Termostable and effective immobilized invertase for sucrose determination in fruit juices.

In this study, immobilization of invertase enzyme was performed on a previously synthesized and characterized poly(N-vinylpyrrolidone-co-butylacrylate-co-N hydroxymethylacrylamide) terpolymer membranes by covalent bonding method. Glutaraldehyde(GA) was used as the crosslinker and Bovine Serum Albumin(BSA) was used as the binding agent. Optimum pH, temperature, amount of polymer, substrate concentration, amount of BSA and amount of GA values were determined for both free and immobilized enzyme. Optimum pH and temperature values were found as pH = 5.0, T = 55 °C, pH = 7.0 and T = 80 °C for free and immobilized enzyme, respectively. In particular, the optimum temperature of 80 °C for the immobilized enzyme provides its potential to be used commercially. The kinetic parameters of the free enzyme and the immobilized enzyme were determined using the well known Lineweaver-Burk method. The Vmax values for free (13.4 µM/min) and immobilized enzyme (12.2 µM/min) were found as close to each other, while the Km value of the immobilized enzyme (8.33 mM) was much lower than that of the free enzyme (29.41 mM). In reuse studies conducted with peach and orange juices, it was determined that the immobilized enzyme retained approximately 90% of its activity even after 30 reuses within 1 month.

An immobilized invertase enzyme for the selective determination of sucrose in fruit juices.

Poly(N-vinylpyrrolidone-co-butylacrylate-co-N-hydroxymethylacrylamide) has been synthesized by free radical polymerization at 70 °C. Copolymer were characterized by FT-IR, elemental analysis and viscometric methods. Invertase was immobilized onto poly(N-vinyl pyrrolidone-co-butyl acrylate-co-N-hydroxymethyl acrylamide) by entrapment method. Optimum parameters (pH, temperature, substrate concentration, amount of polymer) for immobilization to obtain maximum activity were investigated. Kinetic parameters, Km and Vmax, of the free and immobilized invertases were also assayed. Results showed that immobilization enhanced the enzyme stability against changes of pH and temperature and immobilized enzyme showed lower Km value than free enzyme. One of the most interesting results is that the optimum operational temperature of the immobilized enzyme was 15 °C higher than that of the free enzyme. The next is the activity of the immobilized enzyme at the optimum temperature (70 °C) was approximately the same as the activity of the free enzyme at its optimum temperature (55 °C). Finally, immobilized invertase were used for determination of sucrose in commercial fruit juices. A new method and equation based on immobilized invertase were derived for determination of sucrose in commercial cheryy and pomegranate juices.

Protein A immunosensor for the detection of immunoglobulin G by impedance spectroscopy.

A novel highly sensitive electrochemical impedimetric Protein A immunosensor for the determination of immunoglobulin G (IgG) was developed by immobilization of Protein A within a newly synthesized, and characterized polymer, poly(maleicanhydride-alt-decene-1). TiO2 nanoparticles (10-30 nm) were synthesized, characterized with X-ray diffraction, transmission electron microscopy and Brunauer-Emmett-Teller surface analysis. The electron transfer between IgG and the poly(maleicanhydride-alt-decene-1)-TiO2-Protein A is quasireversible with a formal potential of 225 mV vs Ag|AgCl. The response of the poly(maleicanhydride-alt-decene-1)-TiO2-Protein A immunosensor was proportional to IgG concentration with a correlation coefficient of 0.9963. The detection limit and linear range was 0.57 ng mL(-1) and 0.0062-500 µg mL(-1), respectively. Impedance measurments showed that synthesized TiO2 nanoparticles have better conducting properties compared with commercial degussa P25 TiO2 nanoparticles. The nonspecific binding of anti-MBP was 10 %. The label-free impedimetric immunosensor provided a simple and sensitive detection method for the specific determination of IgG in human serum.

Preparation and properties of invertase immobilized on a poly(maleic anhydride-hexen-1) membrane.

Poly(maleic anhydride-alt-hexen-1)(poly(MA-alt-H-1)) has been synthesized by radical polymerization and characterized by DSC, FT-IR, acid number determination, viscometric and NMR methods. Data showed that the co-polymer is an alternating co-polymer whose composition does not depend on the monomer feed composition. Invertase was immobilized onto a poly(MA-alt-H-1) membrane via glutaraldehyde and bovine serum albumin. The Km value of poly(MA-alt-H-1)-invertase was approximately 4.4-fold higher than the free enzyme, indicating decreased affinity by the invertase for its substrate (sucrose), whereas Vmax was lower for the immobilized invertase. Immobilization improved the pH stability of the enzyme, as well as its temperature stability. Immobilized samples obtained were stable and could be used many times over a period of 2 months without considerable activity loss.

Bioengineering Functional Copolymers. IX. Poly[(maleic anhydride-co-hexene-1)-g-poly(ethylene oxide)].

Amphiphilic bioengineering copolymers having a combination of hydrophilic/hydrophobic linkages and polyelectrolyte behavior, along with an ability to interact with biomacromolecules, in particular with the invertase enzyme, have been synthesized by (a) complex-radical copolymerization of maleic anhydride (MA, the acceptor) and hexene-1 (H-1, the donor) monomers with benzoyl peroxide as the initiator in 1,4-dioxane at 65 degrees C under high-conversion conditions and (b) subsequent grafting (polyesterification) of synthesized poly(MA-alt-H-1) with alpha-methoxy-omega-hydroxy-poly(ethylene oxide) (PEO). Copolymerizations were also carried out in the steady state, in order to essentially reduce the effect of copolymer composition drift. The values of the monomer reactivity ratios (r(1) and r(2)) determined by using the known terminal models of Fineman-Ross (FR) and Kelen-Tüdös (KT), as well as by nonlinear regression (NLR) analysis, are: r(1) = 0.16 and r(2) = 0.30 (FR), r(1) = 0.14 and r(2) = 0.27 (KT), and r(1) = 0.15 and r(2) = 0.29 (NLR), respectively. All the copolymers and graft copolymers were characterized by FTIR spectroscopy, (1)H{(13)C} NMR spectroscopy, viscometric measurements, and chemical (acid number), thermal (DSC and TGA), and X-ray diffraction analyses. Unlike poly(MA-alt-H-1)s, PEO macrobranched graft copolymers exhibit expressed polyelectrolyte and swelling behavior in diluted and concentrated dioxane solutions, respectively. The copolymer and its PEO hyperbranched derivatives can be used as carriers for enzyme immobilization.