Gel Entrapment Research Articles

Pressure Cycling to Enhance an Immobilized Enzymic Reaction by Enzyme Entrapment in a Pressure-Sensitive Gel

The enzyme β-galactosidase was entrapped in poly(N-isopropylacrylamide) gel. The gel swelled or collapsed sharply upon the rise or fall of environmental pressure. The gel beads, prepared by reverse suspension polymerization, were placed in a stirred batch reactor filled with a solution of the substrate o-nitrophenol β-D-galactopyranoside. Operations under constant or cycling pressure were performed. A considerable increase in conversion rate was observed under pressure cycling operation, compared with isobaric operations at either atmospheric or high pressure. This result could be explained by the enhancement of mass transfer inside the gel beads during the pressure cycling operation.

Synthesis of yttrium aluminium garnet by the gel entrapment technique using hexamine

A novel technique has been developed for the preparation of yttrium aluminium garnet (YAG), represented by the chemical formula Y 3Al 5O 12, by achieving the homogeneous precipitation of the constituent metal hydroxides from the stoichiometric mixture of the metal nitrate solution by the addition of hexamethylene tetramine solution. Aluminium ions form a gel at pH 4, entrapping all the liquid, followed by the precipitation of yttrium ions at a higher pH. In spite of the sequential precipitation, the resultant mass is homogeneous because of the gel entrapment. Using this technique, singlephase YAG powder of high specific surface area (80 m 2 g −1) was obtained at 810 °C. Characterisation was done by thermogravimetry, differential thermal analysis, X-ray diffraction and scanning electron microscopy to ascertain the high quality of the product. The technique was subsequently used for the production of highpurity neodymium-doped YAG powder with excellent microhomogeneity on an increased batch size of 1 kg.

In vitro function of porcine hepatocyte spheroids in 100% human plasma

To assess their functional stability, porcine hepatocyte spheroids, which have great potential in bioartificial liver systems and/or xenogeneic liver transplantation, were cultured in vitro in 100% human plasma, and their ammonium detoxification capacity was measured. In a short-term functional study, collagen-gel entrapped and rotationally cultured porcine hepatocyte spheroids showed almost the same functional expression, which was approximately twice that of monolayers. No significant functional deterioration was observed up to day 10 as compared with those in a synthesized medium without collagen gel entrapment. In a longer term study (4 wk) using rotational culture, morphological and functional deterioration presumably due to immunorejection involving complement proteins and natural antibodies progressed gradually with culture time. On the termination of the culture, the detoxification ability for the residual cells was approximately two-thirds that on day 2.

Xylitol production by immobilized recombinant Saccharomyces cerevisiae in a continuous packed‐bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3–11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al3+ to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L · h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads. © 1996 John Wiley & Sons, Inc.

Xylitol production by immobilized recombinantSaccharomyces cerevisiae in a continuous packed-bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al(3+) to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L . h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads.

Immobilization of Pseudomonas sp. BA2 by entrapment in calcium alginate and its application for the production of l-alanine

The conditions for immobilizing the new l-aminoacylase-producing bacterial strain Pseudomonas sp. BA2 by entrapment in calcium alginate gel were investigated. The optimal gel concentration and cell loading were determined. It was demonstrated that the addition of the substrate n-acetyl- l-alanine to the immobilizing matrix enhanced l-aminoacylase activity. The enzymatic properties of immobilized Pseudomonas sp. BA2 were investigated to ascertain which conditions were suitable for the enzymatic reaction. Optimal pH, temperature, and concentration of Tris-maleate buffer were determined. The influence of adding CoCl 2 on the enzymatic reaction rate was studied and the optimal concentration of the activator was determined. Stability studies showed that the immobilized cell preparation is not adequate for use in repeated batch processes. Continuous operation in a stirred tank reactor allowed us to determine the biocatalyst half-life (7 h approximately) but, due to the high l-aminoacylase activity, the reactor productivity (24.5 mmol of l-alanine in 8 h) was noticeably higher than that previously obtained in a packed bed reactor with Aspergillus ochraceus pellets. The results reported in this paper show the potential for using the immobilized Pseudomonas sp. BA2 in calcium alginate to produce l-alanine; however, before large scale production can be undertaken, further biocatalyst stabilization studies have to be made.

