelling showed the same order as that of release. Among the beads, only alginate/P(NIPAM- co -MAA- co -ODA) bead exhibited pH-dependent release. At acidic condition, inter- and intraelectrostatic repulsion is weak and P(NIPAM- co -MAA- co -ODA) could readily be assembled into an aggregate due to the prevailing hydrophobic interaction of ODA. Thus, it could block the pore of bead matrix, leading to a suppressed release. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source]


Intracellular pH homeostasis in the filamentous fungus Aspergillus niger

FEBS JOURNAL, Issue 14 2002
Stephan J. A. Hesse
Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+ -alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ,pH over the cytoplasmic membrane of 6.6,6.7 was reached (pHex 0.7,0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ,pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ,pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m -chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 µm CCCP), transient (2 µm CCCP) or permanent (10 µm CCCP) collapse of the vacuolar membrane ,pH. Nonlethal levels of the metabolic inhibitor azide (N3,, 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid-secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively). [source]


pH-controlled cell release and biomass distribution of alginate-immobilized Lactococcus lactis subsp. lactis

JOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2001
G. Klinkenberg
Aims:,To investigate the growth and release of Lactococcus lactis subsp. lactis in gel beads and to affect rates of cell release by changing the growth conditions. Methods and Results:,The rate of release and the distribution of immobilized L. lactis subsp. lactis in alginate beads were studied in continuous fermentations for 48 h. A change in operating pH from 6·5 to 9·25 initially reduced the ratio of the rates of cell release to lactate production by almost a factor of 105. Compared with fermentations at pH 6·5, growth at pH 9·25 also increased the final internal bead biomass concentration by a factor of 5 and increased the final rate of lactate production by 25%. After 48 h, the ratio of the rates of cell release to lactate production was still 10 times lower than in fermentations at pH 6·5. Conclusions:,A change in the operating pH from 6·5 to 9·25 reduced rates of cell release throughout 48 h of fermentation and increased the final rates of lactate production and internal bead biomass concentration. Significance and Impact of the Study:,These data illustrate that diffusional limitations and corresponding pH gradients can be exploited in affecting the distribution of immobilized growing cells and their concomitant release. [source]


Transcriptional and proteolytic regulation of the insulin-like growth factor-I system of equine articular chondrocytes by recombinant equine interleukin-1,

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006
Ryan M. Porter
Interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I), which have opposing effects on matrix metabolism within articular cartilage, are thought to play prominent roles in the pathogenesis of osteoarthritis. To better understand the link between these anabolic (IGF-I) and catabolic (IL-1) stimuli, we examined exogenous IL-1 regulation of the IGF-I signaling system of articular chondrocytes (ACs). Equine ACs from non-arthritic stifle joints were expanded in monolayer culture, encapsulated for 10 days in alginate beads, and stimulated as high-density monolayers with recombinant equine IL-1, (0, 1, 10 ng/ml) for 48 h. IL-1, enhanced expression of IGF-IR levels, as determined by both [125I]-IGF-I binding studies and Western blotting, while reducing the concentration of endogenous IGF-I detected in conditioned media by radioimmunoassay. Western ligand blotting revealed that chondrocytes primarily secreted IGF binding proteins (IGFBPs) with molecular weights of 28,30 and 32,34 kDa, which were identified as IGFBPs 5 and 2, respectively, and that IL-1, treatment diminished IGFBP-2, the prominent homolog in conditioned media. Northern blot analysis suggested IL-1, regulation of IGF-I and, to some extent, IGF-IR was mediated by transcription; however, the cytokine did not affect IGFBP-2 expression. To test for evidence of proteolysis by matrix metalloproteinases (MMPs), additional cultures were co-incubated with inhibitors for MMPs 2/9, 3, and 8. IGFBP-2 suppression was partially reversed by gelatinase (MMP-2/9) inhibition. In summary, these findings further delineate the role of IL-1 as a key regulator of the IGF-I system within articular cartilage, demonstrating that regulation occurs through both direct (transcriptional) and indirect (proteolytic) mechanisms. J. Cell. Physiol. 209: 542,550, 2006. © 2006 Wiley-Liss, Inc. [source]


