Preparation and activity of carbonic anhydrase immobilized on porous silica beads and graphite rods

Techniques for the immobilization of bovine carbonic anhydrase (BCA) on porous silica beads and graphite are presented. Surface coverage on porous silica beads was found to be 1.5 x 10(-5) mmol BCA/m(2), and on graphite it was 1.7 x 10(-3) mmol BCA/m(2) nominal surface area. Greater than 97% (silica support) and 85% (graphite support) enzyme activity was maintained upon storage of the immobilized enzyme for 50 days in pH 8 buffer at 4 degrees C. After 500 days storage, the porous silica bead immobilized enzyme exhibited over 70% activity. Operational stability of the enzyme on silica at 23 degrees C and pH 8 was found to be 50% after 30 days. Catalytic activity expressed as an apparent second-order rate constant K'(Enz) for the hydrolysis of p-nitrophenyl acetate (p-NPA) catalyzed by BCA immobilized on silica beads and graphite at pH 8 and 25 degrees C is 2.6 x 10(2) and 5.6 x 10(2) M(-1)s(-1) respectively. The corresponding K(ENZ) value for the free enzyme is 9.1 x 10(2) M(-1)s(-1). Activity of the immobilized enzyme was found to vary with pH in such a manner that the active site pK, on the porous silica bead support is 6.75, and on graphite it is 7.41. Possible reasons for a microenvironmental influence on carbonic anhydrase pK(a), are discussed. Comparison with literature data shows that the enzyme surface coverage on silica beads reported here is superior to previously reported data on silica beads and polyacrylamide gels and is comparable to an organic matrix support. Shifts in BCA-active site pK(a) values with support material, a lack of pH dependent activity studies in the literature, and differing criteria for reporting enzyme activity complicate literature comparisons of activity; however, immobilized BCA reported here generally exhibits comparable or greater activity than previous reports for immobilized BCA.     

"Glutaraldehyde-P", a stable, reactive aldehyde matrix for affinity chromatography

A high-performance affinity chromatography support based on silica has been developed for the immobilization of proteins containing primary amino groups. A hydrophilic polymer covalently bound to the silica surface minimizes nonspecific protein binding to the support while preserving high binding capacity. The Schiff base reaction involved in the coupling of a ligand to the affinity medium is rapid, allows the use of mild conditions during the coupling process, and results in a very stable linkage. Reaction parameters were studied for protein coupling to the affinity support to determine optimum binding conditions and dynamic capacity as a function of protein size. The stability of the ligand-matrix bond was determined. The performance and reproducibility of the affinity support are demonstrated by its use in the analysis of nitrophenyl sugar derivatives, purification of glycoproteins, and isolation of anti-bovine immunoglobulin G developed in rabbit.     

Immobilization of a (dextran-adamantane-COOH) polymer onto beta-cyclodextrin-modified silica

Adamantane-modified compounds are known to form stable complexes with beta-cyclodextrins (beta-CD) by host-guest interactions. In this study, the inclusion complex formed between beta-CD cavities and the adamantane group was evaluated for the elaboration of a cation-exchange support. The synthesis of the chromatographic supports involved three steps: (i) a polymer of beta-CD was grafted to diol-modified silica, (ii) a dextran polymer was modified by both adamantane groups and ionizable COOH functions, (iii) the dextran derivative (Ad-Dex-COOH) was bound to the chromatographic support by complexation between the adamantane groups of the dextran and beta-CD cavities of the support. The polymer immobilization on the beta-CD support was successful as the resulting support exhibited weak cation-exchange properties. The stationary phase was easy to prepare under mild conditions (aqueous media, room temperature) and was quite stable when using aqueous mobile phases. The chromatographic behaviour of model proteins was studied in isocratic elution by examining the effect of salt concentration in the buffer on retention. A mixed retention mode was found for lysozyme, revealing both electrostatic and hydrophobic interactions with the stationary phase.     

