Purificação de l-asparaginase de aspergillus caespitosus ccdca 11593 em criogel de troca iônica

Abstract

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of L-asparagine into aspartic acid and ammonia, thereby reducing the formation of acrylamide, a potentially carcinogenic compound generated during the Maillard reaction between reducing sugars and L-asparagine. Chromatographic techniques are widely employed for enzyme purification, and polymeric cryogels have emerged as well-established matrices for this purpose due to their high porosity, elevated adsorption capacity, and ability to process large sample volumes. Nevertheless, structural modifications are required to enhance the efficiency of target biomolecule capture. In this study, polyacrylamide cryogels were functionalized with nicotinamide and applied to the purification of L-asparaginase produced by Aspergillus caespitosus CCDCA 11593 using ion-exchange chromatography. The effects of cryogel functionalization strategies (glutaraldehyde method and Schiff base method), sodium phosphate buffer concentration (0.05 and 0.10 mol·L⁻¹), and pH (3.0, 5.0, and 8.0) on enzyme adsorption were evaluated in order to maximize the adsorption capacity (q, mg·g⁻¹). The cryogel functionalized with niacinamide via the glutaraldehyde method (Glu-Nic) exhibited superior performance (q = 161.32 ± 2.36 mg·g⁻¹ and residual activity in the supernatant of 42,716.36 ± 10,856.18 U·mL⁻¹), and was therefore selected as the optimal matrix for enzyme adsorption under conditions of pH 5.0 and ionic strength of 0.05 mol·L⁻¹. Although the cryogel functionalized with hexanediamine followed by glutaraldehyde and niacinamide addition (Hex-GluNic) showed statistically comparable results, the Glu-Nic matrix was preferred due to the reduced number of functionalization steps while maintaining high adsorption efficiency. Physicochemical characterization of the Glu-Nic cryogels demonstrated the preservation of the macroporous structure, with total porosity (φT = 0.795 ± 0.035), macropore porosity (φM = 0.528 ± 0.054), swelling capacity of 11.33 ± 2.67, and epoxy group density of 352.28 ± 57.93 µmol·g⁻¹, ensuring the availability of active sites for functionalization. The point of zero charge (pHpzc ≈ 6.5) indicated a predominance of positive charges under acidic conditions, favoring electrostatic interactions with L-asparaginase, particularly at pH 5.0. During the ion-exchange chromatography purification step, the Glu-Nic cryogel reduced the total protein concentration from 1.084 to 0.074 mg·mL⁻¹, accompanied by an increase in specific activity to 441.94 U·mg⁻¹, confirming the high selectivity and efficiency of the purification process. Adsorption isotherms were best described by the Langmuir model (qₘₐₓ = 68.7 mg·g⁻¹), suggesting homogeneous monolayer adsorption. Thermodynamic analysis indicated that the adsorption process was spontaneous (ΔG° < 0) and exothermic (ΔH° < 0). Furthermore, SDS-PAGE analysis confirmed the selectivity of the Glu-Nic cryogel for the chromatographic capture of L-asparaginase. Overall, the Glu-Nic cryogel demonstrated high stability and selectivity, highlighting its potential as a promising matrix for biotechnological applications in the purification of therapeutic enzymes such as L-asparaginase. Keywords: Adsorption; acrylamide; cryogel; niacinamide; purification; enzyme