Chlorophenol dehalogenation in a magnetically stabilized fluidized bed reactor

AICHE JOURNAL, Issue 3 2006
Lisa J. Graham
Abstract Aromatic halocarbons are often present in contaminated aquifers, surface waters, wastewater streams, soils, and hazardous wastes. The dehalogenation of p-chlorophenol as a model compound in both the aqueous phase and in slurries of contaminated solids using a magnetically stabilized fluidized bed (MSFB) reactor is discussed. Composite palladium-iron (Pd/Fe) media are employed as both catalyst and sacrificial reactant for the reductive dechlorination of p-chlorophenol. Calcium alginate beads impregnated with Pd/Fe granules are fluidized in a recirculating aqueous stream containing either dissolved p-chlorophenol or a slurry of soil contaminated with this chlorocarbon. Magnetic stabilization of the fluidized bed allows substantially higher rates of mass transfer than would otherwise be achievable, and allows circulation of contaminated solids while fluidization media are retained. Anoxic conditions are sustained under a nitrogen purge and the solution pH of 5.8 is maintained by active control to minimize surface fouling by hydroxides, and to minimize mass-transfer resistances resulting from the surface accumulation of hydrogen bubbles. A model of this process is described and the resulting predictions are compared to the experimentally derived data. © 2005 American Institute of Chemical Engineers AIChE J, 2006 [source]


Sustained hypoxia enhances chondrocyte matrix synthesis

JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2009
Christian H. Coyle
Abstract Articular cartilage is an avascular tissue with chondrocytes in the deeper zones existing under conditions of sustained hypoxia. Using a hypoxic chamber to provide controlled hypoxia, this study was performed to determine whether sustained hypoxia enhances the production of cartilage matrix proteins. Freshly isolated primary bovine articular chondrocytes were encapsulated in three-dimensional alginate beads and maintained at 2% oxygen with media changes using media pre-equilibrated to 2% oxygen. Immunolocalization of HIF-1, was performed to verify hypoxic conditions. Sustained hypoxia resulted in an increase in proteoglycan synthesis after only 1 day, as measured by 35S-sulfate incorporation. This increase was maintained for the duration of the 17 day study. After 17 days of hypoxic culture, increases in total type II collagen and COL2A1 gene expression were probed by indirect immunofluorescence, type II collagen ELISA, and real-time qPCR; in addition, increased glycosaminoglycan deposition was observed as determined by chemical analysis. These studies show that sustained hypoxia enhances articular chondrocyte matrix synthesis and viability in three-dimensional alginate culture. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 793,799, 2009 [source]


Controlled release tamsulosin hydrochloride from alginate beads with waxy materials

JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2005
Min-Soo Kim
The objective of this study was to develop oral controlled release delivery systems for tamsulosin hydrochloride (TSH) using alginate beads with various waxy materials, such as Compritol 888 ATO, Precirol ATO 5 and Gelucires. The beads were prepared from sodium alginate,waxy material,TSH slurry dropped onto calcium chloride to form spherical beads. The effects of the addition of various waxy materials to alginate beads on the drug encapsulation efficiency, bead size and morphology were investigated. The drug encapsulation efficiency significantly increased with the addition of waxy materials. The TSH-loaded alginate beads with and without waxy materials were almost spherical particles with an average diameter of 1.44 and 1.22mm, respectively. In dissolution study, the TSH-loaded alginate beads with waxy materials exhibited controlled release behaviour over a 6-h period, while beads without waxy materials showed release of 100% TSH within 2h. These results may be attributed to the formation of a more rigid alginate matrix structure due to incorporated waxy materials. From the Dunnett's t -test and the f2 factor, the release of TSH from alginate beads, a similar dissolution pattern to that of the marketed product (Harunal capsules) could be achieved by adding Gelucire 50/13 into TSH-loaded alginate beads. From these results, oral controlled release of TSH could be achieved with loading in alginate beads with waxy materials, such as Compritol 888 ATO, Precirol ATO 5 and Gelucires. [source]