Evaluation of a silica-coated magnetic nanoparticle for the immobilization of a His-tagged lipase

Magnetic particles of size 10 nm have been coated with silica to a mean diameter of 40 nm and charged with Cu²⁺ ions via a multidentate ligand, iminodiacetic acid (IDA), for the immobilization of His-tagged Bacillus stearothermopilus L1 lipase. Microporous (average pore diameter of 60 Å) silica gel with a mean particle diameter of 115 µm has been used as a comparative support material. The molar ratio of Cu²⁺ to IDA was found to be 1:1.14 and 1:1.99 in the silica gel and the silica-coated magnetic nanoparticles (SiMNs), respectively. The specific activity of the immobilized enzyme was found to conform to the following order: Cu²⁺-charged SiMN>SiMN>Cu²⁺-charged silica gel>silica gel. When it was immobilized on the Cu²⁺-charged SiMNs, over 70% of the initial activity of the lipase remained after it had been reused five times. However, only 20% of the initial activity remained after the enzyme immobilized on the Cu²⁺-charged silica gel had been reused five times. For the enzyme immobilized on supports without Cu²⁺ cations, all activity was lost after threefold reuse. The differences in the specific activities and the efficiencies of reuse of the enzymes immobilized on the various support materials are discussed in terms of immobilization mechanisms (physical adsorption vs. coordination bonding), mass transfer of a substrate and a product of the enzyme reaction, and the status of the Cu (Cu bound to the IDA on the silica layer vs. Cu directly adsorbed on the silica layer).     

Invertase covalent grafting onto corn stover

The covalent coupling of an invertase from baker's yeast onto an agricultural by-product, corn grits, has been developed. The optimal conditions for each step of the chemical modification of the support have been determined: oxidation with sodium metaperiodate, amination with ethylenediamine, reduction with sodium cyanoborohydride, and activation with glutaraldehyde. Activities up to 7.2 x 10(4) mumol reducing sugars produced/min g support could thus be achieved. Invertase coupling onto corn grits yields a derivative with a 25 times higher activity than when coupling this enzyme onto porous silica. The operational stability of invertase immobilized onto corn stover was found to be very high, with a half-life of up to 365 days at 40 degrees C when using a 2M sucrose solution as substrate. This immobilization method could be easily scaled up to the preparation of 10 kg of invertase derivative.     

Three-dimensional immobilization of beta-galactosidase on a silicon surface

Many alternative strategies to immobilize and stabilize enzymes have been investigated in recent years for applications in biosensors. The entrapment of enzymes within silica-based nanospheres formed through silicification reactions provides high loading capacities for enzyme immobilization, resulting in high volumetric activity and enhanced mechanical stability. Here we report a strategy for chemically associating silica nanospheres containing entrapped enzyme to a silicon support. beta-galactosidase from E. coli was used as a model enzyme due to its versatility as a biosensor for lactose. The immobilization strategy resulted in a three-dimensional network of silica attached directly at the silicon surface, providing a significant increase in surface area and a corresponding 3.5-fold increase in enzyme loading compared to enzyme attached directly at the surface. The maximum activity recovered for a silicon square sample of 0.5 x 0.5 cm was 0.045 IU using the direct attachment of the enzyme through glutaraldehyde and 0.16 IU when using silica nanospheres. The immobilized beta-galactosidase prepared by silica deposition was stable and retained more than 80% of its initial activity after 10 days at 24 degrees C. The ability to generate three-dimensional structures with enhanced loading capacity for biosensing molecules offers the potential to substantially amplify biosensor sensitivity.     

A patch coating method for preparing biocatalytic films of Escherichia coli

A method has been developed for immobilizing viable but nongrowing Escherichia coli in highly uniform patches. The patches consist of a thin layer of bacteria in acrylate vinyl acetate covered with a thin layer of the same polymer devoid of bacteria and sealed by the edges. This method permits study of immobilized cell physiology in biocatalytic films by the assay methods used for suspended cells. Large numbers of patches of immobilized E. coli can be generated on metal or polyester sheets. Those described here are 12.7 mm in diameter; in them the cell layer is 30 microm thick and contains more than 5 x 10(8) viable cells. The method allows the cell-plus-polymer layer and the polymer sealant to be varied in thickness from 5 to 60 microm and from 7 to 80 microm, respectively. No leakage of cells was detected from 87% of the patches during 15 days of rehydration. Culturability of the immobilized cells, released by shaking the cells out of the porous polymer layer, was 80% of pre coating culturability. E. coli beta-galactosidase activity and measurements of total RNA and DNA from immobilized and suspended cells indicated that cells immobilized in the thin polymer layer have higher specific beta-galactosidase activity and a slower total RNA degradation rate than suspended cells over 15 days.     

