We prepared two constructs of the gamma-lactamase gene from Sulfolobus solfataricus P2 and heterogenously expressed these as two separate proteins. The first construct had His tags fused at both the N and C terminals of the y-lactamase gene (double fusion enzyme). The second had a single His tag fused at the N terminal of the gene (single fusion enzyme). Enterokinase treatment of the two fusion enzymes produced two additional proteins. The kinetic parameters, absorption capacity on nickel-chelating agarose, and immobilization stability of the four proteins were compared. The double fusion enzyme was chosen as the on-column transformation catalyst. It was purified to electrophoretic homogeneity on nickel-chelating agarose and immobilized on the same matrix in the meantime. The optimal temperature of the immobilized enzyme was 10 degrees C higher than that of the free enzyme. The optimal pH was also 2.0 units higher. The immobilized enzyme maintained approximately 80% of its maximum activity at pH 5-11. The activity of the free enzyme decreased rapidly at pH values below 6.0 or above 8.0. The immobilized enzyme was used for on-column transformation reactions for 5 h each day. It retained approximately 75% of its original activity after repeated transformation cycles over a period of 30 days. (C) 2010 Elsevier Ltd. All rights reserved.