Mixed Ion Exchange Supports as Useful Ion Exchangers for Protein Purification:  Purification of Penicillin G Acylase from Escherichia coli

Abstract

A support having similar amounts of carboxymethyl and amino groups has been prepared and evaluated as an ion exchanger. It has been found that this support was able to adsorb a high amount of protein from a crude extract of proteins (approximately 55%) at pH 5. Moreover, it was able to adsorb approximately 60% of the protein that did not become adsorbed on supports bearing just one kind of ionic groups. The use of divalent cations reinforced the adsorption of proteins on these supports. These results suggest that the adsorption of proteins on supports bearing almost neutral charge is not driven by the existence of opposite charges between the adsorbent and the biomacromolecule but just by the possibility of forming a high number of enzyme-support ionic bonds. This support has been used to purify the enzyme penicillin G acylase (PGA) from Escherichia coli. PGA was not significantly adsorbed at any pH value on either amino- or carboxyl-activated supports, while it can be fully adsorbed at pH 5 on this new carboxyl-amino matrix. Thus, we have been able to almost fully purify PGA from crude extracts with a very high yield by using these new supports.

Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization

“…Moreover, the enzyme should have the capacity to become adsorbed to anion exchangers. Considering that this is a multipoint process (several ion bridges need to be produced to fix the enzyme to the support), [122][123][124] the strategy presents the problem that the support should never be physically inert. Furthermore, the inertness of the support surface may be in many instances a desired feature of the support, to prevent uncontrolled support-enzyme interactions that may affect enzyme stability (sometimes in a positive sense, but in another may have a negative impact in the enzyme stability).…”

Immobilization of enzymes on heterofunctional epoxy supports

“…Table 2 shows some of the most relevant results obtained in our laboratory. Some enzymes are not significantly immobilized on certain supports 19 ; for example, PGA from Escherichia coli is not immobilized on EDA-epoxy supports (this enzyme did not become adsorbed on mild anionic exchangers) 47,48 . The lack of external Cys on the enzyme means that the immobilization on the disulfide-epoxide support was very poor.…”

Synthesis and modification of supports with an alkylamine and their use in albumin adsorption

“…The aim of most of the research in the field of ion exchange is to enhance functional properties such as selectivity, capacity, and rate, which have direct impact on the applicability of the resins 1. These supports can also be employed as anion exchangers2–5 or in affinity chromatography, after attaching a ligand 6–9. Thus, matrix derivatives with alkyl amines are used in affinity chromatography for the purification of amine oxidases10 and other proteins with affinity for the amino group 11, 12.…”