B-512F dextransucrase is an industrial enzyme used in commercial production of dextran and prebiotics. Several researchers have studied the covalent immobilization of this enzyme with little success due to dextran masking the reactive groups on the enzyme and inactivation of the enzyme during immobilization. A novel dextransucrase was designed and produced successfully to eliminate problems faced in covalent immobilization. In production of the novel enzyme, B-512F dextransucrase was truncated at N and C terminals and fused to glutathione S-transferase. The novel dextransucrase was fully active and carried out dextran biosynthesis and acceptor reactions effectively. The novel enzyme was immobilized covalently onto Eupergit C 250L, giving rise to 100% immobilization and 83.3% activity yields. Immobilized enzyme was used successfully for the production of acceptor products and low molecular weight dextran. The immobilized enzyme showed no decrease in activity for 15 batch reactions and retained its initial activity at storage (4 degrees C) for 35 days. Optimal conditions were not affected by the immobilization. The kinetic parameters for the free and immobilized enzyme were determined. The acceptor reactions using fructose, glucose, maltose, and lactose were also studied. The novel method developed could also be used in immobilization of some other biomolecules. (C) 2012 Elsevier B.V. All rights reserved.