Aplicação de criogéis funcionalizados com L-fenilalanina na adsorção de lisozima por cromatografia de afinidade hidrofóbica

Abstract

Supermacroporous polyacrylamide cryogels are polymeric materials that have stood out among the new adsorbents developed for use in chromatographic processes. Its application has extended to the purification of several biomolecules such as lysozyme. Due to its effective antimicrobial action against some bacteria, this enzyme is of great industrial interest. This work aimed to synthesize polyacrylamide cryogels, functionalize them with the amino acid L-phenylalanine and apply them to lysozyme adsorption by hydrophobic affinity chromatography under different conditions of ionic strength, pH and type of salt. The best experimental condition for lysozyme adsorption was used to capture this enzyme, in continuous mode, from a solution containing chicken egg white. Subsequently, the cryogel functionalized with L-phenylalanine (cryogel-Phe) was chemically characterized by Fourier transform infrared spectroscopy (FTIR), its mechanical and morphological properties (macro, micro and mesoporous fraction, bound water fraction and dry polymer, swelling capacity and degree of expansion), crystallinity, thermal properties (differential scanning calorimetry, DSC and thermogravimetry, TGA) and hydrodynamic properties of the column were also analyzed. The best experimental condition for lysozyme adsorption was obtained by using sodium thiocyanate saline solution 0.025 mol L-1

at pH 5.0. In this

condition, the adsorptive capacity (q) of the cryogel-Phe was 67.65 ± 1.52 mg g- 1

. FTIR confirmed the presence of the amino acid L-phenylalanine immobilized in the cryogen-Phe, since peaks at 1716 cm-1

, 1475 cm-1

, 1510 cm-1

and 1355 cm- 1

indicated the presence of this amino acid and a reduction in the peak at 1100 cm- 1 was observed indicating that part of the epoxy groups available for

immobilization of ligands on the cryogel surface were filled. In addition, Kjeldahl's analysis of L-phenylalanine solutions before and after contact with the cryogel indicated an immobilization of 458.65 ± 40.59 mgphenylalanine gcriogel -1 . The cryogel-Phe showed a Young's modulus of 526.71 ± 27.21 kPa. Morphological analyzes indicated high porosity (0.95) of the material with pores between 10 μm and 75 μm. By XRD the cryogel-Phe showed characteristics of an amorphous material. DSC indicated a glass transition temperature of 17.80 ° C and the TGA indicated that the onset of thermal degradation occurred after 200 ° C. The column exhibited a hydraulic permeability of 4.15 x 10-13 m2, an axial dispersion coefficient ranging from 10-7

to 10-6 m2 s -1 and height equivalent to a theoretical plate (HETP) between 0.10 and 0.21 cm. It was concluded that the cryogel-Phe is an effective material in the capture of lysozyme by hydrophobic affinity chromatography, because a purification factor of 11.49, a recovery of 31.66% of lysozyme with a purity of 87.92% and enzymatic activity of 1294.17 U mL-1 was obtained. In addition, this support showed high mechanical and thermal resistance, high porosity, low axial dispersion and high hydraulic permeability, being an attractive adsorbent to be used in the purification of this biomolecule.