Glutaraldehyde cross-linking of lipases adsorbed on aminated supports in the presence of detergents leads to improved performance.

Lipases from Candida rugosa (CRL) and lipase isoforms A and B from Candida antarctica (CAL-A and CAL-B) were adsorbed on aminated supports in the presence of detergents to have individual lipase molecules. Then, one fraction was washed to eliminate the detergent, and both preparations were treated with glutaraldehyde. The presence of detergent during the cross-linking of the lipases to the support permitted an increase in the recovered activity (in some instances, even by a 10-fold factor). This activity was higher even than that exhibited by the just adsorbed lipases, suggesting that it was not a result of some protective effect of the detergent in the enzyme activity during glutaraldehyde chemical modification. Moreover, the enantioselectivity of the different enzyme preparations was very different if the glutaraldehyde was offered in the presence or in the absence of detergent, in some cases increasing the E value (even by a 7-fold factor in the case of CAL-A in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester), in other cases even inverting the enantio preference (e.g., in the case of CRL). The irreversible chemical inhibition of the enzyme that was immobilized and cross-linked with glutaraldehyde in the presence of detergents was more rapid than that in the other preparations (by more than a 10-fold factor). This experiment reveals an exposition degree of the active serine in the preparation cross-linked with the support in the presence of detergent that is higher than that in the other preparations. The results suggested that different enzyme structures were "stabilized" by the glutaraldehyde treatment if performed in the presence or in the absence of detergent, and that, in the presence of detergent, a form of the lipase with the serine residue more exposed to the medium and much more active could be obtained. This strategy seems to be of general use to improve the lipase activity to be used in macroaqueous media.

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports.

In this work, we have used supports activated with m-amino-phenylboronic groups to "reversibly" immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.

Selective and mild adsorption of large proteins on lowly activated immobilized metal ion affinity chromatography matrices. Purification of multimeric thermophilic enzymes overexpressed in Escherichia coli.

A strategy to selectively adsorb large proteins on immobilized metal ion affinity chromatography supports is presented. It is based on the fact that large proteins have a large surface that permits the long distance interaction with groups placed quite far apart (very dispersed onto the support surface) in the support, therefore, even using lowly activated supports, these proteins may be able to yield multiple interactions with the support, which is not possible for smaller proteins. This has been shown using a crude extract from Escherichia coli, where only large proteins were adsorbed on supports having 0.25 micromol of metallic groups/g of support. Then, these lowly activated supports have been used for purifying multimeric enzymes from thermophilic organisms (alpha- and beta-galactosidases from Thermus sp. strain T2) cloned and over-expressed in mesophilic ones. A previous heating step of the crude extract destroyed the quaternary structure of all multimeric enzymes from the host (E. coli). Thus, the only large protein remaining in the supernatant of this heated extract are the cloned multimeric thermophilic enzymes, permitting their very simple purification by using only one chromatographic step.

Ion exchange using poorly activated supports, an easy way for purification of large proteins.

Ion-exchange chromatography using commercial ionic supports is a commonly used technique for protein purification. However, selective adsorption of a target protein from a given extract onto commercial ion exchangers seems to be quite complex since they are designed to adsorb the maximum percentage of proteins with the opposite charge. In this paper, ion-exchanger supports with different activation degrees (from 1 to 40 micromol of amino groups per g of agarose) have been prepared and used for the purification of large proteins. These kinds of proteins have large surfaces to interact by many points with the support. Therefore, it was possible to purify large proteins as beta-galactosidase from Thermus sp. strain T2 from a crude extract from Escherichia coli or bovine liver catalase from a commercial preparation, with tailor-made ion-exchanger supports. A simple step of adsorption/desorption on lowly activated supports rendered both enzymes rather pure as confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Moreover, this strategy makes also easy the desorption step that requires rather low NaCl concentrations, which may become a serious problem for desorption of large proteins when using conventional supports, due to their ability of generating a very strong adsorption.

Detection and purification of two antibody-antigen complexes via selective adsorption on lowly activated anion exchangers.

Taken advantage of the mechanism of adsorption of macro-molecules on ionic exchangers, (a multipoint interaction between the protein and the support), it is possible to selectively adsorb large proteins leaving small ones in the supernatant. Associated proteins should present a significant difference in its size as compared to the non-associated forms. Thus, the protein complexes may have much larger surfaces to interact with the support. Here, by selecting the support with the highest activation degree that was unable to adsorb the non-associated proteins, we have shown the simple and selective adsorption of immuno complexes (as a model), while antibodies and antigens remained in the supernatant. Therefore, it was possible to selectively adsorb on lowly activated supports (e.g., agarose 4BCL having only 1 micromol of amino groups per g of support) rabbit IgG/anti-rabbit immunoglobulins (immuno complex), while these supports were unable to adsorb the individual immunoglobulines. Similarly, horseradish peroxidase (HRP)/anti-HRP were selectively adsorbed on lowly activated supports, while the individual proteins were not adsorbed at all. Afterwards, the adsorbed associated proteins (purified at least from the non-associated counterparts and concentrated by the adsorption on the support) may be cross-linked with aldehyde-dextran and be desorbed from the matrix for their analysis. This strategy may permit very simple experiments to detect the presence of protein-protein complexes. Finally, we have shown the advantages of this technique compared to the use of one of the proteins previous immobilized on a support.