Polyaniline as a support for urease immobilization

Polyaniline synthesized by chemical oxidative polymerization was used as an immobilization support for jack bean urease. Such immobilized enzyme has a good catalytic activity, storage stability, and reusability. Properties of free and immobilized urease were compared. Blends of polystyrene, cellulose acetate and poly(methyl methacrylate) with polyaniline were used for urease immobilization as well.     

Polyionic hydrogels as support for immobilization of lipase

Summary Polyionic hydrogels have been prepared by complexation of chitosan and xanthan. These hydrogels have been used to immobilize lipase from porcine pancreas (E.C. 3.1.1.3). Immobilization efficiency varied between 90 and 99% of initial activity. Immobilized lipase retained its activity towards hydrolysis of olive oil in water as an emulsion and of olive oil in isooctane.     

Natural silk fibroin as a support for enzyme immobilization

Silk fibroin derived from Bombyx mori cocoon is being developed and utilized for purposes besides traditional textile material. Fibroin can be easily made up into various forms, several of which can serve as enzyme-immobilized supports. There are numerous reports on immobilized enzymes using these forms of silk fibroin as supports in which the enzyme-immobilized fibroin membranes were characterized in detail by means of spectrophotometry, infrared spectra, NMR, ESR. Enzyme-immobilized fibroin membranes have been successfully used in several biosensors for the determinations of glucose, hydrogen peroxide and uric acid in which glucose and urate biosensors in a flow injection system were able rapidly to analyze various biosamples including human whole blood or serum.     

Grape skins as a natural support for yeast immobilization

Grape skins were used to immobilize Saccharomyces cerevisiae. In repeated batch fermentations of grape by immobilized and free cells, the maximum specific rate of alcohol production on glucose decreased from 7.98 h^–1 at 25 °C to 0.7 h^–1 at 5 °C. The rate was approximately twice as high as that on fructose. The rates for free cells were very low. The maximum alcohol yield (0.45 g g^–1) was obtained at 5 °C when the immobilized biocatalyst was used.     

Use of porous glass fiber as a support for biocatalyst immobilization

Summary Porous glass fiber has a very high surface area and good mechanical properties that make it an excellent support for biocatalyst immobilization. By packing aligned glass fibers in a tubular reactor such that the fibers are all parallel to the axis of the tube, the resulting pressure drop is considerably smaller than for a similar bed of packed beads. The utility of this support was demonstrated by immobilizing -glucoamylase by silane-glutaraldehyde coupling, and measuring its activity toward converting maltose to glucose. Using optimized immobilization conditions, an enzyme loading of 1.5 mg protein perm ² surface area was obtained, with an activity of 370 units/g glass at 50°C. The half-life of the immobilized glucoamylase was more than twice as long as that of the free enzyme.     

Rice straw as a support for immobilization of microbial lipase.

Candida rugosa lipase was covalently immobilized on rice straw activated with glutaraldehyde using poly(ethylene glycol) (PEG) as the stabilizing agent. The effects of PEG molecular weight and enzyme loading were studied according to a full 2(2) factorial design. Higher immobilization yields (>70%) were attained when the lipase loading was 95 units/mg of dry support, independent of PEG molecular weight. All derivatives showed high hydrolytic and synthetic activities. This work provides preliminary results on the use of agricultural residues as a support matrix for immobilizing lipase and on the application of the resulting derivatives to butyl butyrate synthesis as a study model.     

Ionic liquid-reconstituted cellulose composites as solid support matrices for biocatalyst immobilization

Preparation of cellulose-polyamine composite films and beads, which provide high loading of primary amines on the surface allowing direct one-step bioconjugation of active species, is reported using an ionic liquid (IL) dissolution and regeneration process. Films and bead architectures were prepared and used as immobilization supports for laccase as a model system demonstrating the applicability of this approach. Performance of these materials, compared to commercially available products, has been assessed using millimeter-sized beads of the composites and the lipase-catalyzed transesterification of ethyl butyrate.     

Development of ultraporous fired bricks as support for yeast cell immobilization

Ultraporous fired bricks (porosities from 56 to 72%) were developed from materials locally available in Nigeria. The grog particle size was used to modulate the porosity of the bricks. The porous bricks produced were then employed as supports for the immobilization of a yeast strain isolated from a local alcoholic beverage, palm wine. The influence of a brick's porosity and particle size on the cell-loading capacity and cell growth inside the fired brick support were studied. The study revealed that a brick's porosity varied linearly with the mean particle size of the grog, increasing from a porosity of 56% at a particle size of 0.805 mm to 72% at a particle size of 0.075 mm. Cell saturation of the surface area available within the support matrix was completed within four hours of contact between the cell and the adsorbing surface especially for the most porous samples. Cell growth was therefore not observed in such cases; however, the less porous samples supported some cell growth upon incubation. Cell holdup was also observed to increase exponentially when either the porosity was increased or the particle size was decreased. The influence of particle size, however, became insignificant at very high porosities.     

