Calcium alginate entrapped preparations of Aspergillus oryzae beta galactosidase: its stability and applications in the hydrolysis of lactose

Insoluble concanavalin A-beta galactosidase complex was obtained by using jack bean extract and this complex was crosslinked with glutaraldehyde, in order to maintain the integrity of complex in the presence of its substrate or products. Concanavalin A-beta galactosidase complex retained 92% of the initial enzyme activity whereas crosslinked complex showed 88% activity. Entrapment of concanavalin A-beta galactosidase complex into calcium alginate beads provided suitability to use this preparation in reactors. Temperature- and pH-optima of the various immobilized beta galactosidase preparations were the same as its soluble counterpart. Entrapped crosslinked concanavalin A-beta galactosidase complex retained more than 50% activity after 1h exposure with 4.0 M urea at room temperature. Moreover, entrapped crosslinked concanavalin A-beta galactosidase complex retained 81 and 62% of the original enzymatic activity in the presence of 5% calcium chloride and 5% galactose, respectively. Entrapped crosslinked concanavalin A-beta galactosidase complex preparation was more superior in the continuous hydrolysis of lactose in a batch process as compared to the other entrapped preparations. This entrapped crosslinked concanavalin A-beta galactosidase complex retained 95% activity after seventh repeated use and 93% of its original activity even after 2 months storage at 4 degrees C.     

Calcium alginate entrapped preparation of α-galactosidase: its stability and application in hydrolysis of soymilk galactooligosaccharides

Thermostable α-galactosidase from Aspergillus terreus _GR was insolubilized using concanavalin A obtained from jack bean extract and in order to maintain the integrity of complex in the presence of its substrate or products, this complex was crosslinked with glutaraldehyde. Soluble α-galactosidase entrapped in calcium alginate retained 82% of enzyme activity whereas, Con A-α-galactosidase complex entrapped in calcium alginate and crosslinked Con A-α-galactosidase complex entrapped calcium alginate retained 74 and 61% activity, respectively. A fluidized bed reactor was constructed for continuous hydrolysis of galactooligosaccharides in soymilk using crosslinked Con A-α-galactosidase complex entrapped calcium alginate. Optimum conditions such as pH (5.0) and temperature (65°C) were the same for all immobilized enzyme preparations and soluble enzyme. Crosslinked Con A-α-galactosidase entrapped complex exhibited enhanced thermostability and showed 62% of activity (38%) after 360 min at 65°C. Entrapped crosslinked Con A-α-galactosidase complex preparation was superior in the continuous hydrolysis of oligosaccharides in soymilk by batch processes compared to the other entrapped preparations. The entrapped crosslinked concanavalin A-α-galactosidase complex retained 95% activity after eight cycles of use.     

Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Survival of Bifidobacterium longum Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Biological control of phytophagous ladybird beetles Epilachna vigintioctopunctata (Col., Coccinellidae) by chitinolytic phylloplane bacteria Alcaligenes paradoxus entrapped in alginate beads

The chitinase secreting strain KPM‐012A of Alcaligenes paradoxus was isolated from tomato leaves and vitally entrapped in sodium alginate gel beads to provide a new method for biocontrol of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, the peritrophic membrane was dissected from the adult ladybird beetles that ingested the suspension of KPM‐012A after starvation to observe degradation of the midgut surface by the bacteria under electron microscopy. The peritrophic membrane around the bacteria was degraded, suggesting the release of chitinase from the ingested bacteria. Large amounts of chitinase were successfully released from KPM‐012A‐entrapped calcium alginate beads. This chitinase release from the microbial beads was sustained for 1 week and was sufficient to digest the peritrophic membrane. Daily supply of tomato leaves treated with the microbial beads caused considerable suppression of leaf feeding and oviposition by the adult ladybird beetles, indicating that this method is effective for decreasing population of insect pests in the subsequent generation. Thus, the present study provided an experimental basis for the biocontrol measures of herbivorous insect pests by the chitinolytic bacteria entrapped in alginate beads.     

Characterization of calcium alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate

Calcium alginate (CA), chitosan-coated calcium alginate (CCA-I), and chitosan–calcium alginate complex (CCA-II) gel beads, in which an oil-in-water emulsion containing allyl isothiocyanate (AITC) was entrapped, were prepared and characterized for efficient oral delivery of AITC. The AITC entrapment efficiency was 81% for CA gel beads, whereas about 30% lower values were determined for the chitosan-treated gel beads. Swelling studies showed that all the gel beads suddenly shrunk in simulated gastric fluid (pH 1.2). In simulated intestinal fluid (pH 7.4), CA and CCA-I gel beads rapidly disintegrated, whereas CCA-II gel beads highly swelled without degradation probably due to the strong chitosan–alginate complexation. Release studies revealed that most entrapped AITC was released during the shrinkage, degradation, or swelling of the gel beads, and the chitosan treatments, especially the chitosan–alginate complexation, were effective in suppressing the release. CCA-II gel beads showed the highest bead stability and AITC retention under simulated gastrointestinal pH conditions.     

