Alginate as immobilization material: III. Diffusional properties

The rate of diffusion of serum albumin (MW 6.9 x 10(4) D) out of beads of calcium alginate gels depends upon the concentration and uronic acid composition of the alginate (ManA/GulA ratio), the conditions under which the beads are produced, the pH, and the temperature. The diffusion coefficient decreases with increasing alginate concentration, and (ManA/GulA) ratio and with decreasing pH. Diffusion out of the beads, in which the alginate is uniformly distributed (homogeneous gel), is faster than out of the beads in which the alginate is concentrated at the surface (inhomogeneous gel). The temperature dependence of the diffusion coefficient follows the Arrhenius law, with an activation energy of approximately 23 kJ x mol(-1).     

Photophosphorylation in bacterial chromatophores entrapped in alginate gel: Improvement of the physical and biochemical properties of gel beads with barium as gel-inducing agent

The immobilization of Rhodopseudomonas capsulata chromatophores by entrapment in an alginate gel is described. Alginate beads were prepared with Ba²⁺, Sr²⁺ and Ca²⁺ as gel-forming agents and compared for their mechanical strength, chemical resistance against disruption by phosphate-induced swelling, and yield of photophosphorylation activity. Barium alginate beads proved to have better physico-chemical properties than the more commonly used calcium alginate beads. After embedding in barium alginate gel, R. capsulata chromatophores retained a high yield (up to 70%) of their photophosphorylation capacity. Alginate entrapment did not cause a large increase in the Michaelis constant for ADP and phosphate, the substrates of adenosinetriphosphatase (ATPase). These constants were $\text{K}{}^{\mathrm{ADP}}{}_{\mathrm{<mtext>m</mtext>}}\text{= 1.4 × 10}{}^{\mathrm{?5}}\text{m}$ and $\text{K}{}^{\mathrm{P}}{}_{\mathrm{i}}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.2 × 10}{}^{\mathrm{?4}}\text{m}$ for free chromatophores and $\text{K}{}^{\mathrm{ADP}}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.3 × 10}{}^{\mathrm{?4}}\text{m}$ and $\text{K}{}^{\mathrm{P}}{}_{\mathrm{i}}{}_{\mathrm{<mtext>m</mtext>}}\text{= 5.6 × 10}{}^{\mathrm{?4}}\text{m}$ for chromatophores entrapped in barium alginate gel. However, embedding gave no additional protection against rapid inactivation of chromatophores upon storage at 3°C. Preliminary results with a batch reactor for continuous ATP regeneration are presented. The barium alginate method has two features which are not generally encountered at the same time, extremely mild conditions for entrapment and excellent physical properties of the gels beads, which make this method a suitable tool for the construction of bioreactors with immobilized cells or organelles.     

Binary immobilization of tyrosinase by using alginate gel beads and poly(acrylamide-co-acrylic acid) hydrogels

The use of the immobilized and the stable enzymes has immense potential in the enzymatic analysis of clinical, industrial and environmental samples. However, their widespread uses are limited due to the high cost of their production. In this study, binary immobilization of tyrosinase by using Ca-alginate and poly(acrylamide-co-acrylic acid) [P(AAm-co-AA)] was investigated. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the free and binary immobilized enzymes. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized tyrosinase were also examined. For the free and binary immobilized enzymes on Ca-alginate and P(AAm-co-AA), optimum pH was found to be 7 and 5, respectively. Optimum temperature of the free and immobilized enzymes was observed to be 30 and 35 degrees C, respectively. Reuse number, storage and thermal stability of the free tyrosinase were increased by a result of binary immobilization.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads.

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel 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.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

Diffusivity of Cu2+ in calcium alginate gel beads: recalculation

Calculations of the diffusivity of Cu(2+) in calcium alginate gel beads using the shrinking core model were checked by us. Corrected results are reported here. Diffusivity was still found to increase with increasing alginate concentration, but at a lower rate than reported in the cited paper. The diffusivity increased by a factor of 2 over the range of alginate concentrations studied rather than 10. The original data is included with sample calculations. (c) 1994 John Wiley & Sons, Inc.     

