Biomimetic‐dye affinity adsorbents for enzyme purification: Application to the one‐step purification of Candida boidinii formate dehydrogenase

Abstract

Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4 degrees C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( K(D)) of the complex between immobilized BM6 and FDH was found to equal 0.05 microM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30 degrees C).

BSA adsorption onto nanospheres: Influence of surface curvature as probed by electrophoretic light scattering and UV/vis spectroscopy

“…Moreover, it is well known that the simplest theoretical model that can be used to describe the adsorption of a monolayer is the Langmuir model [23][24][25][26]. This model is based on a kinetic approach, assumes an energetically uniform surface and disregards the interaction between adsorbed molecules.…”

“…the Langmuir, Freundlich and Langmuir-Freundlich adsorption isotherm models, were used to fit and describe the experimental data. The Langmuir model assumes a dynamic adsorption process with reciprocation between adsorbed and unadsorbed protein molecules and disregards the interaction between adsorbed molecules [23][24][25][26]. The Freundlich isotherm, empirical in origin, is suitable for heterogeneous surfaces and allows interactions between adsorbed molecules and between these and the surface being involved in the dynamic adsorption process [25][26][27][28].…”

Influence of varying surface hydrophobicity of chitosan membranes on the adsorption and activity of lipase

“…26 However, the Langmuir model with the ideal assumption such as monolayer coverage of adsorbate over a homogenous adsorbent surface, no interactions existing between adsorbate molecules, etc., is hard to meet the practical process in this case. 27 Therefore, in this work, all the isotherms were fitted to the following LangmuirFreundlich model 28 : Figure 1 Effect of molar ratio of carboxyl group to amido group on amidation degree. …”

Affinity Chromatography: Principles and Applications

“…Covalent attachment of the dye can be achieved through nucleophilic displacement of the dye's chlorine atom by hydroxyl groups on the support's surface (Labrou, 2000;Labrou et al, 1995;Labrou, 2002). Chlorotriazine polysulfonated aromatic molecules (triazine dyes) have been used for the purification of albumin, oxidoreductases, decarboxylases, glycolytic enzymes, nucleases, hydroloases, lyases, synthetases, and transferases (Labrou, 2000;Labrou et al, 1995). The main advantages of using dye-ligands and biomimetic dye-ligands are their low cost and resistance to chemical and biological degradation.…”