Laboratory of Enzyme Technology, Department of Biotechnology, School of Applied Biology and Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece.
Biomolecules. 2020 May 27;10(6):822. doi: 10.3390/biom10060822.
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 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.
在本研究中，我们报告了一种基于纤维素的亲和吸附剂的开发及其在利用鱼类副产物纯化蛋白酶方面的应用。亲和吸附剂是使用纤维素微纤维作为基质合成的，使用酸沉淀法从回收的报纸中分离出来。作为亲和配体，使用三嗪染料 Cibacron Blue 3GA（CB3GA）并将其直接固定在纤维素微纤维上。采用吸附平衡研究和前沿亲和层析法，使用牛血清白蛋白（BSA）和溶菌酶（LYS）作为模型蛋白来评估吸附剂的色谱性能。吸附平衡研究表明，两种蛋白质的吸附均符合朗缪尔等温线模型。吸附动力学符合准二级模型。该亲和吸附剂被应用于从副产物（胃和胰腺）中开发蛋白酶的纯化程序。开发并优化了用于胰蛋白酶和糜蛋白酶的单步纯化方案。该方案提供了适合技术和工业用途的高收率的酶。
