Modulation of global stability, ligand binding and catalytic properties of trypsin by anions.

Specific salts effect is well-known on stability and solubility of proteins, however, relatively limited knowledge is known regarding the effect on catalytic properties of enzymes. Here, we examined the effect of four sodium anions on thermal stability and catalytic properties of trypsin and binding of the fluorescent probe, p-aminobenzamidine (PAB), to the enzyme. We show that the specific anions effect on trypsin properties agrees with the localization of the anions in the Hofmeister series. Thermal stability of trypsin, Tm, the affinity of the fluorescent probe to the binding site, Kd, and the rate constant, kcat, of trypsin-catalyzed hydrolysis of the substrate N-benzoyl-L-arginine ethyl ester (BAEE) increase with increasing kosmotropic character of anions in the order: perchlorate

Correction: Reactive Green 5-Decorated Polyacrylamide/Chitosan Cryogel: an Affinity Matrix for Catalase.

The original version of this article unfortunately contained a mistake.

Reactive Green 5-Decorated Polyacrylamide/Chitosan Cryogel: An Affinity Matrix for Catalase.

Acrylamide/chitosan-based cryogel was fabricated, and a triazine dye, Reactive Green 5, was attached to the cryogel by nucleophilic substitution to build a dye affinity support for adsorption of catalase enzyme. Characterization of cryogel was performed using FTIR, SEM, EDX, BET, and swelling test. Synthesized cryogel beared pores with ~ 200 µm in size and the surface area of 11.8 m2/g. Maximum catalase adsorption was (17.6 ± 0.29 mg/g) measured at pH 4.0 and 25 °C. The adsorption sites on the cryogel were saturated at 0.75 mg/mL enzyme concentration. Increased ionic strength caused a decrease in adsorption capacity. Desorption of catalase from cryogel was enabled using 0.5 M NaSCN solution. Consecutive adsorption experiments were carried out fifteen times to evaluate the reusability of the cryogel. Thermal, storage, and operational stabilities of immobilized catalase were higher than the free one. The data produced implicate that catalase-adsorbed dye-affinity cryogel may be used for H2O2 detection or removal when necessary. Graphical Abstract.

Covalent immobilization of papain onto poly(hydroxyethyl methacrylate)-chitosan cryogels for apple juice clarification.

Enzyme immobilization appears as a remarkable technique to safely attach enzymes for several applications and cryogels stand as promising support materials to be used in such investigations. In this work, papain enzyme was immobilized onto an interpenetrating network obtained by cryogelation of N,N'-methylenebisacrylamide cross-linked 2-hydroxyethyl methacrylate and glutaraldehyde cross-linked chitosan. Cryogels were modified with -NH2 functionality and glutaraldehyde in order to attach papain covalently. Immobilization was carried out at 25 ℃ in 0.1 M pH 7.0 phosphate buffer at 1.0 mg/ml enzyme concentration for 5 h. The amount of papain immobilized onto cryogel was calculated to be 15.2 ± 2.54 mg/g cryogel. Macroporous structure and surface area were determined by scanning electron microscopy and Brunauer-Emmett-Teller techniques, respectively. Energy dispersive X-ray analysis showed that papain was bound to the cryogel and cryogel structure was composed of 2-hydroxyethyl methacrylate, chitosan, and glutaraldehyde. Proteolytic activities of free and immobilized papain were measured using casein as substrate. Optimum pH values and temperatures were 8.0 and 65 ℃ for free and immobilized enzymes and kinetic parameters were calculated at these conditions. Reusability and storage stability results indicated that immobilization enhanced the stability of papain compared to free form. Efficiency of immobilized papain was demonstrated by apple juice clarification study as an industrial use of the enzyme. Phenolic compound, protein, total soluble solid contents, and viscosity of apple juice before and after clarification were determined.

Hofmeister effect on catalytic properties of chymotrypsin is substrate-dependent.

Effect of Hofmeister sodium salts, sulfate, chloride, bromide and perchlorate, on catalytic properties and stability of chymotrypsin has been studied by absorbance and circular dichroism spectroscopies. To address Hofmeister effect on activity of chymotrypsin, two different substrates, N-benzoyl-L-tyrosine ethyl ester and amide N-succinyl-L-phenylalanine-p-nitroanilide, were used. Catalytic activity of chymotrypsin is dependent on salt concentration and position of anion in Hofmeister series. The enzyme activity for both substrates is only slightly affected by chaotropic anions and increases with kosmotropic nature of anions. While the trend of Hofmeister effect on chymotrypsin catalysis is similar for both substrates, the amplitude of the effect significantly differs. In the presence of 1 M sulfate, catalytic efficiency increased by ~2-fold for the ester but ~20-fold for the amide substrate. Positive correlation between stability and activity of chymotrypsin indicates the interdependence of these enzyme properties and is in agreement with recently developed macromolecular rate theory suggesting an important role of protein dynamics in enzyme catalysis. Linear dependencies of catalytic properties of chymotrypsin with partitioning of anions at bulk water/air as well as at hydrocarbon surface strongly indicate that the modulated enzyme properties are results of direct interaction of anions with protein surface.