Immobilization of lipoxygenase in an alginate-silicate solgel matrix: formation of fatty acid hydroperoxides

A method for the immobilization of lipoxygenase (LOX) in an alginate-silicate gel matrix was developed. In this method, a mixture of calcium alginate beads and LOX in borate buffer are dispersed into a hexane solution of tetramethoxy-ortho-silicate (TMOS). Hydrolysis of the TMOS gives products that permeate and co-polymerize with the alginate gel to form a colloid within the beads that entraps the LOX. Optimum reaction conditions for sol-gel entrapment of LOX are at pH 9.0 in 0.2M borate buffer. The composite gel, after isolation and vacuum drying, had excellent protein retention that has good enzyme activity and stability at room temperature. The activity of the entrapped LOX was less than the activity of the free enzyme. However, the activity of the immobilized LOX can be restored by the addition of borate buffer and glycerol, or borate buffer saturated with an organic solvent. In contrast to the free enzyme in solution, which loses its activity in less than one day, sol-gel entrapped LOX retains its activity at ambient temperature for at least 25 days and can be recycled. This report demonstrates that the sol-gel entrapment method for immobilizing LOX can be useful in developing a process for the oxidation of polyunsaturated fatty acids.     

Alginate/carbon composite beads for laccase and glucose oxidase encapsulation: application in biofuel cell technology

Alginate–carbon beads were prepared in order to develop a biocompatible matrix for laccase and glucose oxidase immobilization for application in biofuel cell technology. The enzyme loading capacity was high (91%) in pure alginate beads for glucose oxidase. For laccase, the loading capacity was enhanced from 75% to 83% by introducing carbon. Desorption out of the matrix was controlled by the enzymes’ diffusion and reached a plateau after 40 h for laccase and 70 h for glucose oxidase. Two-thirds of both enzymes was irreversibly retained inside the alginate beads. This proportion increased to 80% for laccase in combined alginate/carbon beads. Half-life of the adsorbed enzyme was enhanced to 74 days for laccase in carbon/alginate beads and 45 days for glucose oxidase in pure alginate as compared to 38 days and 23 days for free enzymes, respectively.     

Photoinduced Decarboxylation-dependent Transamination of Pyridoxal 5′-Phosphate-α-Amino Acid Schiff Bases

Bilirubin oxidase was purified from the culture filtrate of Myrothecium verrucaria MT-1 by a procedure involving ammonium sulfate precipitation, charcoal treatment, and QAE-Sephadex A-50 and Sephadex G-100 column chromatographies. The purified enzyme was homogeneous on disc gel electrophoresis.

Copper and carbohydrate were contained in the enzyme. The enzyme was inhibited by Fe²⁺ and compounds that complex with copper. Bilirubin, biliverdin, hemin and chlorophyllin which consist of tetrapyrrole, and substrates of laccase were oxidized by the enzyme. Bilirubin was oxidized more rapidly than other substances. Bilirubin oxidase differed from laccase in reactivity with substances consisting of tetrapyrrole. Substances consisting of tetrapyrrole were oxidized only a little by laccase but rapidly oxidized by bilirubin oxidase. The apparent Km value for bilirubin was calculated to be 190 μm.     

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.     

Development of simple methods for preparation of yeast and plant protoplasts immobilized in alginate gel beads

A simple method for preparation of yeast and plant protoplasts immobilized in alginate gel beads was developed. Yeast cells were first immobilized in strontium alginate gel beads and then treated with protoplast isolation enzyme so that the protoplasts are formed inside the beads. In the case of plant cells, degassing treatment was necessary in order to facilitate enzyme penetration into the cell aggregates. A mixture of the degassing treated plant cells and sodium alginate solution was dropped into SrCl2 containing the protoplast isolation enzymes. Thus protoplasts isolation and gel solidification proceeded simultaneously. With these methods, the required time was shorter while the viability of the immobilized protoplasts were higher than when the conventional method is used.     

Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties

Thermoalkalophilic esterase enzyme from Bal?ova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7 M CaCl(2) solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization.     

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.     

An immobilized enzyme reactor for the detoxification of bilirubin

An immobilized enzyme reactor has been developed for the degradation of bilirubin as a potential treatment for neonatal jaundice. It utilizes the enzyme bilirubin oxidase from Myrothecium verrucaria, which in the presence of molecular oxygen converts bilirubin to biliverdin and other products that are much less toxic than bilirubin. Bilirubin oxidase was covalently attached to agarose beads using cyano transfer activation. Forty percent of the specific activity of bilirubin oxidase was retained after immmobilization, and preparations with 20 units of enzymatic activity per gram of drained wet weight of gel were obtained. The stability of bilirubin oxidase at pH 7.4 and 37 degrees C was improved fivefold by immobilization. A 15-mL column containing immobilized bilirubin oxidase, through which a 37 degrees C solution of 332muM bilirubin and 450muM human serum albumin in 0.05M phosphate buffer (pH 7.4) was passed at 1 mL/min, converted more than 60 percent of the bilirubin per pass. The substrate specificity of the enzyme and the small volume of the reactor are important characteristics for this clinical application where it is desirable to remove only one compound from the blood and to minimize the volume of blood in the extracorporeal circuit. This reactor, by detoxifying the jaundiced infant's blood of bilirubin, would eliminate the risks associated with the use of donor blood as is done currently in treating severe neonatal jaundice.     