Calcium alginate beads as a slow-release system for delivering angiogenic molecules in vivo and in vitro.

A method previously used in this laboratory for entrapment of tumor cells in alginate beads has been extended to provide a slow release delivery system for growth factors with known in vivo angiogenic activity. Protein growth factors were entrapped in alginate beads in amounts sufficient to cause incorporation of 3H-thymidine by COMMA-D cells in vitro, and in vivo neovascularization when injected subcutaneously into Balb/c mice. Entrapment of 125I-labelled growth factors showed that the amount of molecule entrapped in alginate beads may vary with the charge of the molecule. In vitro cell proliferation studies showed that entrapment in alginate beads may provide a slow-release system or a stabilizing environment for the protein. In some cases biological activity of the growth factor in solution was increased by the presence of control alginate beads. When alginate-entrapped growth factors were injected into Balb/c mice, induction of new blood vessels could be monitored qualitatively by macroscopic photography and assessed quantitatively by measuring the pooling of radiolabelled red blood cells at the experimental site. Subcutaneous injection of purified angiogenic factors not entrapped in alginate beads did not cause neovascularization. Diffusion of 125I-labelled growth factors from alginate beads in the animal showed that release in vivo may depend on the charge of the protein molecule. These results indicate that injection of purified molecules entrapped in alginate beads provides an effective localized and slow-release delivery of biologically active molecules. This delivery system may extend the time of effectiveness of biologically active molecules in vivo compared to direct injection without alginate entrapment. The method of entrapment and injection has potential for identifying active factors in tumor-induced angiogenesis and testing new compounds as modulators of neovascularization.     

Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.     

Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.     

Immobilization of carbonic anhydrase in alginate and its influence on transformation of CO2 to calcite

Present investigation entails carbonic anhydrase (CA) immobilization and its influence on transformation of CO₂ to calcite. CA enzyme was immobilized in alginate beads, subsequently maintained its catalytic efficiency after sequential operational cycles. The immobilized beads showed better operational stability by retaining nearly 67% of its initial activity even after six cycles. Batch scale studies with free and immobilized enzyme revealed that the entrapped CA hydrates CO₂ to bicarbonate and/or carbonate which was then made to react with Ca²⁺ ions to transform into calcite. Calcite was characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The entrapped CA was employed for the performance evaluation with respect to several operational parameters including the influence of enzyme concentration in free and immobilized condition. It was concluded that immobilized CA in alginate beads would have the potential for CO₂ sequestration by biomimetic route.     

Immobilization of glycoenzymes using crude concanavalin A and glutaraldehyde

Insoluble concanavalin A complexes of glucose oxidase and amyloglucosidase have been prepared by mixing the glycoenzyme solutions with either pure concanavalin A or a buffer extract of jackbean meal. The complexes obtained using excess lectin exhibited relatively low specific activity compared with those obtained with low lectin concentration. The crosslinked complexes, however, exhibited relatively higher thermostability. In addition, glutaraldehyde treated concanavalin A-glucose oxidase complexes, especially those prepared using excess lectin, exhibited significantly broader pH-activity profiles compared to the soluble enzyme. Electrophoresis of the concanavalin A-glucose oxidase and concanavalin A-amyloglucosidase from pure lectin and from crude extract showed similar protein composition. The concanavalin A-amyloglucosidase complex was crosslinked to obtain relatively large (0.15–0.17 mm diameter) and homogeneous particles by treatment with 2% glutaraldehyde followed by homogenization. Glutaraldehyde treatment, however, caused a marked decrease in the retained activity of the particles, especially on starch. The crosslinked concanavalin A-amyloglucosidase preparations were, however, only slightly more stable against thermal denaturation than the soluble enzyme.     

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.     

Covalent stabilization of alginate gel for the entrapment of living whole cells

Summary Three methods were developed for preparing alginate gels containing cells that are stable in phosphate containing media. In Method I preformed alginate beads containing entrapped cells were treated with polyethyl eneimine followed by glutaraldehyde. In Method II alginate sol was treated with a carbodiimide and N-hydroxysuccinimide (to form active esters), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine. In Method III alginate so] was treated with periodate (to form aldehyde groups), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine. Saccharomyces cerevisiae cells, immobilized in such stabilized gels, exhibited almost the same fermentation activity as the standard preparation. The viability of the immobilized cells was retained during the stabilization procedure as judged from their ability to multiply in the presence of nutrients.The preparations remained stable in phosphate buffer for at least ten days without substantial release of cells. The extent of cross-linking was controlled by varying the time and the concentration of reactants, thus giving preparations ranging from beads with a thin stabilized shell to beads homogeneously stabilized.     