Production of Fructooligosaccharides by Immobilized Mycelium of Aspergillus japonicus

β-Fructofuranosidase (EC 3.2.1.26) in Aspergillus japonicus mycelium was immobilized by entrapment in calcium alginate gel. After immobilization, the enzyme was active over a wider pH range, and had improved thermostability. The total amount of fructooligosaccharides produced by immobilized enzyme was similar to that produced by a free enzyme system. A packed-bed reactor was employed for production of fructooligosaccharides at 42°C using the immobilized enzyme. The reactions were continued for 35 days and only 17% of enzyme activity was lost during this period.

Large-Scale Preparation and Function of Porcine Hepatocyte Spheroids

To obtain a large number of porcine hepatocyte aggregates (spheroids) that have great potential in a bioartificial liver (BAL), we performed spheroid formation at a high cell density in a 1-L-scale spinner flask fitted with a silicon tubing apparatus for oxygen supply. We thereby obtained, within 24 hours, approximately fifty times more porcine hepatocyte spheroids as compared with the results of previous reports. The amount obtained corresponds to 2.5x10(9) cells and to roughly one-sixth of the cell number required for a BAL for a human patient. When we cultured spheroids continuously in suspension, they expressed three times more albumin secretion and twice the ammonium removal as compared with conventional monolayers during 10 days culture. Collagen gel entrapment of spheroids particularly lowered albumin secretion. We therefore conclude that the supension culture vessel of porcine hepatocyte spheroids is one of the most promising module types.

Effects of immobilization on biomass production and acetic acid fermentation of Acetobacter aceti as a function of temperature and pH

Acetobacter aceti cells were immobilized using entrapment in Ca-alginate gel and adsorption on preformed cellulose beads. The cell number within the supports showed no significant alterations on changing temperature or pH, whereas the acetic acid production was slightly increased by immobilization.

Cultured rat neuronal and glial cells entrapped within hydrogel polymer matrices: a potential tool for neural tissue replacement

Cultured Schwann cells, neonatal astrocytes or cells dissociated from embryonic cerebral hemispheres were dispersed within poly-[ N-(2-hydroxypropyl)-methacrylamide]-based hydrogel matrices by gel entrapment and maintained in vitro for 1–6 days. Glial cells were pre-labelled with Hoechst 33342. Cell differentiation and viability were studied by immunocytochemistry. Up to 15% of Schwann cells initially entrapped within the polymer matrices were immunopositive for the low affinity nerve growth factor receptor, S100, glial fibrillary acidic protein (GFAP) and laminin; up to 10% of pre-labelled astrocytes were immunopositive for GFAP and laminin. Embryonic neurons displayed immunostaining for neurofilaments. Hydrogels containing entrapped Schwann cells were implanted into the rat neocortex. These polymers supported cellular and axonal ingrowth within parts of the polymer matrix.

Production of ethyl butyrate using gel-entrapped Candida cylindracea lipase

Candida cylindracea lipase was immobilized by entrapment in gels formed from photo-crosslinkable prepolymers for synthesis of ethyl butyrate by direct esterification of ethanol and butyric acid in hexane. Enzyme immobilization enhanced the enzyme activity up to 2.8 fold, improved the ester yield, and alleviated alcohol inhibition of the enzyme activity. The hydrophobic gel was better than a hydrophilic one in terms of overall performance, and the entrapped enzyme retained 70% of its initial activity after 15 cycles of batch reaction.

Thermal stabilization of lipase by sol-gel entrapment in organically modified silicates formed on kieselguhr

The catalytic activity and thermal stability of lipase entrapped in organically modified silicates by the sol-gel method have been studied in ester synthesis in organic solvents. When methyltrimethoxysilane (MTrMOS) was used as the organic silane precursor which was mixed with tetramethoxysilane (TMOS) at a molar ratio of 3 : 1, the entrapped lipase exhibited a higher esterification activity than the lipase deposited on Celite 545 at temperatures as high as 65°C. This esterification activity was further enhanced by forming the lipase-entrapping gel on the surface of Celite 545. Thermal stabilization of lipase by entrapment in hybrid gels seems to be closely related to the mechanism of the sol-gel transition accompanied by microscopic phase separation. As a result of screening of different organic silanes, and of optimization of the preparation conditions, the hybrid gel-entrapped lipases on Celite 545 derived from an equimolar mixture of either propyltrimethoxysilane or trimethylmethoxysilane and TMOS were found to be the most thermostable, retaining their full activities up to 75°C, and 100 to 200 times higher activity at 95°C compared to the lipase deposited on the same support.

