Comparative study of controlled pore glass, silica gel and poraver for the immobilization of urease to determine urea in a flow injection conductimetric biosensor system

This study compared the responses of three enzyme reactors containing urease immobilized on three types of solid support, controlled pore glass (CPG), silica gel and Poraver. The evaluation of each enzyme reactor column was done in a flow injection conductimetric system. When urea in the sample solution passed though the enzyme reactor, urease catalysed the hydrolysis of urea into charged products. A lab-built conductivity meter was used to measure the increase in conductivity of the solution. The responses of the enzyme reactor column with urease immobilized on CPG and silica gel were similar and were much higher than that of Poraver. Both CPG and silica gel reactor columns gave the same limit of detection, 0.5 mM, and the response was still linear up to 150mM. The analysis time was 4-5 min per sample. The enzyme reactor column with urease immobilized on CPG gave a slightly better sensitivity, 4% higher than the reactor with silica gel. The life time of the immobilized urease on CPG and silica gel were more than 310h operation time (used intermittently over 7 months). Good agreement was obtained when urea concentrations of human serum samples determined by the flow injection conductimetric biosensor system was compared to the conventional methods (Fearon and Berthelot reactions). These were statistically shown using the regression line and Wilcoxon signed rank tests. The results showed that the reactor with urease immobilized on silica gel had the same efficiency as the reactor with urease immobilized on CPG.     

Spherical vs. granular immobilization support selection and performance on an optical flow cell immunosensor based on the fluorescence of Cyanine-5

A spherical porous glass support Trisoperl (TRISO) with four pore diameters (? 47.8; 55.9; 102.6, and 108.8 nm) was characterized and selected for application in an optical flow cell immunosensor, in comparison with controlled pore glass (CPG). The TRISO support was functionalized with aldehyde and isothiocyanate (-NCS) groups to attach bovine serum albumin and alkaline phosphatase (AP). The TRISO isothiocyanate pore diameter 47.8 nm (TRISO(-NCS) 47.8 nm) showed the better potential to be used in the immunosensor. It immobilized more protein (19.3 mg AP per g support) while presenting an optical performance comparable to the CPG. CPG(-NCS) and TRISO(-NCS) 47.8 nm were tested in the immunosensor model where the saturation of the Goat IgG immobilized in the supports with Monoclonal Anti-Goat IgG conjugated with Cyanine-5 was reached, followed by regeneration with the elution buffer modified PBS pH 2.0. The TRISO(-NCS) 47.8 nm presented lower fluorescence intensity at saturation (around 39 AU) than CPG(-NCS) (150 to 104 AU), but revealed a major advantage related to the uniform arrangement of the spherical particles in the flow cell, generating no significant fluorescence differences between gravity and flow package.     

Studies on immobilized Saccharomyces cerevisiae. III. Physiology of growth and metabolism on various supports

In earlier communications general analyses of rapid ethanol fermentation by Saccharomyces cerevisiae immobilized on inert supports were described. In this article physiology of growth and metabolism (parameters like rates of CO(2) evolution and O(2) uptake, respiratory quotient, and generation time) of Saccharomyces cerevisiae immobilized on different supports are reported. Values of the ratio of specific oxygen uptake rate for immobilized cells to free cells have been found to be 0.732, 0.781 and 0.785 for carrier A, carrier B, and covalently crosslinked controlled pore glass (CPG, specific surface area of 439 m(2) g(-1)), respectively. Rates of specific CO(2) evolution for immobilized cells to free cells for these supports are 0.784, 0.822, and 0.783, respectively. Marked reduction in generation time of Saccharomyces cerevisiae on all the supports has been observed. No change in size (4.8-5 mum) and specific growth rate (mu(m) = 0.275 h(-1)) of cells leaving the reactor has been observed.     

Immobilized Clostridium perfringens neuraminidase. Substrate cleavage and enzyme release during incubation.

Pure Clostridium perfringens neuraminidase was immobilized on Sepharose 4 B, azido-Sepharose 4 B and controlled pore glass (CPG)- glycophase using different coupling procedures. The immobilized enzyme showed increased stability under various conditions relative to the soluble enzyme. The low release of active enzyme from the supports under incubation conditions was quantitated using a highly sensitive radioactive assay. The activity of the immobilized enzyme was dependent on the nature of the support and the substrate. Activity decreased with increasing substrate molecular weight, but the enzyme showed improved cleavage with GD1a micelles and human erythrocytes, substrates having ordered surface properties. Uses of immobilized neuraminidase in biochemistry and cell biology are considered and evaluated relative to the measured release of enzyme from the supports reported and to the molecular size and organization of possible substrates.     

Immobilization of amyloglucosidase on alkylamine derivatives of metal-link-activated inorganic supports

A new process to couple amyloglucosidase (AG) to inorganic supports is described. The technique consists in activating the support with a transition metal salt according to the metal-link method and subsequent amination and linkage of the alkylamine derivative using glutaraldehyde. The various parameters susceptible of influencing the properties of the immobilized enzyme (IME) preparation are investigated. The best result are obtained when 100 mg of 1000-Å controlled porous glass (CPG) are treated with 45 mg of TiCl₄ and the activated carrier aminated using a 10-g/L solution of hexamethylenediamine (HMDA) in carbon tetrachloride (10 Ml/100 mg). Preparation obtained according to the process here described show operational stabilities much superior to those of AG immobilized on the same support by the traditional metal-link method or its variations. The mechanism involved in the preparation of the amino derivative of CPG is proposed.     