A facile and efficient method of enzyme immobilization on silica particles via Michael acceptor film coatings: immobilized catalase in a plug flow reactor

A novel method was developed for facile immobilization of enzymes on silica surfaces. Herein, we describe a single-step strategy for generating of reactive double bonds capable of Michael addition on the surfaces of silica particles. This method was based on reactive thin film generation on the surfaces by heating of impregnated self-curable polymer, alpha-morpholine substituted poly(vinyl methyl ketone) p(VMK). The generated double bonds were demonstrated to be an efficient way for rapid incorporation of enzymes via Michael addition. Catalase was used as model enzyme in order to test the effect of immobilization methodology by the reactive film surface through Michael addition reaction. Finally, a plug flow type immobilized enzyme reactor was employed to estimate decomposition rate of hydrogen peroxide. The highly stable enzyme reactor could operate continuously for 120 h at 30 °C with only a loss of about 36 % of its initial activity.     

Enhancing the catalytic properties of porcine pancreatic lipase by immobilization on SBA-15 modified by functionalized ionic liquid

The mesoporous silica SBA-15 was modified by carboxyl-functionalized ionic liquid (COOH-IL-SBA). The prepared support was used to immobilize porcine pancreatic lipase (PPL) by physical adsorption (PPL-COOH-IL-SBA) and covalent attachment (PPL-CON-IL-SBA). Enzymatic properties of the immobilized PPL were investigated in the triacetin hydrolysis reaction. It was found that carboxyl functionalized ionic liquid modification of the support surface was an effective method to improve the properties of immobilized PPL. Incorporating into the functionalized SBA-15 made PPL more resistant to temperature and pH changes, compared with PPL immobilized on parent SBA-15 (PPL-SBA). Especially, after the covalent attachment to a functionalized support, the stability of PPL was improved obviously, which retained 81.25% and 52.50% of the original activity after incubation for 20 days and four times recycling, respectively, whereas PPL-SBA exhibited only 58.80% and 27.78% of the original activity under the same conditions. In addition, physical and chemical properties of the supports and immobilized PPL were characterized by small-angle X-ray powder diffraction (SAXRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), nitrogen adsorption, nuclear magnetic resonance (NMR) and thermogravimetry (TG). The images and data confirmed chemical modification in SBA-15 and PPL immobilization on the tested support.     

Facile synthesis of C8-functionalized magnetic silica microspheres for enrichment of low-concentration peptides for direct MALDI-TOF MS analysis

In this study, novel C8-functionalized magnetic polymer microspheres were prepared by coating single submicron-sized magnetite particle with silica and subsequent modification with chloro (dimethyl) octylsilane. The resulting C8-functionalized magnetic silica (C8-f-M-S) microspheres exhibit well-defined magnetite-core-silica-shell structure and possess high content of magnetite, which endow them with high dispersibility and strong magnetic response. With their magnetic property, the synthesized C8-f-M-S microspheres provide a convenient and efficient way for enrichment of low-abundance peptides from tryptic protein digest and human serum. The enriched peptides/proteins were subjected for MALDI-TOF MS analysis and the enrichment efficiency was documented. In a word, the facile synthesis and efficient enrichment process of the novel C8-f-M-S microspheres make them promising candidates for isolation of peptides even in complex biological samples such as serum, plasma, and urine.     

Enzyme-containing Michael-adduct-based coatings

A two-step method was developed to homogeneously insert carbonic anhydrase (CA, E.C. 4.2.1.1) into Michael-adduct-based coatings. CA was first covalently coupled to an N-vinylformamide-based water-soluble polymer. Unlike native CA, the resulting polymer/CA system could be dispersed within a film matrix. The enzyme-containing coating (ECC) hydrolyzes p-nitrophenyl propionate in buffered media at high rates retaining approximately 7% apparent activity. In comparison, other two-step techniques for the chemical coupling of CA to the coating surface were less efficient and led to coatings with significantly less activity. A three-step immobilization process coupling the enzyme to the surface of a partially hydrolyzed coating also raised retention of activity after coating synthesis. CA-ECC is stable under ambient conditions retaining 45% activity after 90 days of storage at room temperature.     