Statistical optimization of one-step immobilization process for recombinant endoglucanase from Clostridium thermocellum

Endoglucanase CelA from Clostridium thermocellum (CtCelA) is a thermophilic endo-β-1,4-glucanase and has a low solubility when expressed in Escherichia coli. To make industrial application of CtCeA more appealing, artificial oil bodies (AOBs) was implemented for one-step renaturation and immobilization of recombinant CtCelA. CtCelA was first fused with oleosin (Ole-CtCelA), a structural protein of plant seed oils. Ole-CtCelA was overexpressed in E. coli, and its insoluble form was recovered and mixed with plant oils to assemble AOBs. Moreover, the Box–Behnken design and the central composite design were employed to optimize the condition for assembly of AOBs and the enzymatic reaction condition, respectively. Consequently, the approach led to the resumption of active CtCelA on AOBs. CtCelA-bound AOBs exhibited an optimum activity at 69 °C and pH 6.3 while the immobilized protein remained stable for several hours at 70 °C and after 5 repeated uses. Overall, it indicates a promise of this novel approach for direct processing and immobilization of recombinant CtCelA.     

Functionalized nanocomposite coating of a glass surface for oligonucleotide immobilization

A new type of coating for manufacturing DNA chips was constructed on the basis of an organicinorganic nanocomposite based on the polyvinylbutyral-tetraethoxysilane copolymer. The organosilicon composite was functionalized by introduction of ethanolamine vinyl ether copolymers, which contain amino groups and anchor vinyloxide units capable of reacting with silanol groups of the nanocomposite. The resulting coatings form a film on glass slides with a high surface density of amino groups (up to 700 groups/nm²) suitable for three-dimensional immobilization of oligonucleotides. The use of bifunctional reagents (e.g., phenylene diisothiocyanate) for the attachment of oligonucleotides bearing amino linkers to the amino-containing surface provides an immobilization density of 0.5–1.6 pmol/mm². Immobilization with a higher density (10–12 pmol/mm²) was achieved for attachment to amino-containing glass slides upon the use of oligonucleotides containing a selectively activated terminal phosphate group. The activation of oligonucleotides was carried out with the triphenylphosphine-dithiodipyridine pair in the presence of dimethylaminopyridine N-oxide. The resulting DNA chips were shown to be useful in principle for DNA detection.     

Functionalized nanocomposite coating of a glass surface for oligonucleotide immobilization

A new type of coating for manufacturing DNA chips was constructed of the basis of an organic-inorganic nanocomposite based on the polyvinylbutyral-tetraethoxysilane copolymer. The organosilicon composite was functionalized by introduction of ethanolamine vinyl ether copolymers, which contain amino groups and anchor vinyloxide units capable of reacting with silanol groups of the nanocomposite. The resulting coatings form a film on glass slides with a high surface density of amino groups (up to 700 groups/nm2) suitable for three-dimensional immobilization of oligonucleotides. The use of bifunctional reagents (e.g., phenylene diisothiocyanate) for the attachment of oligonucleotides bearing amino linkers to the amino-containing surface provides an immobilization density of 0.5-1.6 pmol/mm2. Immobilization with a higher density (10-12 pmol/mm2) was achieved for attachment to amino-containing glass slides upon the use of oligonucleotides containing selectively activated terminal phosphate groups. The activation of oligonucleotides was carried out with the triphenylphosphine-dithiodipyridine pair in the presence of dimethylaminopyridine N-oxide. The resulting DNA chips were shown to be useful in principle for DNA detection.     

Chitosan from Syncephalastrum racemosum used as a film support for lipase immobilization

Chitosan from a native Mucoralean strain, Syncephalastrum racemosum, isolated from herbivorous dung (Northeast-Brazil), was used as a film support for lipase immobilization. S. racemosum showed highest chitosan yield (152 mg g dry mycelia weight(-1); 15.2% of dry mycelia weight) among the nine strains screened, which presented 89% D-glucosamine. A chitosan film was used for lipase (EC 3.1.1.3) immobilization using glutaraldehyde as a bifunctional agent. The immobilized lipase retained 47% (12.6 micromol s(-1) m(-2)) of its initial catalytic activity after four cycles of reaction. This result is comparable (same order of magnitude) to that of the enzyme immobilized on film made from commercially available crustacean chitosan.     

Modified hydroxyalkyl methacrylate gel as a support for immobilization of yeast cells

Summary Whole cells of Zygosaccharomyces lactis have been covalently linked to fine-grained hydroxyalkyl methacrylate gel Spheron P 1000 E which was prepared by treatment with epichlorhydrin and modified by an amine spacer. Experiments on the coupling of permeabilized and non-permeabilized cells to this gel support have shown that immobilized cell agregates may be obtained by the immobilization of thermally permeabilized cells. Cell clustering can be bypassed by immobilizing non-permeabilized cells. This immobilization procedure makes additional permeabilization possible.     

