Optimization of a pseudo-affinity process for penicillin acylase purification L.P. Fonseca, J.M.S. Cabral Abstract A pseudo-affinity process for penicillin acylase (EC 3.5.1.11) purification using an affinity ligand (Ampi- cillin) attached on Sepharose 4B-CNBr was optimized. The enzyme adsorption on this affiant (Amp-Seph) is inde- pendent of pH between 5.5 and 8.8, in 100 mM phosphate containing 22% (w/v) ammonium sulphate. The desorp- tion of the penicillin acylase from the affinity gels was carried out, the best desorption results being obtained through a non specific eluent, 100 mM phosphate pH 4.6 with 15% (w/v) ammonium sulphate. The best purification results were obtained with an enzymatic extract, produced through osmotic shock of Escherichia coli cells (3.7 IU/mg prot). With this extract and an affinity gel of Sepharose 4B- CNBr derivatized with ampicillin (3.8 lmol/cm 3 gel), a maximum activity capacity adsorbed of 20 IU/cm 3 gel was obtained for initial values of activity and protein concentration of 1.7 IU/cm 3 and 0.4 mg prot/cm 3 , re- spectively. With the optimized eluent it was possible to obtain penicillin acylase in only one purification step with a desorption yield of enzyme activity higher than 90%. The penicillin acylase produced with this process was charac- terized by a maximum purity of 34 IU/mg prot, corre- sponding to a purification degree higher than 150 in relation to the lowest pure enzymatic extract. The enzyme purity of the eluted fractions was certified by SDS gel electrophoresis and liquid chromatography through a Mono Q column in a FPLC apparatus. The gel electro- phoresis presented 4 main stained bands with 2 corre- sponding to a and b subunits of the penicillin acylase with equivalent molecular weights of 27 and 63 kDa. No ex- ternal diffusion resistance on penicillin acylase and total protein adsorption on this affiant (Amp-Seph 3.8 lmol/ cm 3 gel) were observed for continuous adsorption pro- cesses performed at two different agitation speeds (120 and 400 rpm). 1 Introduction Several commercial supports with different activated groups have been developed for its further derivatization with affinity ligands [1], which include dyes, concanavalin A, polynucleotides, cofactors, protein A, protein G, phenyl boric acid, heparin, amino acids, and other affinity ligands [2]. Several of them have been tested in the purification of proteins, antibodies and other biological products by af- finity chromatography [3]. In the affinity technique, a biospecific recognition is involved between the target biomolecule (e.g. protein) and the affinity ligand. How- ever, its general implementation at large scale has found some difficulties due to the high cost of these supports and low adsorption capacity, reproducibility, stability, and leakage of affinity ligands after several cycles of operation. Some of these negative characteristics are due to the ma- trix, space arm and affinity ligand itself that hinder an ideal complex formation e.g. between the protein and af- finity ligand [4]. Some authors have been studied the application of pseudo-affinity techniques in protein purification with some success [5–7]. In this case, the interaction, between the protein and affinity ligands, resulted from hydropho- bic interactions promoted by high salt concentrations [8]. This type of interactions occurred through the hydro- phobic residues of amino acids localized on the protein surface – the hydrophobic domains [8], which represent in several proteins an amino acid composition higher than 50% in relation to amino acids with hydrophilic residues [9]. Therefore for enzymes, involving in or near the active site hydrophobic interactions with their substrates and/or products, it could be profited to establish a protein puri- fication strategy through a pseudo-affinity process. The adsorption of those proteins, which needs high salt con- centrations particularly ammonium sulphate, has the ad- ditional advantage to reduce the proteolytic action of proteases, eliminating bacterial contamination and si- multaneously decreasing the ionic interactions between the support and the proteins [9, 10]. On the other hand, chromatographic gels involving hydrophobic interactions have shown high adsorption capacity of proteins (10– 100 mg/cm 3 gel), low adsorption for some contaminants present in crude extracts and also high yields for different processes of protein purification [11]. Affinity and hydrophobic ligands have been tested in the purification of penicillin acylase [12–21]. Fonseca and Cabral, 1996 [21] verified that the pseudo-affinity process presented some advantages in relation to affinity processes Bioprocess Engineering 20 (1999) 513 – 524 Ó Springer-Verlag 1999 513 Received: 18 June 1998 L.P. Fonseca, J.M.S. Cabral Laborato´rio de Engenharia Bioquı ´mica, Centro de Engenharia Biolo´gica e Quı ´mica, Instituto Superior Te´cnico, Av. Rovisco Pais, 1000 Lisboa Portugal Correspondence to: Professor L.P. Fonseca