Induction of CD44 cleavage in articular chondrocytes

ARTHRITIS & RHEUMATISM, Issue 5 2010
Nobunori Takahashi
Objective The hyaluronan receptor CD44 provides chondrocytes with a mechanism for sensing and responding to changes in the extracellular matrix. The purpose of this study was to document the fragmentation and loss of CD44 and to determine the likely mechanisms involved. Methods A polyclonal anti-CD44 cytotail antibody was generated to detect CD44 fragmentation by Western blot analysis. Chondrocytes were isolated from human or bovine articular cartilage. Primary articular chondrocytes were treated with interleukin-1, (IL-1,), hyaluronan oligosaccharides, or phorbol myristate acetate or were passaged and subcultured in monolayer to induce dedifferentiation. Conditions that altered the capacity of CD44 to transit into lipid rafts, or pharmacologic inhibitors of metalloproteinase or ,-secretase activity were used to define the mechanism of fragmentation of CD44. Results Chondrocytes from osteoarthritic cartilage exhibited CD44 fragmentation as low molecular mass bands, corresponding to the CD44-EXT and CD44-ICD bands. Following dedifferentiation of chondrocytes or treatment of primary chondrocytes with hyaluronan oligosaccharides, IL-1,, or phorbol myristate acetate, CD44 fragmentation was enhanced. Subsequent culture of the dedifferentiated chondrocytes in 3-dimensional alginate beads rescued the chondrocyte phenotype and diminished the fragmentation of CD44. Fragmentation of CD44 in chondrocytes was blocked in the presence of the metalloproteinase inhibitor GM6001 and the ,-secretase inhibitor DAPT. Conclusion CD44 fragmentation, consistent with a signature pattern reported for sequential metalloproteinase/,-secretase cleavage of CD44, is a common metabolic feature of chondrocytes that have undergone dedifferentiation in vitro and osteoarthritic chondrocytes. Transit of CD44 into lipid rafts may be required for its fragmentation. [source]


Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen,induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures

ARTHRITIS & RHEUMATISM, Issue 12 2009
K. Blumbach
Objective Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril,associated proteins type IX collagen and cartilage oligomeric matrix protein (COMP) during cartilage matrix formation. Methods Primary chondrocytes from mice deficient in type IX collagen and COMP (double-deficient) were cultured in monolayer or alginate beads. Anchorage of matrix proteins, proteoglycan and collagen content, collagen crosslinks, matrix metalloproteinase activity, and mechanical properties of the matrix were measured. Electron microscopy was used to study the formation of fibrillar structures. Results In cartilage lacking both type IX collagen and COMP, matrilin 3 showed decreased matrix anchorage. Less matrilin 3 was deposited in the matrix of double-deficient chondrocytes, while larger amounts were secreted into the medium. Proteoglycans were less well retained in the matrix formed in alginate cultures, while collagen deposition was not significantly affected. Electron microscopy revealed similar cartilage collagen fibril diameters in the cultures of double-deficient and wild-type chondrocytes. In contrast, a larger fibril diameter was observed in the matrix of chondrocytes deficient in only type IX collagen. Conclusion Our results show that type IX collagen and COMP are involved in matrix assembly by mediating the anchorage and regulating the distribution of other matrix macromolecules such as proteoglycans and matrilins and have counteracting effects on collagen fibril growth. Loss of type IX collagen and COMP leads to matrix aberrations that may make cartilage more susceptible to degeneration. [source]


Prostaglandin E2 and its cognate EP receptors control human adult articular cartilage homeostasis and are linked to the pathophysiology of osteoarthritis

ARTHRITIS & RHEUMATISM, Issue 2 2009
Xin Li
Objective To elucidate the pathophysiologic links between prostaglandin E2 (PGE2) and osteoarthritis (OA) by characterizing the catabolic effects of PGE2 and its unique receptors in human adult articular chondrocytes. Methods Human adult articular chondrocytes were cultured in monolayer or alginate beads with and without PGE2 and/or agonists of EP receptors, antagonists of EP receptors, and cytokines. Cell survival, proliferation, and total proteoglycan synthesis and accumulation were measured in alginate beads. Chondrocyte-related gene expression and phosphatidylinositol 3-kinase/Akt signaling were assessed by real-time reverse transcription,polymerase chain reaction and Western blotting, respectively, using a monolayer cell culture model. Results Stimulation of human articular chondrocytes with PGE2 through the EP2 receptor suppressed proteoglycan accumulation and synthesis, suppressed aggrecan gene expression, did not appreciably affect expression of matrix-degrading enzymes, and decreased the type II collagen:type I collagen ratio. EP2 and EP4 receptors were expressed at higher levels in knee cartilage than in ankle cartilage and in a grade-dependent manner. PGE2 titration combined with interleukin-1 (IL-1) synergistically accelerated expression of pain-associated molecules such as inducible nitric oxide synthase and IL-6. Finally, stimulation with exogenous PGE2 or an EP2 receptor,specific agonist inhibited activation of Akt that was induced by insulin-like growth factor 1. Conclusion PGE2 exerts an antianabolic effect on human adult articular cartilage in vitro, and EP2 and EP4 receptor antagonists may represent effective therapeutic agents for the treatment of OA. [source]