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.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Use of polyelectrolyte complex-stabilized calcium alginate gel for entrapment of beta-amylase

Calcium alginate gel stabilized with a polyelectrolyte complex (PEC) consisting of potassium poly(vinyl alcohol) sulfate (KPVS) and trimethylammonium glycol chitosan iodide (TGCI) was used for the immobilization of beta-amylase. The immobilization was made by gelling aqueous droplets of enzyme solution including both sodium alginate and KPVS in a CaCl(2) solution containing TGCI. The activity of the enzyme entrapped into the stabilized gel beads was evaluated by studying the batch reaction kinetics of enzyme-catalyzed hydrolysis of maltotetraose. Repeated kinetic measurements, totaling 18, were carried out at fixed time intervals. After each measurement the beads were stirred for 1 day in a freshly prepared 10 mM NaCl solution at 3 degrees C. It was found that the immobilized system remained stable without leading to a serious loss of the activity or to a large leakage of the enzyme from the support. This was explained as being due to a PEC-crosslinked contracted network structure of the stabilized gel 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.     

Determination of the radial distribution of Saccharomyces cerevisiae immobilised in calcium alginate gel beads

Summary The dissolution of alginate gel beads in 20 g sodium citrate /l produces a linear decrease in bead diameter. The rate of dissolution is dependent on the concentration of CaCl₂ within the gel beads. This method allows the controlled release of Saccharomyces cerevisiae from alginate gel beads and permits the simple and rapid determination of the radial distribution of cell concentration.     

Comparison of some properties of free and immobilized alpha-amylase by Aspergillus sclerotiorum in calcium alginate gel beads

Some properties of immobilized alpha-amylase by Aspergillus sclerotiorum within calcium alginate gel beads were investigated and compared with soluble enzyme. Optimum pH and temperature were found to be 5.0 and 40 degrees C, respectively, for both soluble and immobilized enzymes. The immobilized enzyme had a better Km value, but kcat/Km values were the same for both enzymes. Entrapment within calcium alginate gel beads improved, remarkably, the thermal and storage stability of alpha-amylase. The half life values of immobilized enzyme and soluble enzyme at 60 degrees C were 164.2, and 26.2 min, respectively. The midpoint of thermal inactivation (Tm) shifted from 56 degrees C (for soluble enzyme) to 65.4 degrees C for immobilized enzyme. The percentages of soluble starch hydrolysis for soluble and immobilized alpha-amylase were determined to be 97.5 and 92.2% for 60 min, respectively.     

Alginate as immobilization matrix and stabilizing agent in a two-phase liquid system: application in lipase-catalysed reactions.

Alginate was evaluated as an immobilization matrix for enzyme-catalyzed reactions in organic solvents. In contrast to most hydrogels, calcium alginate was found to be stable in a range of organic solvents and to retain the enzyme inside the gel matrix. In hydrophobic solvents, the alginate gel (greater than 95% water) thus provided a stable, two-phase liquid system. The lipase from Candida cylindracea, after immobilization in alginate beads, catalysed esterification and transesterification in n-hexane under both batch and continuous-flow conditions. The operational stability of the lipase was markedly enhanced by alginate entrapment. In the esterification of butanoic acid with n-butanol, better results were obtained in the typical hydrophilic calcium alginate beads than in less hydrophilic matrices. The effects of substrate concentration, matrix area, and polarity of the substrate alcohols and of the organic solvent on the esterification activity were examined. The transesterification of octyl 2-bromopropanoate with ethanol was less efficient than that of ethyl 2-bromopropanoate with octanol. By using the hydrophilic alginate gel as an immobilization matrix in combination with a mobile hydrophobic phase, a two-phase liquid system was achieved with definite advantages for a continuous, enzyme-catalysed process.     

Tuning structural durability of yeast-encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross-linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross-links in an ionically cross-linked alginate gel bead significantly enhances the bead's structural durability. The interpenetrating network (IPN) was prepared by first cross-linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross-linking reaction. The resulting methacrylic alginate gel beads (IPN-MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross-linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN-MA gel beads remained more metabolically active in ethanol production than those in calcium cross-linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes.     

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.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

Elucidation of optimum conditions for immobilization of viable cells by using calcium alginate

Different factors which affect the stability of calcium alginate gel beads entrapping viable cells during fermentation were investigated. It was found that among others, the initial population of cells per ml of gel beads, the length of period of incubation in CaCl₂ solution, and the concentration of sodium alginate used for the immobilization were the most important factors affecting the stability of the gel beads during fermentation. By using an initial cell population of about 10⁵ cells per ml of 2.0% sodium alginate, and incubating the beads for at least 22 h in a CaCl₂ solution after immobilization, the percentage of beads which developed cracks during fermentation was highly reduced. Also, without the addition of CaCl₂ into the fermenting broth, the gel beads were stable for nine consecutive batch fermentations.     

Drying and Rehydration of Calcium Alginate Gels

In this paper, we study the rehydration properties of air-dried calcium alginate gel beads. Rehydration is shown to depend on alginate source (i.e. mannuronic to guluronic acid ratio) and the salt concentration in the rehydration medium. Rehydration curves are described adequately by the empirical Weibull equation. Wide-angle X-ray diffraction measurements are performed to obtain information on the microstructure of dried alginate gels. The X-ray diffraction patterns provide evidence for formation of ordered domains in which alginate polymers are laterally associated. Formation of ordered structures during drying is found to have a large impact on rehydration properties. Lateral association of alginate chains is reduced (and rehydration improved) by removing excess calcium ions from the gel beads in a washing step prior to air drying. In addition, rehydration properties of mixed alginate–carboxymethyl cellulose (CMC) gel beads are investigated. The presence of CMC in the gel matrix is found to reduce lateral association of alginate chains during drying and to improve rehydration properties.