Immobilization of a proteinase from the extremely thermophilic organism Thermus Rt41A

An extracellular proteinase from Thermus strain Rt41A was immobilized to controlled pore glass (CPG) beads. The properties of the free and CPG-immobilized enzymes were compared using both a large (azocasein) and a small (peptidase) substrate. The specific activity of the immobilized proteinase was 5284 azoU/mg with azocasein and 144 sucU/mg for SucAAPFpNA. The percentage recovery of enzyme activity was unaffected by pore size when it was immobilized at a fixed level of activity/g of beads, whereas it increased with increasing pore size when added at a fixed level/m(2) of support. Saturation of the CPG beads was observed at 540 azoU/m(2) of 105-nm beads. Lower levels (50 azoU/m(2) of 50-nm beads) were used in characterization experiments. The pH optimum of the immobilized Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA, compared with the free proteinase which was 10.5 for both substrates. The immobilized enzyme retained 65% of its maximum activity against azocasein at pH 12, whereas the free proteinase retained less than 10% under the same conditions. Stability at 80 degrees C increased on immobilization at all pH values between 5 and 11, the greatest increase in half-life being approximately 12-fold at pH 7.0. Temperature-activity profiles for both the free and immobilized enzymes were similar for both substrates. The stability of the immobilized proteinase, however, was higher than that of the free enzyme in the absence and presence of CaCl(2). Overall, the results show that low levels of calcium (10 muM) protect against thermal denaturation, but that high calcium or immobilization are required to protect against autolysis. (c) 1994 John Wiley & Sons, Inc.     

A phosphate bound universal linker for DNA synthesis.

A uridine-based linker immobilized onto polystyrene beads at the 5' terminus via a phosphodiester group and then used as a universal DNA synthesis support gives post synthesis DNA cleavage in 8 hrs or less without alkali metal salts. DNA produced with the new support was analyzed by HPLC, MALDI mass spectroscopy and PAGE. Each analysis showed DNA of equivalent quality to that produced with standard CPG supports, without contaminating materials resulting from linker or support backbone decomposition.     

Immobilization of acetate kinase and phosphotransacetylase on derivatized glass beads

The enzymes acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1) and phosphotransacetylase (acetyl coenzyme A: orthophosphate acetyltransferase, EC 2.3.1.8) were separately immobilized onto controlled pore glass CPG and silica beads (pore size 50 nm). Different coupling techniques were screened and immobilized enzymes were subjected to storage stability tests. The selected method, the CPG γ-aminopropyl glutaraldehyde succinate dihydrazide, was further optimized to improve the activity of the enzyme-loaded glass beads.     

Immobilization of laccase from Cerrena unicolor on controlled porosity glass

White-rot basidiomycete Cerrena unicolor grown in non-induced and induced conditions was tested for production of laccase, lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP). A typical correlation between the concentration of phenolic compounds in the culture fluid and the extracellular laccase activity was observed. The heterogeneous crude laccase preparation obtained after the non-induced fermentor cultivation was immobilized both on controlled porosity glass (CPG) activated by γ-aminopropyltriethoxysilane (APTES) and on CPG with its surface covered by dextran layers. The laccase activities were tested in the aqueous solution for the native and immobilized preparations using different pH and temperature conditions. Laccase activities were additionally examined for native and immobilized forms of laccase preparations in the aqueous solution containing organic solvents. The greatest activity toward the substrate used in the presence of organic solvents was shown by the laccase preparation coupled with the CPG covered by a dextran layer. Potential inhibitors such as thioglycolic acid, thiourea and EDTA used in 1-mM concentration did not show inhibiting properties towards the laccase preparations.     

A Phosphate Bound Universal Linker for DNA Synthesis

Abstract

A uridine-based linker immobilized onto polystyrene beads at the 5′ terminus via a phosphodiester group and then used as a universal DNA synthesis support gives post synthesis DNA cleavage in 8 hrs or less without alkali metal salts. DNA produced with the new support was analyzed by HPLC, MALDI mass spectroscopy and PAGE. Each analysis showed DNA of equivalent quality to that produced with standard CPG supports, without contaminating materials resulting from linker or support backbone decomposition.     

Silica carriers for immobilization of thermostable α-amylase

The possibilities for immobilization of thermostable α-amylase from Bacillus licheniformis 44MB82 on silica carriers activated by different methods have been studied. Immobilization on Ti(IV)-activated CPG, Chromosorb P, quartz powder and pumice stone was sufficiently effective. The preparation immobilized on CPG showed a shift in pH and temperature-profiles of enzyme action. They were found to be changed from pH 6,0–6,5 to pH 8,5 and from 90°C to 80°C, respectively, when compared to these parameters of the soluble enzyme. Immobilization leads to enhancement of thermostability. Possibilities for batchwise use of the immobilized preparation were established.     