A novel immobilised design for the production of the heterologous protein lysozyme by a genetically engineered Aspergillus niger strain

Abstract A novel immobilisation design for increasing the final concentration of the heterologous protein lysozyme by a genetically engineered fungus, Aspergillus niger B1, was developed. A central composition design was used to investigate different immobilised polymer types (alginate and pectate), polymer concentration [24% and 4% (w/v)], inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration [CaCl₂, 2% and 3.5% (w/v)]. Studies of the kinetics of production showed that optimum lysozyme productivity occurred after 10 days. Lysozyme production was significantly affected by polymer type, polymer concentration, and inoculum support ratio. Overall, immobilisation in Ca-pectate resulted in higher lysozyme production compared to that in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. Regardless of polymer type and concentration, increasing the fungal inoculum level increased lysozyme production. A significantly higher lysozyme yield was achieved with Ca-pectate in comparison to Ca-alginate (approximately 20–23 mg l^–1 and 0.5–2 mg l^–1, respectively). The maximum lysozyme yield achieved was about 23 mg l^–1 by immobilisation in Ca-pectate 2% (w/v) with 33% (v/v) mycelium and 3.5% (w/v) gel-inducing agent (CaCl₂). Response surface methodology was used to investigate the effect of pH and water activity (a_w). The best medium pH was 4.5–5.0, and bead a_w for optimum lysozyme yield was 0.94, regardless of polymer type.     

Enzymic interesterification of fats: Factors influencing the choice of support for immobilized lipase

Three grades of diatomaceous earth (Celite 560, Filtercel and Hyflo Supercel) and a controlled-pore silica have been examined for their suitability as support materials for lipase (triacyglycerol acylhydrolase, EC 3.1.1.3) catalysing the interesterification of fats. The controlled-pore silica gave a preparation with a low activity. Although all three Celites gave preparations with similar lipolytic activities, Hyflo Supercel gave the highest interesterification activity. The distribution of enzyme protein in Hyflo Supercel was examined by transmission electron microscopy.     

Immobilization of Candida cylindracea lipase on poly lactic acid,polyvinyl alcohol and chitosan based ternary blend film: Characterization,activity, stability and its application for N-acylation reactions

The ecofriendly ternary blend polymer film was prepared from the chitosan (CH), polylactic acid (PLA) and polyvinyl alcohol (PVA). Immobilization of Candida cylindracea lipase (CCL) was carried out on ternary blend polymer via entrapment methodology. The ternary blend polymer and immobilized biocatalyst were characterized by using N₂ adsorption–desorption isotherm, SEM, FTIR, DSC, and (%) water content analysis through Karl Fischer technique. Biocatalyst was then subjected for the determination of practical immobilization yield, protein loading and specific activity. Immobilized biocatalyst was further applied for the determination of biocatalytic activity for N-acylation reactions. Various reaction parameters were studied such as effect of immobilization support (ratio of PLA:PVA:CH), molar ratio (dibutylamine:vinyl acetate), solvent, biocatalyst loading, time, temperature, and orbital speed rotation. The developed protocol was then applied for the N-acylation reactions to synthesize several industrially important acetamides with excellent yields. Interestingly, immobilized lipase showed fivefold higher catalytic activity and better thermal stability than the crude extract lipase CCL. Furthermore various kinetic and thermodynamic parameters were studied and the biocatalyst was efficiently recycled for four successive reuses. It is noteworthy to mention that immobilized biocatalyst was stable for period of 300 days.     

Temperature-responsive size-exclusion chromatography using poly(N-isopropylacrylamide) grafted silica

Silica-based packing materials induce non-specific interactions with proteins in aqueous media because of the nature of their surface, mainly silanol groups. Therefore, the silica surface has to be modified in order to be used as stationary phase for the High Performance Size-Exclusion Chromatography (HPSEC) of proteins. For this purpose, porous silica beads were coated with hydrophilic polymer gels (dextrans of different molecular weights) carrying a calculated amount of diethylaminoethyl groups (DEAE). Actually, as shown by HPSEC, these dextran modified supports minimize non-specific adsorption for proteins and pullulans in aqueous solution. Then, in order to change the pore size in response to temperature, temperature responsive polymer of poly(N-isopropylacrylamide) (PIPAAm) was introduced into the surface of dextran-DEAE on porous silica beads. The structure of these supports before and after modification was alternately studied by Scanning Electronic Microscopy (SEM) and Scanning Force Microscopy (SFM). An adsorption of radiolabelled albumin was performed to complete our study. Silica modifications by dextran-DEAE and PIPAAm improve the neutrality of the support and minimize the non-specific interactions between the solid support and proteins in solution. At low temperature, the support having PIPAAm exhibits a high resolution domain in HPSEC and finally permits a better resolution of proteins and pullulans. At higher temperature, hydrophobic properties of PIPAAm produce interactions with some proteins and trigger off a slight delay of their elution time.     