Differential expression of ,B-crystallin and evidence of its role as a mediator of matrix gene expression in osteoarthritis

ARTHRITIS & RHEUMATISM, Issue 1 2009
Stijn Lambrecht
Objective Alpha B,crystallin belongs to the family of small heat-shock proteins (HSPs). The role of this protein family in chondrocytes is not well understood. The present study was undertaken to investigate expression levels of ,B-crystallin in chondrocytes isolated from healthy subjects and patients with osteoarthritis (OA), and to explore the functional role of this potentially interesting protein in chondrocyte metabolism. Methods Western blot and real-time reverse transcriptase,polymerase chain reaction (RT-PCR) analyses were performed to determine expression levels of ,B-crystallin in healthy and OA chondrocytes cultured in alginate beads. RNA interference,mediated gene knockdown was used to explore the role of this small HSP in chondrocyte biology, by transfecting low concentrations of small interfering RNA (siRNA) in cultured chondrocytes. Results We initially identified ,B-crystallin as a small HSP that was differentially expressed between healthy and OA-affected chondrocytes. The decreased abundance of this protein in OA chondrocytes was confirmed by Western blotting. Moreover, real-time RT-PCR confirmed the differential expression between chondrocytes isolated from visibly intact and visibly damaged zones of OA cartilage. The proinflammatory cytokines interleukin-1, and tumor necrosis factor , both down-regulated ,B-crystallin expression. Transfection of low concentrations of siRNA in cultured chondrocytes resulted in efficient knockdown of ,B-crystallin gene expression. This was accompanied by altered expression of the chondrocyte-specific bone morphogenetic protein 2, aggrecan, and type II collagen genes. Conclusion The present findings identify the small HSP ,B-crystallin as a novel mediator of chondrocyte matrix gene expression that may contribute to altered chondrocyte metabolism during the development of OA. [source]


Intraportal Transplantation of Allogenic Pancreatic Islets Encapsulated in Barium Alginate Beads in Diabetic Rats

ARTIFICIAL ORGANS, Issue 11 2003
Stephan Schneider
Abstract:, The survival of microencapsulated islets transplanted into the unmodified peritoneal cavity is limited, even if capsular overgrowth is restricted to a minimum, due to an insufficient oxygen supply to the islets. Therefore, research efforts should focus on finding or creating a transplantation site, which permits a closer contact between the encapsulated islets and the blood. For this reason, the liver could be an interesting candidate. The aim of the present study was to test the hypothesis that the intraportal transplantation of allogenic islets encapsulated in small-sized barium alginate beads is safe and succeeds to induce normoglycemia in diabetic rats. The intraportal transplantation of 1,500 islets encapsulated in barium alginate beads leads within 10 h and up to 24 h to blood sugar concentrations below 40 mg/dL, most likely due to an acute cell lysis of the graft. Afterwards, the reappearance of the diabetic state could be detected in these animals. Most likely these findings are induced by a sudden hypoxia to the islets. We believe that the occlusion of small- and medium-sized portal venules by the alginate beads is responsible for this effect. Therefore, in forthcoming studies, barium alginate beads, with a diameter below 350 µm, stabilized with medical approved additives should be used. [source]


