A new precursor for the immobilization of enzymes inside sol-gel-derived hybrid silica nanocomposites containing polysaccharides

Tetrakis(2-hydroxyethyl) orthosilicate (THEOS) introduced by Hoffmann et al. (J. Phys. Chem. B., 106 (2002) 1528) was first used to prepare hybrid nanocomposites containing various polysaccharides and immobilize enzymes in these materials. Two different types of O-glycoside hydrolyses (EC3.2.1), 1-->3-beta-D-glucanase LIV from marine mollusk Spisula sacchalinensis and alpha-D-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, were taken for the immobilization. To reveal whether the polysaccharide inside the hybrid material influences the enzyme entrapment and functioning, negatively charged xanthan, cationic derivative of hydroxyethylcellulose and uncharged locust bean gum were examined. The mechanical properties of these nanocomposites were characterized by a dynamic rheology and their structure by a scanning electron microscopy. It was found that 1-->3-beta-D-glucanase was usually immobilized without the loss of its activity, while the alpha-D-galactosidase activity in the immobilized state depended on the polysaccharide type of material. An important point is that the amount of immobilized enzymes was small, comparable to their content in the living cells. It was shown by the scanning electron microscopy that the hybrid nanocomposites are sufficiently porous that allows the enzymatic substrates and products to diffuse from an external aqueous solution to the enzymes, whereas protein molecules were immobilized firmly and not easily washed out of the silica matrix. A sharp increase of the enzyme lifetime (more than a hundred times) was observed after the immobilization. As established, the efficient entrapment of enzymes is caused by few advantages of new precursor over the currently used TEOS and TMOS: (i) organic solvents and catalysts are not needed owing to the complete solubility of THEOS in water and the catalytic effect of polysaccharides on the sol-gel processes; (ii) the entrapment of enzymes can be performed at any pH which is suitable for their structural integrity and functionality; (iii) a gel can be prepared at reduced concentrations of THEOS (1-2%) in the initial solution that excludes a notable heat release in the course of its hydrolysis.     

A new method of whole microbial cell immobilization

Summary A simple method for producing gelatin-immobilized microbial cells is described. The microorganism used as an example was baker's yeast (Saccharomyces cerevisiae). The gel particles containing these cells were utilized as an immobilized enzyme (invertase) both in stirred batch and packed bed systems.     

Live diatom silica immobilization of multimeric and redox-active enzymes

Living organisms are adept in forming inorganic materials (biominerals) with unique structures and properties that exceed the capabilities of engineered materials. Biomimetic materials syntheses are being developed that aim at replicating the advantageous properties of biominerals in vitro and endow them with additional functionalities. Recently, proof-of-concept was provided for an alternative approach that allows for the production of biomineral-based functional materials in vivo. In this approach, the cellular machinery for the biosynthesis of nano-/micropatterned SiO₂ (silica) structures in diatoms was genetically engineered to incorporate a monomeric, cofactor-independent (“simple”) enzyme, HabB, into diatom silica. In the present work, it is demonstrated that this approach is also applicable for enzymes with “complex” activity requirements, including oligomerization, metal ions, organic redox cofactors, and posttranslational modifications. Functional expression of the enzymes β-glucuronidase, glucose oxidase, galactose oxidase, and horseradish peroxidase in the diatom Thalassiosira pseudonana was accomplished, and 66 to 78% of the expressed enzymes were stably incorporated into the biosilica. The in vivo incorporated enzymes represent approximately 0.1% (wt/wt) of the diatom biosilica and are stabilized against denaturation and proteolytic degradation. Furthermore, it is demonstrated that the gene construct for in vivo immobilization of glucose oxidase can be utilized as the first negative selection marker for diatom genetic engineering.     

A new method for immobilization of pectic enzymes

Summary Polygalacturonase and pectinesterase have been successfully immobilized on gamma alumina by activation of the support with glutaraldehyde at pH 3.0. The half life of the enzymes increased by four and two fold compared to the immobilization on gamma alumina without activation.     

