Maria L. Villalonga

Preparation and functional properties of trypsin modified by carboxymethylcellulose

Abstract Trypsin from bovine pancreas was modified by the polyaldehyde derivative of carboxymethylcellulose (CMC) via reductive alkylation with NaBH 4 . The modified enzyme contained 57% carbohydrate by weight, resulting from the modification of 52% of the amino groups of the protein. In comparison with the native protease, the modified trypsin retained 62% and 42% of the esterolytic and proteolytic activity, respectively. The value of K m for CMC–trypsin complex was 2.2 times lower than for the native enzyme. The thermostability and pH stability was improved for trypsin by this modification. The conjugate was also more resistant to the action of the anionic surfactant sodium dodecylsulphate and denaturing agents such as 8 M urea and 6 M guanidinium chloride. This modification also protected the enzyme against autolysis at alkaline pH and improved the stability of the enzyme in the presence of methanol.

Decorating carbon nanotubes with polyethylene glycol-coated magnetic nanoparticles for implementing highly sensitive enzyme biosensors

Superparamagnetic Fe3O4 nanoparticles were coated with (3-aminopropyl)triethoxysilane and further branched with monomethoxypolyethylene glycol chains. These nanoparticles were employed for the non-covalent surface modification of single walled carbon nanotubes, conferring them magnetic properties. This nanomaterial was employed to immobilize the enzyme xanthine oxidase in order to construct magnetically modified disposable gold screen-printed electrodes as bioelectrodes for the determination of xanthine. The electroanalytical properties of the biosensor were modulated by the nanomaterial composition, being optimal at a carbon nanotubes : magnetic nanoparticles ratio of 1 : 27. The resulting biosensor showed a linear dependence on the xanthine concentration in the 0.25–3.5 μM range with a fast amperometric response in 12 s. The biosensor also showed a noticeable high sensitivity of 1.31 A M−1 cm−2 and a very low detection limit of 60 nM, which can be compared advantageously with other biosensor designs for xanthine.

Stabilization of α-amylase by chemical modification with carboxymethylcellulose

Carboxymethylcellulose activated by periodate oxidation was covalently linked to porcine pancreatic α-amylase (EC 3.2.1.1). The specific activity of the conjugate prepared was 54% of the native enzyme. The carbohydrate content was estimated to be 62% by weight as a result of the modification of 67% of the amino groups from the protein. In comparison with the native enzyme, the thermostability and pH stability were improved for α-amylase by this modification. The conjugate was also more resistant to the action of denaturant agents such as urea and sodium dodecylsulfate. We conclude that modification of enzymes by the anionic polysaccharide carboxymethylcellulose might be a useful method for improving enzyme stability under various denaturing conditions.

Stabilization of trypsin by chemical modification with β-cyclodextrin monoaldehyde

The monoaldehyde derivative of β-cyclodextrin was attached to trypsin via reductive alkylation with NaBH4. The thermostability was enhanced from 49.5 °C to 60 °C for modified trypsin. The activation free energy of thermal inactivation at 50 °C was increased by 3.2 kJ mol−1. The conjugated enzyme retained 100% of its initial activity after 3 h incubation at pH 9.

Supramolecular immobilization of redox enzymes on cyclodextrin-coated magnetic nanoparticles for biosensing applications.

Mono-6-formyl-β-cyclodextrin moieties were attached to (3-aminopropyl)triethoxysilane-coated superparamagnetic Fe(3)O(4) nanoparticles by reductive alkylation with NaBH(3)CN. The oligosaccharide-capped core-shell nanoparticles were employed as support for the supramolecular immobilization of two different adamantane-modified enzymes, tyrosinase and xanthine oxidase, through host-guest interactions. The enzyme-modified nanomaterial was further used to magnetically modify carbon paste electrodes for constructing amperometric biosensors toward cathecol and xanthine. The tyrosinase and xanthine oxidase based biosensors showed excellent electroanalytical behaviours, with linear ranges of 100 nM-12 μM cathecol and 5.0-120 μM xanthine, sensitivities of 12 mA/M and 130 mA/M, and low detection limits of 22 nM and 2.0 μM, respectively. The supramolecular nature of the immobilization approach was confirmed by electroanalytical methods.

