Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor).

Immobilization of lipases by selective adsorption on hydrophobic supports.

The preparation of immobilized derivatives of lipases that may be useful to develop industrial processes of organic synthesis is an exciting field of research in which three main features have to be simultaneously considered: (a) immobilized derivatives have to be compatible with very different reaction requirements (e.g. continuous adjustment of pH with concentrated alkali, use of aqueous media or organic solvents, etc.); (b) Sometimes, some activity/stability properties of lipases should be improved during immobilization; and (c) because of a complex mechanism of action, lipases are poorly active in the absence of hydrophobic interfaces. In this paper, we will review different approaches for lipase immobilization mainly related to the further use of immobilized derivatives to carry out enantio and regioselective hydrolysis in high water-activity systems. Special emphasis is paid to the selective adsorption of lipases on tailor-made strongly hydrophobic support surfaces. This new immobilization procedure is based on the assumption that the large hydrophobic area that surrounds the active site of lipases is the one mainly involved in their adsorption on strongly hydrophobic solid surfaces. Thus, lipases recognize these surfaces similarly to those of their natural substrates and they suffer interfacial activation during immobilization. This immobilization method permits: (a) promote a dramatic hyper-activation of most of lipases after their immobilization. That is, adsorbed lipases show very enhanced esterase activity in the absence of additional hydrophobic interfaces; (b) promote highly selective adsorption of lipases, at very low ionic strength, from impure protein extracts. That is, we can associate immobilization and purification of lipases; (c) promote interesting improvements of enantioselectivity after immobilization; and (d) promote a strong but reversible immobilization that enables us to recover these expensive supports after inactivation of immobilized lipases.

A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports

A number of bacterial lipases can be immobilized in a rapid and strong fashion on octyl-agarose gels (e.g., lipases from Candida antarctica, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa, Mucor javanicus, and Rhizopus niveus). Adsorption rates in absence of ammonium sulfate are higher than in its presence, opposite to the observation for typical hydrophobic adsorption of proteins. At 10 mM phosphate, adsorption of lipases is fairly selective allowing enzyme purification associated with their reversible immobilization. Interestingly, these immobilized lipase molecules show a dramatic hyperactivation. For example, lipases from R. niveus, M. miehei, and H. lanuginosa were 6-, 7-, and 20-fold more active than the corresponding soluble enzymes when catalyzing the hydrolysis of a fully soluble substrate (0.4 mM p-nitrophenyl propionate). Even higher hyperactivations and interesting changes in stereospecificity were also observed for the hydrolysis of larger soluble chiral esters (e.g. (R,S)-2-hydroxy-4-phenylbutanoic ethyl ester). These results suggest that lipases recognize these "well-defined" hydrophobic supports as solid interfaces and they become adsorbed through the external areas of the large hydrophobic active centers of their "open and hyperactivated structure". This selective interfacial adsorption of lipases becomes a very promising immobilization method with general application for most lipases. Through this method, we are able to combine, via a single and easily performed adsorption step, the purification, the strong immobilization, and a dramatic hyperactivation of lipases acting in the absence of additional interfaces, (e.g., in aqueous medium with soluble substrate). Copyright 1998 John Wiley & Sons, Inc.

Use of dextrans as long and hydrophilic spacer arms to improve the performance of immobilized proteins acting on macromolecules.

New dextran-agarose supports, suitable for covalent immobilization of enzymes and proteins acting on macromolecular substrates, were prepared. The thick internal fibers of agarose gels were covered by a low-density layer of long, flexible, hydrophilic, and inert dextran molecules. Rennin and protein A were immobilized on these novel supports and the resulting derivatives exhibited a very high capacity for biological recognition of soluble macromolecular substrates. Caseinolytic activity of this immobilized enzyme was 15-fold higher than activity of directly immobilized rennin, through short spacer arms, on agarose gels. Similarly, the new derivatives of immobilized protein A were able to adsorb up to 2 molecules of immunoglobulin per each molecule of immobilized protein A. When the immobilized proteins were secluded away from the support surface by using these new long and hydrophilic spacer arms, they exhibit minimal steric hindrances that could be promoted by the proximity of the support surface.