The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels.

Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was deomonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days. Immobilized K. marxianus, inulase, and glucose oxidase preparations were used to demonstrate the porosity and retraining properties of calcium alginate gels. Calcium alginate-immobilized chloroplasts were shown to perform the Hill reaction. Some experiments with immobilized mitochondria are reported.     

A new immobilization of microbial cells

Summary A new immobilization of microbial cells based on the growth of cells in gel is presented. The cells grew very well in carrageenan gel when fed nutrients required for growth. The growing cells immobilized in gel formed a dense layer of cells near the gel surface. Because the cells were near the gel surface, they efficiently catalyzed single enzyme reactions. In addition, the immobilized growing cell system was applied to the complicated multienzyme reactions since large numbers of cells in the gel could constantly be maintained for long periods.     

Reaction of colloidal silica with membranes of intact mammalian cells

Colloidal silical particles were produced at a size that permitted reaction with human erythrocytes and rat macrophages without affecting cell integrity. Binding of colloid was shown by increased electrophoretic mobility of red cells and also resulted in changes in the surface topography of red cells as seen with scanning electron microscopy. The degree to which colloid binds to red cells was determined by microprobe analysis of single intact cells. Furthermore, the capacity of red cells to bind silica was increased if sialic acid residues were removed enzymatically from the cell surface.     

New immobilization method of mammalian cells using alginate and polyacrylate

Mouse-mouse hybridoma cells were immobilized in polyacrylate-alginate gels. The immobilized hybridoma cells were cultured semi-continuously using a fluidized bed reactor, and allowed continuous antibody production without any gel destruction for one month. It has been proved that the polyacrylate-alginate gels were tolerant against physical stress. The composition of the gels suitable for cell growth and antibody production was given as follows; viscosity of alginate at 1% solution: 60–100 cP, alginate concentration: 0.8%, and polyacrylate concentration: 0.2%. In the semi-continuous culture using gels prepared under suitable conditions, the viable cell number was estimated as 2.5×10⁷ cells/ml-gel, and the antibody production rate was 2.2 mg/ml-gel/d, at maximum.     

Development of new alginate fiber for the immobilization of yeast

Summary Stabilities of Ca-alginate fiber and ACP(Alginate + Cclite R-634 + Pectin) gel fibers containing different concentrations of alginate were tested. The results showed that the stability of ACP gel fiber containing 5% alginate(5% ACP gel fiber) was better than those of other fibers tested, considering the degree of release of yeast from gel fiber. Scanning electron microscopic observation proved the above results, and concentrations of cell and ethanol obtained by 5% ACP gel fiber were higher than those of others, respectively.     

Properties of spherical calcium pectate and alginate gels and their use in diffusion chromatography,solids separations and immobilization of enzymes and cells

Water-soluble acidic polysaccharides—deesterified pectins and carboxy-derivatives of starch—precipitated with calcium ions were tested as precursors of spherical calcium gels. Pectates prepared from apple or citrus pectin, similarly to alginates, are compounds forming spherical calcium gels stable in aqueous medium which have a relatively highly reproducible mass, particle size, water content, shape, mechanical strength and shearing. Both the liquid-solid partition of low- and high-molar-mass solutes and its kinetics proved to be reasonable features. Distribution of pore size in the above materials was estimated. Detailed pictures of surface and of the interior of calcium beads in the scanning electron microscope are presented. The possible use of calcium beads as enzyme carriers, as affinity matrixes and entrapment materials for diffusion chromatography, solids separations and bioindication of a specific water pollution was evaluated. Calcium alginate beads were always used as reference material.     