Combined physical and chemical immobilization of glucose oxidase in alginate microspheres improves stability of encapsulation and activity

Chemical sensors utilizing immobilized enzymes and proteins are important for monitoring chemical processes and biological systems. In this study, calcium-cross-linked alginate hydrogel microspheres were fabricated as enzyme carriers by an emulsification technique. Glucose oxidase (GOx) was encapsulated in alginate microspheres using three different methods: physical entrapment (emulsion), chemical conjugation (conjugation), and a combination of physical entrapment and chemical conjugation (emulsion-conjugation). Nano-organized coatings were applied on alginate/GOx microspheres using the layer-by-layer self-assembly technique in order to stabilize the hydrogel/enzyme system under biological environment. The encapsulation of GOx and formation of nanofilm coating on alginate microspheres were verified with FTIR spectral analysis, zeta-potential analysis, and confocal laser scanning microscopy. To compare both the immobilization properties of enzyme encapsulation techniques and the influence of nanofilms with uncoated microspheres, the relationship between enzyme loading, release, and effective GOx activity (enzyme activity per unit protein loading) were studied over a period of four weeks. The results produced four key findings: (1) the emulsion-conjugation technique improved the stability of GOx in alginate microspheres compared to the emulsion technique, reducing the GOx leaching from microsphere from 50% to 17%; (2) the polyelectrolyte nanofilm coatings increased the GOx stability over time, but also reduced the effective GOx activity; (3) the effective GOx activity for the emulsion-conjugation technique (about 3.5 x 10(-)(5) AU microg(-)(1) s(-)(1)) was higher than that for other methods, and did not change significantly over four weeks; and (4) the GOx concentration, when compared after one week for microspheres with three bilayers of poly(allylamine hydrochloride)/sodium poly(styrene sulfonate) ({PAH/PSS}) coating, was highest for the emulsion-conjugation technique. As a result, the comparison of these three techniques showed the emulsion-conjugation technique to be a potentially effective and practical way to fabricate alginate/GOx microspheres for implantable glucose biosensor application.     

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.     

Immobilization of flax protoplasts in agarose and alginate beads. Correlation between ionically bound cell-wall proteins and morphogenetic response.

Linum usitatissimum protoplast-derived colonies that are cultured in auxin-supplemented medium and immobilized in Ca(2+)-alginate matrix form round colonies that develop into polarized, embryo-like structures. On the other hand, protoplast-derived colonies that are immobilized in agarose do not show an organized morphogenetic response, and unique, ionically bound cell-wall protein patterns match this response. Although only slight differences in neosynthesized or total constitutive polypeptides are observed, dramatic changes in ionically bound cell-wall proteins are seen. In protoplasts grown on Ca(2+)-alginate-solidified, auxin-containing medium, several basic polypeptides were strongly induced and were found tightly bound to the cell wall. In contrast, these basic proteins were found only weakly bound to the walls of protoplasts grown on agarose-solidified, auxin-containing medium or on Ca(2+)-alginate-solidified, auxin-free medium, in which they were released into the medium. Our results suggest that plant cells can perceive and respond to the adjacent extracellular matrix, since we show that the growth of flax cells on Ca(2+)-alginate in the presence of auxin-containing medium may promote the binding of specific proteins to the walls. This establishes a direct correlation of an embryo-like morphogenesis with ionically bound cell-wall basic proteins in flax protoplasts grown on Ca(2+)-alginate-solidified, auxin-containing medium.     