Functionalized polymeric liposomes. Efficient immobilization of alpha chymotrypsin

A polymerizable phospholipid has been synthesized having a latent aldehyde moiety in the head group (1). Photopolymerized vesicles which have been prepared from this phosphatidylcholine have been successfully conjugated to alpha chymotrypsin. A high degree of loading was achieved with significant retention of enzymatic function.     

Improving the properties of chitosan as support for the covalent multipoint immobilization of chymotrypsin

Changing gel structure and immobilization conditions led to a significant improvement in the covalent multipoint attachment of chymotrypsin on chitosan. The use of sodium alginate, gelatin, or kappa-carrageenan, activation with glutaraldehyde, glycidol, or epichlorohydrin, and addition of microorganisms followed by cellular lysis allowed the modification of the gel structure. Immobilization yields, recovered activities, and stabilization factors at 55 and 65 degrees C were evaluated. Enzyme immobilization for 72 h at pH 10.05, 25 degrees C and reduction with NaBH 4 in chitosan 2.5%-carrageenan 2.5%, with addition of S. cerevisiae 5% and activation with epichlorohydrin led to the best derivative, which was 9900-fold more stable than the soluble enzyme. This support allowed an enzyme load up to 40 mg chymotrypsin x g gel (-1). The number of covalent bonds, formed by active groups in the support and lysine residues of the enzyme, can explain the obtained results. SEM images of the gel structures corroborate these conclusions.     

Application of preformed cellulose beads as a support in cell immobilization

Summary A new cell immobilization technique, using preformed cellulose beads, has been developed. Corynebacterium sp. and Saccharomycss cerevisiae cells were grown on the beads and were used for tryptophan and ethanol production.     

Optimization by experimental design of polyacrylamide gel composition as support for enzyme immobilization by entrapment

We have developed a methodology based on experimental design, to optimize a polyacrylamide gel as the support for enzyme immobilization, taking advantage of all the properties which this type of gel has. Monomer and crosslinking agent proportions are responsible for both the porous structure and pore size of the gel. A correct selection of those variables and suitable synthesis conditions leads to an increase in the activity retained by the gel. The path of steepest ascent method was used to obtain the relative maximum activity. The maximum retained activity was chosen with a central composite design in terms of the gel composition. The retained activity in the network, loss activity in the wash water, and loss activity due to steric impediment or blockage was modeled in terms of the variables responsible for the gel structure. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 497-506, 1997.     

New cationic exchanger support for reversible immobilization of proteins

New tailor-made cationic exchange resins have been prepared by covalently binding aspartic-dextran polymers (e.g. MW 15 000-20 000) to porous supports (aminated agarose and Sepabeads). More than 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans have been strongly adsorbed on these porous materials at pH 5. This interaction was stronger than in conventional carboxymethyl cellulose (e.g., at pH 7 and 25 degrees C, all proteins previously adsorbed at pH 5 were released from carboxymethyl cellulose, whereas no protein was released from the new supports under similar conditions). Ionic exchange properties of such composites were strongly dependent on the size of the aspartic-dextran polymers as well as on the exact conditions of the covalent coating of the solids with the polymer (optimal conditions: 100 mg aspartic-dextran 20 000/(mL of support); room temperature). Finally, some industrially relevant enzymes (Kluyveromices lactis, Aspergillus oryzae, and Thermus sp. beta-galactosidases, Candida antarctica B lipase, and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recovery and immobilization rates. After enzyme inactivation, the enzyme can be fully desorbed from the support and the support could be reused for several cycles.     

Porous zirconia: a new support material for enzyme immobilization

Four different proteases (trypsin, chymotrypsin, papain and pepsin) were covalently attached to the surface of a new type of porous zirconia, as well as a conventional porous silica, activated with 3-isothiocyanatopropyltriethoxy silane (NCS-silane). The immobilization efficiency onto the porous zirconia material was evaluated in terms of the amount of enzyme attached to the particles and from the biological activity remaining after the immobilization step. The results were compared with the corresponding experiments with a porous silica of similar surface area/g support material. In addition, the storage stability of the modified zirconia and silica biocatalysts were evaluated. These results indicated that specific immobilized enzyme biocatalysts can be achieved with this new zirconia support material which exhibits different properties to those observed with the more conventional silica-based materials. Moreover, the results with the enzyme-zirconia biocatalysts also indicate different characteristics when compared with data for the same enzymes immobilized under similar buffer conditions to organic support materials as previously described by various other investigators. The advantages of zirconia-based immobilized enzyme biocatalysts in terms of their density and chemical robustness are also described relative to other alternative support materials currently in use.