Design of a Fluidized Bed Bioartificial Liver

ARTIFICIAL ORGANS, Issue 7 2000
Cécile Legallais
Abstract: The aim of this study was to design a bioreactor for extracorporeal liver supply containing alginate beads in a fluidized bed regimen. The objective was to achieve a satisfactory mixing into the bioreactor to promote the potential exchanges and mass transfers. First, we checked whether both present phases (solid: alginate beads; liquid: saline solution at 20°C) might allow for this fluidization. Then the optimal design was defined as a function of the required operating conditions, bead volume, and perfusion flow rate; the bioreactor cross section and height especially needed to be adjusted. The efficient fluidization, under optimized conditions, was proven through the follow-up of the head losses generated by the fluidized bed. Criteria for scaling up were also determined. [source]


Protein crystallization in hydrogel beads

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2005
Ronnie Willaert
The use of hydrogel beads for the crystallization of proteins is explored in this contribution. The dynamic behaviour of the internal precipitant, protein concentration and relative supersaturation in a gel bead upon submerging the bead in a precipitant solution is characterized theoretically using a transient diffusion model. Agarose and calcium alginate beads have been used for the crystallization of a low-molecular-weight (14.4,kDa, hen egg-white lysozyme) and a high-molecular-weight (636.0,kDa, alcohol oxidase) protein. Entrapment of the protein in the agarose-gel matrix was accomplished using two methods. In the first method, a protein solution is mixed with the agarose sol solution. Gel beads are produced by immersing drops of the protein,agarose sol mixture in a cold paraffin solution. In the second method (which was used to produce calcium alginate and agarose beads), empty gel beads are first produced and subsequently filled with protein by diffusion from a bulk solution into the bead. This latter method has the advantage that a supplementary purification step is introduced (for protein aggregates and large impurities) owing to the diffusion process in the gel matrix. Increasing the precipitant, gel concentration and protein loading resulted in a larger number of crystals of smaller size. Consequently, agarose as well as alginate gels act as nucleation promoters. The supersaturation in a gel bead can be dynamically controlled by changing the precipitant and/or the protein concentration in the bulk solution. Manipulation of the supersaturation allowed the nucleation rate to be varied and led to the production of large crystals which were homogeneously distributed in the gel bead. [source]


Influence of different magnetites on properties of magnetic Pseudomonas aeruginosa immobilizates used for biosurfactant production

BIOTECHNOLOGY PROGRESS, Issue 6 2009
M. Heyd
Abstract During the last decades, whole-cell immobilization has been used successfully in many bioprocesses. In particular, it is aimed at implementing continuous production processes, reaching higher production rates, and reusing the biocatalyst. In some cases, effective retention of immobilizates in the bioprocess is not feasible by membranes or sieves due to pore plugging or undesired losses of immobilizates. In the present publication, it is reported about the investigation of magnetic immobilizates of Pseudomonas aeruginosa for application in continuous biosurfactant production of rhamnolipids by foam fractionation and retention of entrained immobilizates by high-gradient magnetic separation from foam. Different materials and methods were tested with respect to important parameters, such as stability, diffusion properties or magnetic separation. Good magnetic separation of immobilizates was achieved at 5% (w/w) magnetite loading. Best results in terms of homogeneous embedding, good diffusion properties, and stability enhancement vis-ą-vis pure alginate beads was achieved with alginate beads with embedded Bayoxide® magnetite or MagPrep® silica particles. Although polyurethane immobilizates showed higher stabilities compared with alginate beads, rhamnolipid diffusion in immobilizates was superior in magnetic alginate beads. Regarding bead production, smaller immobilizates were achieved with suspension polymerization compared to droplet extrusion by the JetCutting® technology. In total, magnetic immobilizates are a promising tool for an easier handling of biocatalysts in a continuous biological production process, but they have to be adapted to the current production task.© 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]


A Feasible Enzymatic Process for d -Tagatose Production by an Immobilized Thermostable l -Arabinose Isomerase in a Packed-Bed Bioreactor

BIOTECHNOLOGY PROGRESS, Issue 2 2003
Hye-Jung Kim
To develop a feasible enzymatic process for d -tagatose production, a thermostable l -arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 °C in the immobilized enzyme system and pH 7.5 and 60 °C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 °C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor·h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L·h) and a conversion yield of 46%. [source]


Immobilized Metal Affinity Chromatography without Chelating Ligands: Purification of Soybean Trypsin Inhibitor on Zinc Alginate Beads

BIOTECHNOLOGY PROGRESS, Issue 1 2002
Munishwar N. Gupta
Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL -1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. [source]