Diffusion of Cu2+ in calcium alginate gel beads: further analyses

The diffusivity of Cu(2+), as determined by previous authors from analysis of experimental data in terms of the shrinking core (SCM) and linear absorption (LAM) models, is examined in light of the ability of the models to curve fit all the data. It is concluded from this further analysis that previous conclusions depicting the LAM to have an advantage over the SCM for predictive value are not justified. It is also shown that equally good curve fits can be obtained with a recent absorption/desorption model of diffusion which considers directly, through distribution theory, the effect of heterogeneity of material properties on the rate of diffusion. (c) 1995 John Wiley & Sons, Inc.     

Alginate as immobilization material. II: Determination of polyphenol contaminants by fluorescence spectroscopy, and evaluation of methods for their removal

Alginates, both commercial and laboratory made, are strongly fluorescent due to small amounts of polyphenolic materials. These contaminants can be detected by fluorescence spectroscopy in concentrations lower than 1 ppm. This technique has been used to measure polyphenols in a wide range of alginates and various procedures for preparation of biotechnological-grade alginates have been evaluated.     

Production of alginate beads by emulsification/internal gelation. I. Methodology

Summary Small diameter alginate beads (microspheres) were formed via internal gelation of alginate solution emulsified within vegetable oil. Gelation was initiated by addition of an oil-soluble acid thereby reducing the pH of the alginate solution and releasing soluble Ca²⁺ from the citrate complex. Smooth, spherical, micron-sized beads were formed. The mean diameter ranged from 200 to 1000 m, controlled by the reactor impeller design and rotational speed. The technique has potential for large-scale and continuous applications in immobilization.Correspondence to: R. J. Neufeld     

Transport and consumption rate of O2 in alginate gel beads entrapping hepatocytes

Experiments carried out with hepatocytes entrapped in alginate gel particles with a cell density of about 10⁷ cells ml^–1 showed an oxygen consumption rate of about 2.2×10^–16 mol cell^–1 s^–1. Under these conditions, no inner anoxic core is present in 1.8 mm diameter beads. From these data, the maximum bead size consistent with non-critical O₂ concentration at the bead center has been evaluated for different cell densities and O₂ concentrations in the medium.     

Chondronectin: physical and chemical properties

Chondronectin, the chondrocyte attachment factor, was purified from chicken serum and characterized as to its physical and chemical properties. From sedimentation equilibrium data it was found to have a native molecular weight of 175,800 +/- 800 and a subunit molecular weight of 55,540 +/- 800 in the presence of guanidinium chloride and cysteine, suggesting a trimeric structure linked by disulfide bonds. As visualized by electron microscopy after rotary shadowing, the protein appears compact and globular. The amino acid and carbohydrate compositions of chondronectin are distinct from fibronectin, the fibroblast attachment factor, and laminin, the epithelial cell attachment factor. The activity of chondronectin in promoting attachment of chondrocytes is stable to digestion by collagenase, elastase, and neuraminidase, but is destroyed by trypsin treatment. The data suggest that chondronectin is structurally and chemically distinct from fibronectin and laminin.     

Ribosomal dysentery vaccine. I. Method of production, physical and chemical properties]

Studies of A. and G. Youmans on the experimental tuberculosis led to discovery of a fundamentally new type of vaccines (ribosomal vaccines) which proved to be highly effective in the prophylaxis of many experimental infections. Therefore it seems reasonable to prepare analogous vaccine from Shigellae for the study of its efficiency in experimental shigellosis. Ribosomal preparations from Shigella sonnei were prepared by sonic disruption of microbial cells followed by differential ultracentrifugation according to A. and G. Youmans' method with slight modifications. The yeild of ribosomal fraction was about 2 per cent by weight; all the series had an UV adsorption maximum at 260 nm, the ratio OD260:OD280 being approximately 2. They contained about 55% of RNA, 35% of protein and no more than 8% of saccharides. As shown by centrifugation in sucrose gradient and by analytical ultracentrifugation the preparations were homogeneous. The presence of undissociated ribosomes was confirmed by electron microscopy. Thus, the ribosomal preparations obtained proved to be sufficiently purified for carrying out experimental investigations of their biological activity.