Feasibility study on the use of calcium alginate-entrapped hybridoma cells for IgM production

In order to test the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production, HO-22-1 hybridoma cells entrapped into calcium alginate beads with varying alginate concentrations were cultivated in spinner flasks. It was observed that the IgM produced by the entrapped cells could diffuse out of the calcium alginate beads regardless of alginate concentrations tested (0.8–2.5%). Since the increase in alginate concentrations showed an adverse effect on cell growth and maximum cell concentration, the use of lower alginate concentration was desirable for higher volumetric monoclonal antibody (MAb) productivity. When the entrapped cells in 0.8% alginate beads were cultivated in repeated-fed batch mode, the reduction of serum concentration in the medium from 10% to 1% did not decrease the volumetric IgM production. Taken together, the data obtained here showed the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production.Alginate was generously provided by the Kelco company. This work was supported by the Ministry of Science and Technology, Korea.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

Gel beads composed of collagen reconstituted in alginate

Spherical gel beads of collagen/alginate were prepared by discharging droplets of a mixture containing collagen (1.07-1.9 mg/ml) and alginate (1.2-1.5% w/v) into 1.5% w/v CaCl2 solution at 4°C. Collagen in the gel beads was reconstituted by raising the temperature to 37°C after alginate was liquefied by citrate. Scanning electron microscopy of the beads revealed the characteristic fibrous structure of collagen. To demonstrate the application of this new technique in cell culture, GH3 rat pituitary tumor cells were entrapped and grown in the gel beads. The immobilized cells proliferated to a density of 1.95 x 106 cell/ml which is about an order of magnitude higher than that grown in the alginate beads.     

Suppression of leaf feeding and oviposition of phytophagous ladybird beetles (Coleoptera: Coccinellidae) by chitinase gene-transformed phylloplane bacteria and their specific bacteriophages entrapped in alginate gel beads

The chitinase gene-transformed strain KPM-007E/chi of Enterobacter cloacae was vitally entrapped in sodium alginate gel beads with its specific virulent bacteriophage EcP-01 to provide a new method for microbially digesting chitinous peritrophic membranes of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, chitinase SH1 from a gram-positive bacterium Kurthia zopfii was overproduced by Escherichia coli cells and purified by affinity column chromatography. The purified enzyme effectively digested peritrophic membranes dissected from the ladybird beetles to expose epithelial tissues beneath the peritrophic membrane, and the beetles that had ingested chitinase after submergence in chitinase solution had considerably reduced their feeding on tomato leaves. KPM-007E/chi, entrapped in the alginate beads, released the chitinase. More chitinase was released when KPM-007E/chi was present with their specific virulent bacteriophage EcP-01 in the beads because of lysis of bacterial cells infected with the bacteriophages. This chitinase release from the microbial beads (containing KPM-007E/chi and EcP-01) was sufficient to digest the peritrophic membrane as well as to suppress feeding of bead-sprayed tomato leaves by the ladybird beetles. A daily supply of tomato leaves treated with the microbial beads considerably suppressed leaf feeding and oviposition by the ladybird beetles, suggesting a possible application of chitinase-secreting bacteria for suppressing herbivorous insect pests.     

Formulation and drying of alginate beads for controlled release and stabilization of invertase

Several alternatives to the conventional alginate beads formulation were studied for encapsulation of invertase. Pectin was added to the alginate/enzyme solution while trehalose and β-cyclodextrin were added to the calcium gelation media. The effect of composition changes, freezing, drying methods (freeze, vacuum, or air drying), and thermal treatment were evaluated on invertase stability and its release kinetics from beads. The enzyme release mechanism from wet beads depended on pH. The addition of trehalose, pectin, and β-cyclodextrin modified the bead structure, leading in some cases to a release mechanism that included the relaxation of the polymer chains, besides Fickian diffusion. Enzyme release from vacuum-dried beads was much faster than from freeze-dried beads, probably due to their higher pore size. The inclusion of β-cyclodextrin and especially of pectin prevented enzyme activity losses during bead generation, and trehalose addition was fundamental for achieving adequate invertase protection during freezing, drying, and thermal treatment. Present results showed that several alternatives such as drying method, composition, as well as pH of the relese medium can be managed to control enzyme release.     

Entrapment of biological control agents applied to entomopathogenic nematodes

Summary Hydrogels of alginate, phospho guar gum, carboxymethyl guar gum, k-carrageenan and cellulose sulphate, respectively were tested to find easily redissolvable gels. The entomopathogenic nematode, Heterorhabditis sp., was entrapped in calcium alginate beads, calcium alginate hollow spheres and foils made from different hydrogels. Emigration from calcium alginate beads after 7 days of storage was 100 % at room temperature and was lowered to 6 % at 6 °C, whereas no emigration from calcium alginate hollow spheres was found at either temperature. Highly concentrated polymer foils produced on gauze showed reduced emigration with a survival of 80 % after 24 h compared to foils produced on glass slides. Calcium alginate beads can be used for a controlled release of the nematode into the environment, while hollow spheres and foils are suitable for storage.Dedicated to Prof. Dr. F. Wagner on the occasion of his 65th birthday