Synthesis and characterization of supermacroporous cryogel with immobilized p-aminobenzenesulfonamide as affinity ligand for the purification of lactoperoxidase from whey.

This study proposed the development of a monolithic supermacroporous affinity column for direct capture of lactoperoxidase, a glycoprotein present in milk, whey, and colostrum, with several applications due to its wide antimicrobial activity. A poly(acrylamide)-based cryogel was produced by radical co-polymerization of monomers in frozen aqueous solution and activated with p-aminobenzenesulfonamide as a ligand for specific interaction with the lactoperoxidase. The axial liquid dispersion coefficients at different liquid flow rates were determined by measuring residence time distributions using the tracer pulse-response method. The axial dispersion coefficient was low and the height equivalent to theoretical plate was not dependent on the flow velocity. The adsorptive capacity of affinity cryogel was studied as a function of flow velocity and the best condition was 0.9 cm/min. The response surface methodology was applied to optimize the capture of the enzyme, as a function of pH and salt concentration. Higher purification factor value was found at a salt concentration of 80 mmol/L and pH of 8.0 (p < 0.05). There was no influence of the variables under study on the yield (p > 0.05). The results indicated that affinity cryogel is a promising chromatography support for the use in high-throughput one-step purification of lactoperoxidase from whey.

Adsorption and immobilization of ß-glucosidase from Thermoascus aurantiacus on macroporous cryogel by hydrophobic interaction.

Enzyme immobilization has been reported as a promising approach to improving parameters such as thermal stability, pH and reusability. In this study, a polyacrylamide cryogel functionalized with L-phenylalanine was prepared to be used in the adsorption of ß-glucosidase from Thermoascus aurantiacus, aiming at its separation and also its immobilization on the cryogel matrix. The enzyme was produced by solid state fermentation. First, the adsorption was studied as a function of the pH and the resulting yield (Y, %) and purification factor (PF, dimensionless) were determined (1.57-5.13 and 64.19-91.20, respectively). The PF and yield from eluate samples obtained at pH 3.0 were the highest (5.13 and 91.20, respectively). Then, ß-glucosidase was immobilized on the hydrophobic cryogel and the recovery activities (%) were determined as a function of temperature and in the presence of different saline solutions. The values ranged from 14.45 to 45.97. As expected, salt type and ionic strength affected the activity remained in the immobilized ß-glucosidase. The average bioreactor activity was 39.9 U/g of dry cryogel and its operational stability was measured, with no decrease in activity being observed during seven cycles. Kinetic parameters of free and immobilized enzyme were determined according to different models.

Modification of polysulfone membrane used in the water filtration process to reduce biofouling.

Polysulfone membranes (PSF) were modified with silver nanoparticles obtained by new synthesis (nAgNS), silver nanoparticles obtained commercially (nAgC), silver sulfadiazine (SP), dodecyltrimethylammonium bromide (DOTAB), benzalkonium chloride (CB) or sodium dodecylbenzene sulfonate (DBSS) to improve the efficiency of the water filtration process by reducing biofouling. All membranes had lower hydrophobicity compared with PSF. The zeta potentials of all membranes were negative at pH 7.0, except for CB 10%. In the agar diffusion test, E. coli was considered to be sensitive to the antimicrobial effect of the nAgNS 1%, 3%, 6%, 10% and DOTAB 10%, whereas S. aureus was sensitive to the nAgNS 1%, 3%, 6%, 10%, DOTAB 10%, CB 0.22%, 2% and 10%. The lowest adhesion of E. coli was found in the nAgNS 6% and 10%. In the evaluation of the loss of flow rate during filtration of the E. coli suspension and pure water, nAgNS showed higher flow rate values when compared with PSF. The nAgNS did not release quantities of silver (0.1 mg/l) above the amount considered safe by the World Health Organization. Membranes nAgNS 6% and 10% showed the best anti-biofouling characteristic.

Development of an affinity cryogel for one step purification of lysozyme from chicken egg white.

In this study, a supermacroporous polyacrylamide cryogel was produced by cryo-polymerization and activated with Tris(hydroxymethyl)aminomethane (Tris-cryogel) to be applied as an affinity ligand for a one step purification of lysozyme (LYZ), directly from chicken egg white (EW). The Tris-cryogel presented interconnected pores with size varying in the range of 20-80µm and swelling capacity of 19.6±0.9g/g. The axial dispersion of the Tris-cryogel was analyzed at different flow velocities and mobile phase viscosities. It was verified that higher viscosity resulted in a higher degree of dispersion, causing the HETP values to increase from 0.04cm to 0.8cm. Adsorption isotherms were measured at 15°C and 35°C at pH 7.5. A Langmuir model was fitted to the equilibrium data, with a maximum adsorptive capacity of 285mg/g at 15°C and 363mg/g at 35°C. Thermodynamic analysis based on the Van't Hoff relationship showed that the process was spontaneous and enthalpically driven. Lysozyme was purified directly from egg white in a one step purification process at different pH values (7.5, 8.5 and 9.5). Independent of the pH, the specificity of Tris-cryogel for lysozyme adsorption was confirmed. At pH 7.5, yield and purification fold were higher (30% and 45). In addition, the effect of the dilution rate on egg white and flow velocity were also analyzed and it was shown that flow velocity did not affected purification and column efficiency, and that diluting the egg white increased yield to 70% with a purification fold of 23. Results show Tris-cryogel is a promising matrix for use in high throughput purification of lysozyme from egg white.