Strategies for "minimal growth maintenance" of cell cultures: a perspective on management for extended duration experimentation in the microgravity environment of a Space station.

How cells manage without gravity and how they change in the absence of gravity are basic questions that only prolonged life on a Space station will enable us to answer. We know from investigations carried out on various kinds of Space vehicles and stations that profound physiological effects can and often to occur. We need to know more of the basic biochemistry and biophysics both of cells and of whole organisms in conditions of reduced gravity. The unique environment of Space affords plant scientists an unusual opportunity to carry out experiments in microgravity, but some major challenges must be faced before this can be done with confidence. Various laboratory activities that are routine on Earth take on special significance and offer problems that need imaginative resolution before even a relatively simple experiment can be reliably executed on a Space station. For example, scientists might wish to investigate whether adaptive or other changes that have occurred in the environment of Space are retained after return to Earth-normal conditions. Investigators seeking to carry out experiments in the low-gravity environment of Space using cultured cells will need to solve the problem of keeping cultures quiescent for protracted periods before an experiment is initiated, after periodic sampling is carried out, and after the experiment is completed. This review gives an evaluation of a range of strategies that can enable one to manipulate cell physiology and curtail growth dramatically toward this end. These strategies include cryopreservation, chilling, reduced oxygen, gel entrapment strategies, osmotic adjustment, nutrient starvation, pH manipulation, and the use of mitotic inhibitors and growth-retarding chemicals. Cells not only need to be rendered quiescent for protracted periods but they also must be recoverable and further grown if it is so desired. Elaboration of satisfactory procedures for management of cells and tissues at "near zero or minimal growth" will have great value and practical consequences for experimentation on Earth as well as in Space. All of the parameters and conditions and procedural details needed to meet all the specific objectives will be the basis of the design and fabrication of cell culture units for use in the Space environment. It is expected that this will be an evolutionary process.

Effects of hepatocyte growth factor on viability and biotransformation functions of hepatocytes in gel entrapped and monolayer culture

An extracorporeal bioartificial liver device must maintain viability and differentiated function of hepatocytes cultivated at high cell density. Growth factors, such as hepatocyte growth factor, found in high concentrations in the plasma of patients with fulminant hepatic failure, have the potential to promote hepatocyte dedifferentiation and thus, decrease function. We tested the hypothesis that hepatocyte growth factor would improve viable cell density and decrease biotransformation functions of liver cells in monolayer culture and in hepatocytes entrapped in collagen cylindrical gel "noodles" as found in the extracorporeal bioartificial liver. In vitro, controlled study. University research laboratory. Adult Sprague Dawley Rats. Hepatocytes were harvested by a two-step collagenase technique. Harvested hepatocytes were plated onto type 1 collagen coated plates or entrapped in type 1 collagen cylindrical gels and cultured in different concentrations of hepatocyte growth factor. Interval measurements of 3H-thymidine incorporation, albumin synthesis, biotransformation functions, and viability were made. In monolayer culture, the addition of hepatocyte growth factor caused a dramatic increase in 3H-thymidine incorporation. This increase was accompanied by a decrease in the appearance of the lidocaine metabolite, monoethyglycinexylidide. Albumin production was unchanged. In cylindrical gel entrapment cultures, hepatocyte growth factor caused a significant increase in 2-day viability but had no effect on the metabolite appearance of lidocaine or 4-methyl umbelliferone or albumin production. Hepatocyte growth factor induces dedifferentiation of hepatocytes in monolayer culture. Collagen matrix entrapment appears to abrogate this effect and improve liver cell viability. There may be reciprocal regulation of hepatocyte reproductive and differentiated functions, such as biotransformation, which can be influenced by the entrapment of hepatocytes in an extracellular type 1 collagen matrix.

