Tamara Montes, Fernando López-Gallego, Manuel Fuentes, Cesar Mateo, Valeria Grazu, Lorena Betancor, Jose M. Guisan, and Roberto Fernandez-Lafuente
Chemical modification and immobilization of proteins have been usually utilized as parallel techniques to improve enzyme stability. In this chapter, we show that chemical modification of the protein surface to greatly increase its reactivity with the groups of a support activated with glyoxyl residues may be a very good alternative for greatly increasing the protein stability via multipoint covalent attachment. For this purpose, some of the carboxylic acids of the proteins are transformed into amino groups by reaction with ethylendiamine via the carbodiimide coupling method. The new amino groups have a lower pK than Lys residues, enabling immobilization under milder conditions and a higher degree of stabilization. These results show that the coupling of different stabilization techniques may yield a synergistic effect, higher than any individual strategy.
Key Words: Enzyme stability; protein surface modification; multipoint covalent attachment; glyoxyl supports.
Enzyme stabilization is still one of the critical issues in the design of a biotransformation (1,2). Enzyme inactivation usually starts with reversible conformational changes that finally promote their irreversible inactivation (3). Thus, most of the strategies for protein stabilization are focused on the prevention of the first three-dimensional (3D) distortion of the protein. Multipoint covalent attachment may be one of the techniques that have offered the best results for solving this problem. It has been suggested that this behavior could result from a more rigid conformation generated by numerous bonds linking the enzyme to the insoluble carrier, which could prevent the conformational changes that may vary the relative position of the residues implicated in the immobilization. Glyoxyl supports have been described as a very adequate immobilization system to yield immobilized-stabilized proteins via multipoint covalent attachment. Many enzymes have been stabilized using this technique—e.g., penicillin G acylase from Escherichia coli (4) and Kluyvera citrophila (5), trypsin (6) chymotrypsin (7), alcalase (8), carboxy-peptidase A (9,10), FNR NADP-reductase (11), esterase (12), thermolysin (13), DAAO (14), catalases (15,16), and lipases from different sources (17,18), urokinase (19), L-aminoacylase (20), Chitosinase (21-23).
These good results are the consequence of some glyoxyl-agarose properties, namely, low-steric hindrances for the amino-aldehyde reaction and a short spacer arm that renders high stability at longer reaction times (24). These properties are shared with other groups, like the epoxy ones (25), but glyoxyl supports give in general higher stabilization factors, suggesting some other peculiarity that is important to obtaining this very high degree of stabilization.
Glyoxyl agarose has an apparent drawback as the need of alkaline pH values to achieve a multipoint covalent attachment in the immobilization process and the necessity of using highly activated supports, because of the reversibility of the individual shift's base links that makes that the enzyme only become immobilized via a certain multipoint covalent attachment (26).
However, this apparent problem is what causes glyoxyl-supports to reach such high-stabilization values when applied to most enzymes. It promotes the "auto-oriented" immobilization of the protein by the area/s with the highest density/ies of lysines, where the most intense multipoint attachment may take place (26).
Moreover, the immobilization conditions need to be carefully designed to permit a very intense multi-interaction. They must exhibit good support-enzyme geometrical congruence, moderately high temperature, or high density of reactive groups (5).
It has been suggested recently that the stabilization of enzymes achieved via multipoint covalent attachment on glyoxyl support may be improved if other tools, such as chemical or genetic modification, are used to fulfill this goal (27,28). The genetic additional enrichment in Lys of some very rich areas in Lys of penicillin G acylase permits to greatly improve the immobilized PGA stability (27).
An alternative approach could be to chemically modify the enzyme, not to get a more stable enzyme, but to have a protein surface very enriched in reactive groups and, therefore, with much better possibilities of yielding a more intense multipoint covalent attachment during immobilization (see Fig. 1) (28).
In this chapter, we will propose the use of the chemical amination of the enzyme surface to improve the possibilities of achieving a more intense multipoint covalent attachment
1. Cross-linked 10BCL and 6BCL agarose beads were donated by Hispanagar S.A. (Burgos, Spain).
2. Protein amination solution was 1 M ethylenediamine (ED), pH 4.7, containing different amounts of 1-ethyl-3-(dimethylamino-propyl) carbodiimide (EDAC) (see Note 1).
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