BIOMIMETIC-DYE AFFINITY ADSORBENTS FOR ENZYME-PURIFICATION - APPLICATION TO THE ONE-STEP PURIFICATION OF CANDIDA-BOIDINII FORMATE DEHYDROGENASE

Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boid inii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven biomimetic analogues of the monochlorotriazin e dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichlorotriazine dye, Vilmafix(R) Blue A-R (VBAR), bearing a carboxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose g el, Ultrogel(R) A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evalu ated for their ability to purify FDH from extracts obtained after pres s-disintegration of G. boidinii cells. Optimal, conditions for maximiz ing specific activity of FDH in starting extracts (1.8 U/mg) were real ized when cell growth was performed on 4% methanol, and press disinteg ration proceeded in four consecutive passages before the homogenate wa s 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 b iomimetic moiety mercaptopyruvic acid linked on the chlorotriazine rin g (BM6), displayed the highest purifying ability. Adsorption equilibri um data which were obtained for the BM6 adsorbent in a batch system co rresponded well to the Langmuir isotherm and, in addition, breakthroug h curves were taken for protein and FDH adsorption in a fixed bed of B M6 adsorbent. The dissociation constant (K-D) of the complex between i mmobilized BM6 and FDH was found to equal 0.05 mu M. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of G. boidini i in a single step (60% overall yield of FDH). The purified FDH afford ed a single-band on sodium dodecyl sulphate polyacrylamide gel electro phoresis, and a specific activity of 7.0 U/mg (30 degrees C). (C) 1995 John Wiley & Sons, Inc.