Mixed photo-cross-linked polyvinyl alcohol and calcium-alginate gels for cell entrapment

Living cells may be immobilized by gel entrapment under very mild conditions. The ionotropic gelation of alginate with bivalent cations such as Ca2+, as well as photo-induced gelation of polyvinyl alcohol (PVA) bearing photosensitive stilbazolium (SbQ) groups, are procedures that are compatible with most bioactive materials. In the search for more stable and stronger alginate gel beads, experiments have been carried out to investigate mixed gels from alginate and PVA-SbQ. The swelling capacities, diffusion properties, and potential toxic effect of the binary gel beads have been evaluated. The gel beads of selected PVA-SbQ/alginate mixtures were applied successfully as carriers in a denitrification process with continuous feeding of unsterilized water medium. Under such conditions, the purely synthetic PVA-SbQ network is expected to have a longer lifespan than a natural biopolymer such as alginate.

Immobilization of “Disguised” yeast in chemically crosslinked chitosan beads

A new simple method for the preparation of chemically crosslinked chitosan beads is presented. It consists of the dropwise addition of 2-3% (w/v) low molecular weight chitosan solution containing 2% (w/v) glyoxal in 1% (w/v) tetrasodiumdiphosphate, pH 8.0. Immobilized viable baker's yeast (Saccharomyces cerevisiae) could be obtained via gel entrapment within the new beads when means preventing their direct contact with soluble chitosan were provided, "disguising" the cells until gelation and crosslinking were completed. Such means included cell suspension in castor oil or mixing with carboxymethyl-cellulose powder. Application of these means was shown to be necessary, as cells exposed to soluble chitosan immediately lost their viability and glycolytic activity. Yeast disguised in castor oil was also protected from bead reinforcement by glutaraldehyde treatment, significantly strengthening bead stability while operating under acidic conditions. This capability was demonstrated by continuous ethanol production by chitosan entrapped yeast.

Enzyme Immobilization in Nanoparticles Produced by Inverse Microemulsion Polymerization

This paper deals with the immobilization of alkaline phosphatase by physical gel entrapment within colloidal particles produced by inverse microemulsion polymerization. This approach has been applied first to the production of polyacrylamide nanoparticles and then extended to the preparation of biodegradable latexes based on a polymerizable derivative of dextran. Conditions for production and purification of nanoparticles have been defined in close relation to protein lability. The catalytic activity of the immobilized enzyme has been compared to the enzyme properties in solution.

Thermostable α-amylase production by immobilized Bacillus licheniformis cells in agar gel and on acrylonitrile/acrylamide membranes

Immobilized cells of Bacillus licheniformis 44MB82-G were used for the production of thermostable alpha-amylase. The immobilization was carried out by entrapment in agar gel or by binding to formaldehyde-activated acrylonitrile/acrylamide membranes. The alpha-amylase production after 144 h of cultivation of membrane immobilized cells was 40% higher in comparison with the free cells. The respective value for the agar-entrapped cells was 22%. Similar trends were observed in the repeated batch fermentations performed with the immobilized cells. The scanning electron micrographs (SEM) of the immobilized cells gave additional information about their binding to the respective carriers.

The effect of immobilization on the course of biotransformation reactions by plant cells

The influence of different immobilization methods on the biotransformation of verbenol bySolanum aviculare plant cells has been studied. The biotransformation course was compared using free and immobilized cells. Immobilization techniques used included entrapment in alginate, pectate and carrageenane gels, in polyurethane foam, and on the surface of polyphenylenoxide.

A new enzyme immobilization procedure using copper alginate gel: Application to a fungal phenol oxidase

A new procedure was developed for enzyme immobilization by entrapment in copper alginate gel. The mechanical properties of the copper alginate gel were characterized and compared with those of the most widely used calcium alginate. The system was applied to the immobilization of a fungal phenol oxidase. Optimal conditions for enzyme immobilization were set up: the system immobilized 85% of the enzyme, and the remaining 15% was recovered in the aqueous immobilization medium. The stability and activity of the immobilized enzyme were studied. After immobilization, the enzyme was active in a wider pH range, the temperature of its optimal activity was shifted to lower values, and the possibility of storage at 4°C was greatly improved. The immobilized enzyme generally increased the rate of oxidation of various substrates. The results indicate a potential use of this system for the construction of bioreactors to be used in the detoxification of polluted waste waters.