Abstract
In the present study, we report the development of a cellulose-based affinity adsorbent and its application for the purification of proteases from fish by-products. The affinity adsorbent was synthesized using cellulose microfibers as the matrix, isolated from recycled newspapers using the acid precipitation method. As an affinity ligand, the triazine dye Cibacron Blue 3GA (CB3GA) was used and immobilized directly onto the cellulose microfibers. Absorption equilibrium studies and frontal affinity chromatography were employed to evaluate the chromatographic performance of the adsorbent using as model proteins bovine serum albumin (BSA) and lysozyme (LYS). Absorption equilibrium studies suggest that the adsorption of both proteins obeys the Langmuir isotherm model. The kinetics of adsorption obey the pseudo-second-order model. The affinity adsorbent was applied for the development of a purification procedure for proteases from Sparus aurata by-products (stomach and pancreas). A single-step purification protocol for trypsin and chymotrypsin was developed and optimized. The protocol afforded enzymes with high yields suitable for technical and industrial purposes.
Highlights
The development of chromatographic materials for the separation and purification of proteins has been an essential tool for research and development in biotechnology [1,2,3]
The results indicate that the overall rate of the bovine serum albumin (BSA) and LYS adsorption process is most likely to be Biomolecules 2020, 10, x FOR PEER REVIEW
The results showed that at acidic pH values the binding capacity is enhanced, compared to that at the most crucial parameters in dye-ligand affinity chromatography as it can affect dramatically the chromatographic behavior, such as affinity, selectivity and recovery of the target protein [29,30,41]
Summary
The development of chromatographic materials for the separation and purification of proteins has been an essential tool for research and development in biotechnology [1,2,3]. Affinity chromatography is the most specialized method for the effective purification of proteins, compared to other separation methods [2,3,4,8,9,10,11,12,13]. It offers high selectivity, resolution, and capacity in most protein purification procedures. Affinity chromatography has the advantage of exploiting a protein’s structure or function (molecular recognition) as it is based on the specific and reversible interaction between the target protein with an immobilized ligand [2,3,4,8,9]. The interaction between the target protein and the immobilized ligand is the result of various molecular interactions involved, namely, electrostatic, hydrogen bonding, hydrophobic and van der Waals interactions [14]
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