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

<jats:title>Abstract</jats:title><jats:p>Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from <jats:italic>Candida boidinii</jats:italic> cells in a single step by biomimetic‐dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron® Blue 3GA (CB3GA), and parent dichloro‐triazine dye, Vilmafix<jats:sup>®</jats:sup> Blue A‐R (VBAR), bearing a car‐boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel<jats:sup>®</jats:sup> 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 <jats:italic>C. boidinii</jats:italic> 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°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 ( <jats:italic>K</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub>) of the complex between immobilized BM6 and FDH was found to equal 0.05 μ<jats:italic>M</jats:italic>. Adsorbent BM6 was employed in the purification of FDH from a 18‐L culture of <jats:italic>C. boidinii</jats:italic> 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°C). © 1995 John Wiley & Sons, Inc.</jats:p>