Enzymes are biological catalysts, specific in their reactions and normally tightly packed in cellular organelles or in enzyme cascade enabling catalytic process to take place precisely when and where they are needed. Artificial applications of such compartmentation or packing go back to the 1950s, when immobilized enzymes (enzymes with restricted mobility) were first prepared internationally by inclusion in polymeric matrices or binding onto carrier materials. Since then, numerous methods of immobilization on different materials have been developed.

Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes or alternative technologies, and the steadily increasing number of applications for immobilized enzymes. There are several reasons to use immobilized enzymes. In addition to the convenient handling of enzyme preparations, the two main targeted benefits are (1) easy separation of enzyme from the product and (2) reuse of the enzyme. Easy separation of the enzyme from the product simplifies enzyme applications and permits reliable and efficient reaction technology. Enzyme reuse provides a number of cost advantages, which are often an essential prerequisite for establishing an economically viable enzyme-catalysed process.

The properties of immobilized enzyme preparation are governed by the properties of both the enzyme and the carrier material. The interaction between the two provides an immobilized enzyme with specific chemical, biochemical, mechanical and kinetic properties. As far as the manufacturing costs are concerned, the yield of immobilized enzyme activity is determined by the immobilization method in relation to the amount of soluble enzyme used. Under process conditions, the resulting activity can be further reduced by mass transfer effect. That is, the yield of enzyme activity following immobilization does not only depend on losses caused by the binding procedure but can be further reduced as a result of diminished availability of enzyme molecules within pores or by slowly diffusing substrate molecules. Such limitation lead to lowered efficiency. However, improved stability under working conditions can compensate for such drawbacks, resulting in overall benefits.

In this article we have selected papain, a thiol protease, for immobilization on chelating Sepharose, which can easily be recovered from enzyme-carrier complex, regenerated and reused. This immobilization is based on the ability of protein side chains of cysteine, histidine and tryptophan to substituted weakly bonded ligands in the metal complexes.The need for immobilization of papain has been due to its great industrial and medicinal potential. Papain is used as a chill-proofing agent during beer finishing operation in brewing process. The potential uses of papain include its frequent use as a biocatalyst for amino acid ester and peptide synthesis.

The results of our findings clearly indicate that the immobilization of papain on chelating Sepharose activated with Cu(II) ions exhibited high immobilization and activity yields suggesting that the carrier and metal ion are considered suitable for papain immobilization. After the elution of papain from Cu (II) activated carrier, the regeneration and reactivation with the same metal ion was highly effective as evident by the activity yield of immobilized papain during subsequent cycles of reimmobilization. The immobilized papain also exhibited a marked increase in thermostability at higher temperature.

The cost of carriers used for industrial application is very important. The regenerability of the carrier is, therefore, relevant. The mild conditions used for papain immobilization, the high recovery of immobilized preparations and the regenerability of the matrix are the main features of the method reported in this manuscript.