Dried calcium alginate/magnetite spheres: A new support for chromatographic separations and enzyme immobilization

Dried spheres made from an alginate solution containing magnetite particles have excellent potential as a support for enzyme immobilization and chromatographic applications. The beads were found to be much stronger than gels such as polyacrylamide and dextran, indicating that high flow rates and pressures could be used in column separations. The support withstood not only temperatures of up to 120 degrees C, but also most pH values and common solvents. While some solutions, such as phosphate buffers, dissolved the spheres, stabilization with Tyzor TE(R) eliminated this problem. The physical properties of the beads include a glasslike density of 2.2 g/mL, excellent sphericity, low porosity, and a narrow size distribution. The magnetite present in the support allows the beads to be used for magnetic separations such as high gradient magnetic filtration. Their high degree of microroughness provides a large exposed surface area for enzyme and ligand binding. Mixed Actinomyces fradiae proteases and Aspergillus niger alpha-amylase, two enzymes representative of classes which attack large substrates, were immobilized on the bead's surface with high activity and stability. A cyanuric dye which can be used in chromatographic applications (Cibacron Blue F3GA(R)) was also readily coupled to the surface of this support with good yield. The support should have a wide range of applications in bioseparation and immobilized biochemical technology.     

Properties of heterocysts isolated with colloidal silica

A method is described for the isolation of heterocysts that are virtually free of contaminating cell debris after sonication of aerobically grown Anabaena 7120. Isolated heterocysts reduced acetylene in a light-dependent process in the absence of exogenously provided ATP; heterocysts supplied with ATP and Na₂S₂O₄ reduced acetylene slowly in the dark but still showed a marked light activation. Nitrogenase activity was greatest in fractions containing intact heterocysts. Up to 13% of the activity of the intact filaments was accounted for in the isolated heterocyst preparation.Isolated heterocysts took up O₂ in a light-independent process; O₂ uptake with added NADP⁺ was enhanced by pyruvate, isocitrate and intermediates of the oxidative pentose pathway.     

Efficient immobilization technique for enhancement of cellobiose dehydrogenase activity on silica gel

In this study, cellobiose dehydrogenase (CDH) of Phanerochaete chrysosporium ATCC 32629 was immobilized on silica gel for the further application of CDH in the saccharification process of biomass. To prevent the loss of enzyme activity during enzyme immobilization, the pretreatment of CDH was performed by various pretreatment materials before immobilization. When pretreated enzymes were used in immobilization, the activities of immobilized CDH were higher than non-pretreated CDH even in same amounts of immobilized protein. The specific activity of pretreated immobilized CDH with lactose was about two times higher than that of non-pretreated immobilized CDH. Moreover, the pretreated immobilized CDH showed better reusability than non-pretreated immobilized CDH, with 67.3% of its original activity being retained after 9 reuses.     

A new method for immobilization of microbial cells by cross-linking

A method for immobilization of microbial cells was designed. The method uses generation of reactive aldehyde groups on the cell wall surface under conditions of periodate oxidation. The linking of aldehyde groups by various bifuctional aromatic diamines and then by glutaraldehyde produced immobilized cells, which are promising for use in biocatalysis with high-molecular-weight substrates.     

Optimization of critical parameters for immobilization of yeast cells to alginate gel matrix

The optimum concentrations of sodium alginate (wt. %), calcium chloride (M) and yeast cells (wt. %), and curing time (h) for enhanced gel stability were obtained employing a full factorial search. The results indicate that the concentrations of sodium alginate and CaCl₂, and the curing time of the beads were found to have a pronounced effect on the stability of the beads. The cell concentration, on the other hand, has an adverse influence either individually or in combination with other variables. The path of steepest ascent method has been used to optimize the variables and the resultant gel beads were evaluated for fermentation ability.     

Formation of effective channels in alginate gel for immobilization of anchorage-dependent animal cells

Summary The conditions for formation of effective channels in alginate gels for growth of anchorage-dependent animal cells were examined. Many channels were formed in the gels by adding a low concentration solution of a high molecular weight polymer of alginate to a high concentration solution of divalent cations. It is recommended that an alginate with a high molecular weight and a low mannuronic acid/guluronic acid ratio be gelled by contact with strontium ions for the cultivation of immobilized anchorage-dependent cells because the gels produced have many channels and are mechanically strong.     

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.