Putrescine-polysaccharide conjugates as transglutaminase substrates and their possible use in producing crosslinked films.

Putrescine (1,4-diaminobutane) was covalently linked to alginate and low-methoxyl pectin to synthesize new aminated polysaccharides. Both putrescine-pectin and -alginate conjugates, although the latter at higher concentrations, were found to be able to act as effective acyl acceptor transglutaminase substrates in vitro using both dimethylated casein and soy flour proteins as acyl donors. Monodansylcadaverine, a well known acyl acceptor transglutaminase substrate, dose-dependently counteracted the covalent binding of the aminated polysaccharides to the proteins. Putrescine-pectin conjugate was also tested to prepare, in combination with soy flour proteins, edible films in the presence of purified microbial transglutaminase. Characterization of the enzymatically crosslinked films showed a significant decreased water vapor permeability, with respect to the ones obtained with non-aminated pectin in the presence of transglutaminase, as well as improved mechanical properties, such as high extensibility. Possible biotechnological applications of hydrocolloid films containing putrescine-polysaccharide derivatives enzymatically crosslinked to proteins were suggested.

Novel enzyme biosensor for hydrogen peroxide via supramolecular associations.

A polythiolated-beta-cyclodextrin polymer was synthesized and used as a coating material for gold electrodes. The functionalized electrodes were employed for immobilizing adamantane-modified horseradish peroxidase via supramolecular associations. The enzyme-containing electrode was used as an amperometric biosensor device with 1mM hydroquinone as electrochemical mediator. The biosensor exhibited a fast amperometric response (10s), a good linear response toward H(2)O(2) concentrations between 28 microM and 5.5 mM, and a low detection limit of 7 microM. The biosensor showed a sensitivity of 109 microA/Mcm(2) and retained 98% of its initial electrocatalytic activity after 40 days of storage at 4 degrees C in 50mM sodium phosphate buffer pH 7.0. The host-guest supramolecular nature of the immobilization method was confirmed by cyclic voltammetry.

Hydrogen peroxide biosensor with a supramolecular layer-by-layer design.

A new sensor design is reported for the construction of an amperometric enzyme biosensor toward H (2)O(2). It was based in the supramolecular immobilization of alternating layers of horseradish peroxidase (either modified with 1-adamantane or beta-cyclodextrin-branched carboxymethylcellulose residues) on Au electrodes coated with polythiolated beta-cyclodextrin polymer. The analytical response of the electrodes, using 1 mM hydroquinone as an electrochemical mediator, increases when the number of enzyme layers increases. The biosensor having three enzyme layers showed a sensitivity of 720 microA/M cm (2) and a detection limit of 2 microM and retained 96% of its initial activity after 30 days of storage. The host-guest supramolecular nature of the immobilization method was confirmed by cyclic voltammetry.

Ferrocene branched chitosan for the construction of a reagentless amperometric hydrogen peroxide biosensor.

Chitosan was chemically branched with ferrocene moieties and further used as a support for the immobilization of horseradish peroxidase on a glassy carbon electrode. The reagentless biosensor device showed a linear amperometric response toward hydrogen peroxide concentrations between 35 x 10(-6) M and 2.0 x 10(-3) M. The biosensor reached 95% of the steady-state current in about 20 s and its sensitivity was 28.4 x 10(-3) microA x M(-1). The enzyme electrode retained 94% of its initial activity after 2 weeks of storage at 4 degrees C in 50 x 10(-3) M sodium phosphate buffer at pH 7.0.

Chitosan-whey protein edible films produced in the absence or presence of transglutaminase: analysis of their mechanical and barrier properties.

Chitosan-whey protein edible films with different protein concentrations were prepared in the absence or presence of microbial transglutaminase as cross-linking agent. The films prepared in the presence of the enzyme showed low solubility at a wide range of pH, a lower degree of swelling, and good biodegradability following protease treatments. The presence of transglutaminase induced also an enhancement in film mechanical resistance and a reduction in their deformability. Finally, the barrier efficiency toward oxygen and carbon dioxide was found to be markedly improved in the cross-linked films which showed also a lower permeability to water vapor. Some potential practical applications of transglutaminase-treated chitosan-whey protein films are suggested.