Functional Stabilization of Trypsin by Conjugation with β-Cyclodextrin-Modified Carboxymethylcellulose

Abstract Bovine pancreatic trypsin was chemically modified by a β-cyclodextrin-carboxymethylcellulose polymer using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as coupling agent. The conjugate retained 110% and 95% of the initial esterolytic and proteolytic activity, respectively, and contained about 2 mol of polymer per mol of trypsin. The optimum temperature for trypsin was increased to 8°C after conjugation. The thermostability of the enzyme was increased to about 16°C after modification. The conjugate prepared was also more stable against thermal incubation at different temperatures ranging from 45°C to 60°C. In comparison with native trypsin, the polymer-enzyme complex was more resistant to autolytic degradation at pH 9.0, retaining about 65% of the initial activity after 3 h incubation. In addition, modification protected trypsin against denaturation in the presence of sodium dodecylsulfate.

Chemical glycosidation of trypsin with O‐carboxymethyl‐poly‐β‐cyclodextrin: catalytic and stability properties

The polysaccharide O‐carboxymethyl‐poly‐β‐cyclodextrin was synthesized (molecular mass 13000 Da, 40% carboxy groups) and attached to the surface of bovine pancreatic trypsin. The resulting neoglycoenzyme retained high proteolytic and esterolytic activity and contained approx. 1.0 mol of polymer/mol of enzyme. The optimum temperature for trypsin activity was increased by 10°C after this transformation. Thermostability of the polymer–enzyme complex was increased by about 14°C over 10 min incubation. The conjugate was also more resistant to thermal inactivation at different temperatures, ranging from 45 to 60°C, demonstrating the influence of supramolecular and polymer–protein electrostatic interactions on trypsin thermostabilization. Additionally, the conjugate was 36‐fold more resistant to the action of the anionic surfactant SDS. This modification also protected the enzyme from autolysis at alkaline pH.

Stabilization of α‐chymotrypsin by modification with β‐cyclodextrin derivatives

Bovine pancreatic α‐chymotrypsin was chemically modified with two different β‐cyclodextrin derivatives, named mono‐6‐formyl‐β‐cyclodextrin and mono‐6‐succinyl‐6‐deoxy‐β‐cyclodextrin. The modified enzymes contained approx. 3–5 mol of oligosaccharide/mol of protein, and retained full proteolytic and esterolytic activity. The optimum temperature for α‐chymotrypsin was increased by 8 °C and its thermostability was enhanced by about 4–6 °C after modification. The conjugated enzymes were also more resistant to thermal inactivation at temperatures ranging from 45 to 55 °C. Additionally, the modified enzymes were 7‐fold more stable against incubation at pH 9.0. The possible influence of supramolecular interactions on the thermal stabilization of modified α‐chymotrypsins was also studied.

Supramolecular-mediated Immobilization of Trypsin on Cyclodextrin-modified Gold Nanospheres

Bovine pancreatic trypsin was immobilized on β- and γ-cyclodextrin coated gold nanospheres via supramolecular associations. The enzyme retained 100%–120% of its catalytic activity and its thermal stability at 50°C was 2–2.5 fold increased in the presence of the β- and γ-cyclodextrin modified metal nanoparticles, respectively. The influence of these immobilization processes on the conformational properties of the enzyme was studied by fluorescence spectroscopy. These results open a new perspective to the possible application of cyclodextrin-modified gold nanospheres as water-soluble carriers for enzyme immobilization.

Metal-induced stabilization of trypsin modified with α-oxoglutaric acid †

Abstractα-Oxoglutaric acid was attached to trypsin via reductive alkylation with NaBH4 thereby introducing metal-chelating groups at the protein surface. The thermostability of the modified enzyme was increased by 6.5–13 °C and its resistance to autolytic degradation was improved 2- to 4-fold in 5 mm ZnCl2, MnCl2 or MgCl2.