Synthesis of magnetic silica nanomarkers with controlled physicochemical properties

Magnetic markers which can be detected with an extremely high sensitivity with the method of magnetic particle quantification (MPQ) were synthesized. Using a controlled Stober reaction, a set of magnetic silica markers of different sizes and zeta potentials was obtained. The use of a carboxymethyl dextran polymer to stabilize the magnetite particles during the synthesis made it possible to substantially reduce the detection limit of the obtained construct, which opens up new opportunities for creating effective diagnostic nanoagents.     

Novel purification system for 6xHis-tagged proteins by magnetic affinity separation

We have developed a novel nickel-silica matrix for the generation of magnetic beads for metal-ion affinity chromatography. In contrast to magnetic Ni-NTA agarose beads, the novel particle type (SiMAC) consists of a magnetic core and a nickel-silica composite matrix with the nickel ions tightly integrated in the silica. This results in a much higher number of chelating groups compared with Ni-NTA agarose beads. With the SiMAC beads, greatly improved purification of histidine-tagged proteins from crude bacterial extracts was achieved. The yield was at least twice as high as with conventional materials, the method is faster, since the coupling step is omitted and there is no need for handling toxic Ni(2+) salts.     

Preparation of C60‐functionalized magnetic silica microspheres for the enrichment of low‐concentration peptides and proteins for MALDI‐TOF MS analysis

In this work, for the first time, a novel C60‐functionalized magnetic silica microsphere (designated C60‐f‐MS) was synthesized by radical polymerization of C60 molecules on the surface of magnetic silica microspheres. The resulting C60‐f‐MS microsphere has magnetite core and thin C60 modified silica shell, which endow them with useful magnetic responsivity and surface affinity toward low‐concentration peptides and proteins. As a result of their excellent magnetic property, the synthesized C60‐f‐MS microspheres can be easily separated from sample solution without ultracentrifuge. The C60‐f‐MS microspheres were successfully applied to the enrichment of low‐concentration peptides in tryptic protein digest and human urine via a MALDI‐TOF MS analysis. Moreover, they were demonstrated to have enrichment efficiency for low‐concentration proteins. Due to the novel materials maintaining excellent magnetic properties and admirable adsorption, the process of enrichment and desalting is very fast (only 5 min), convenient and efficient. As it has been demonstrated in the study, newly developed fullerene‐derivatized magnetic silica materials are superior to those already available in the market. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel C60‐f‐MS microspheres makes it a promising candidate for isolation of low‐concentration peptides and proteins even in complex biological samples such as serum, plasma, and urine or cell lysate.     

The validity of the genus Spiniferomonas (Chrysophyceae)

Cells of Spiniferomonas bourrellyi Takahashi have been shown to possess a chloroplast. A procedure for examining the same cells by both light and electron microscopy was developed so that the structure of silica scales (which is diagnostic for the species) could be confirmed for cells clearly possessing chloroplasts. A previously published proposal invalidated the genus Spiniferomonas because the type species ( S. bourrellyi ) was found to be colourless and the new genus Chromophysomonas was erected to include all remaining pigmented species of Spiniferomonas . The findings reported here support reinstatement of S. blurrily as the validated type of the genus; Chromophysomonas is considered a redundant synonym.     

Conformational effects and cooperative interactions in poly[5(6)-vinylbenzimidazole]-catalyzed solvolyses of anionic,long-chain substrates

A new synthetic procedure for the monomer, 5(6)-vinylbenzimidazole, has been developed. Also, a new method for the formation of the benzimidazole moiety is reported. The monomer, 5(6)-vinylbenzimidazole, was subjected to a free radical solution polymerization and a solid state thermal polymerization. Poly[5(6)-vinyl-benzimidazole], prepared by the solution-free radical polymerization, was shown to be a linear addition polymer with pendant benzimidazole groups, whereas the thermally prepared polymer was shown to have a different structure. The solvolyses of negatively charged esters with varying aliphatic chain lengths catalyzed by poly[5(6)-vinylbenzimidazole] and compared to monomeric benzimidazole in 40% 1-propanol-water at 26°C revealed that the polymer was more efficient and that its activity was a function of the degree of ionization. Neutral-neutral and neutral-anionic benzimidazole interactions are suggested. The polymer conformation was found to have a significant effect on the solvolysis reactions.