Multi-component reactions for covalent immobilization of biomolecules: What is the mechanism?

Industrial application of the free form of enzymes remains controversial because of their poor stability and prohibitive cost. Therefore, there is a great interest in methods trying to develop useful biocatalysts for industrial applications by improvement their catalytic properties such as activity, stability or recycling capacity [1]. Numerous attempts have been carried out on the preparation of immobilized enzymes, which involves a variety of immobilization techniques and new support materials [2]. Among them, irreversible covalent attachment of enzymes on a solid support is an efficient and popular method and is usually preferred when leaching of enzyme from the support is a main concern. Moreover, if the immobilization protocol is properly designed to give an intense covalent attachment, the immobilization will be a powerful way to improve the enzyme rigidity, which increases enzyme stability against any distorting agents. However covalent attachment is often time consuming, needs harsh condition (for example high pH) and generally results in reduced activity of the immobilized enzyme. Therefore, it is still a big demand for discovery of new simple strategies for the covalent binding to improve enzyme catalytic functions. Recently we have reported the interesting results of using multi-component reactions for enzyme immobilization on aldehyde, epoxy and acid functionalized supports [3-5]. High capacity of loading, simple and rapid immobilization and significant improvement in activity of the immobilized enzyme are the main advantages of this novel procedure. In this procedure the support supplies the aldehyde, epoxy or acid groups, the enzyme molecules supply the carboxylic or amine groups and the other missing component is added to the reaction medium. However this protocol offers great flexibility to use different functional groups of many supports. In fact by utilizing this method a large number of supports and functional groups can be used for immobilization of enzymes.