New matrices for the purification of pectinases by affinity chromatography

Polygalacturonic acid was used as a ligand in the affinity technique for pectinases purification from the filtrate of Aspergillus niger 71 culture. For this purpose four matrices were examined, namely, alkylamine controlled porous glass (CPG), alkylamine silica gel as well as keratin or polyamide coated silica gel. Good results of pectinase purification was obtained on silanized CPG or keratin coated silica gel supports.     

Keratin and polyamide-coated inorganic matrices as supports for glucoamylase immobilization

Previously solubilized feather keratin and polyamide were used for coating sand, glass beads and silica gel. These new seven supports were employed for comparative studies on pure glucoamylase / EC 3.2.1.3 / immobilization. The immobilization yield of glucoamylase on keratin and polyamide coated supports was comparable with conventional matrices used earlier. The highest activity per 1 g of support was shown by the enzyme bound to polyamide-coated CPG, and the bests operational stability by the enzyme immobilized on polyamide-coated CPG with keratin subsequently deposited on it.     

Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Universal solid supports for the synthesis of oligonucleotides with terminal 3''-phosphates.

The preparation of two types of supports based on controlled pore glass (CPG) is presented. These supports are compatible with established phosphoroamidite chemistry of oligonucleotides synthesis giving rise to an oligonucleotide with terminal 3'-phosphate function during final deprotection. CPG was modified with: (i) methacrylic acid derivatives and 2-mercaptoethanol (1) or (ii) aminoalkylsilane, succinic anhydride and benzidine (2). Support 2 can be also used for the synthesis of partially protected oligonucleotide 3'-phosphates.     

Optimal pyoverdin-CPG composites for development of an optical biosensor to detect iron

Optical fluorescence-quenching-based biosensing cell is described and optimization of covalent binding of highly selective natural iron-chelating peptide secreted by bacteria is suggested. Pyoverdin biosynthesized by Pseudomonas monteilii and having 70% iron chelating activity was immobilized on amino alkylated controlled pore glass (CPG) and cross-linked with glutaraldehyde (2.5%, 28°C, 30 min). The pyoverdin-CPG immobilization was confirmed using fluorescence microscopic images (excitation range, 465–495 nm) for bright green fluorescence and by FTIR spectrum stretching at 3406.4 cm⁻¹ for amino group. The pyoverdin loading capacity of activated CPG matrix was 25 mg g⁻¹ of CPG and its rinsing analysis (leaking profile of the immobilized peptide vs. washing) detected negligible (2–3 μg) pyoverdin in the second wash.     

Covalent attachment of oligonucleotides to solid supports.

Coupling efficiencies for the covalent attachment of oligonucleotides (17-29 bases in length) to solid supports derivatized with alkyl-amino and -carboxylic functionalities have been determined. Attachment efficiencies of 60-80% were obtained for coated long-chain alkylamino controlled pore glass (CPG) supports. Similar efficiencies of immobilization were observed for carboxyl-bearing supports, which additionally exhibited lower levels of non-covalent binding. The extent of terminally linked oligonucleotide was determined to be 50-55% of the overall attachment in the carbodiimide-mediated coupling reaction of a 5'-aminohexyl phosphoramidate derivative of a 29-mer to Sephacryl carboxyl support. While lower overall efficiencies of attachment were obtained in the reaction with Sephacryl N-hydroxysuccinimide-activated carboxyl support, greater than 80% of this coupling results in end-attached oligonucleotides.     

Affinity chromatography as a rapid and convenient method for purification of fungal laccases

A rapid and convenient method for graduation, isolation, and purification of laccase from Trametes versicolor and Fomes fomentarius culture fluids was developed. For purification affinity chromatography on syringyl- and vanillyl-controlled porosity glass (CPG) columns was applied. The purified laccase of F. fomentarius was immobilized on porous glass. Some properties of the immobilized enzyme in comparison to the free one are discussed.     

Thermolytic release of covalently linked DNA oligonucleotides and their conjugates from controlled-pore glass at near neutral pH

The functionalization of long chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and its conversion to the support 7 has led to the synthesis of DNA oligonucleotides and their 3'- or (3',5')-conjugates. Indeed, CPG support 7 has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. Unlike conventional succinylated CPG supports, this distinctively functionalized support allows oligonucleotide deprotection and removal of the deprotection side products to proceed without releasing the oligonucleotide into the aqueous milieu. When freed from deprotection side products, the DNA oligonucleotide is thermolytically released from the support within 2 h under nearly neutral conditions (pH 7.2, 90 degrees C). The quality of these oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated CPG supports in terms of shorter than full length oligonucleotide contaminants and overall yields. The versatility of the thermolytic CPG support 7 is further demonstrated by the synthesis of a DNA oligonucleotide (20-mer) and its conjugation with an azido and alkynyl groups at both 5'-and 3'-termini, respectively. The functionality of the (3',5')-heteroconjugated oligonucleotide 18 is verified by its circularization to the DNA oligonucleotide 19 under "click" chemistry conditions.     

Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.