The hydantoin-hydrolysing enzymes in a sonicated crude extract of Pseudonionas putida strain RUKM3s were stabilised by covalent coupling to the well-known support material, Eupergito C. The activities of the enzymes in the immobilised and non-immobilised state were evaluated on the basis of the yield of their respective products from the substrates hydantoin and N-carbamylglycine in batch reactions, and the immobilisation parameters of the biocatalyst were optimised. The optimum operating pH of hydantoinase and N-carbamoylase (NCAAH) were found to be 9-10 and 9, respectively, while the optimum operating temperature was found to be 40 degrees C for both enzymes, both when unimmobilised and immobilised. Thus, the pH and temperature optima of the enzymes were not affected by the immobilisation. The support matrix bound 63% of the soluble protein from a solution containing 5 mg/mL protein. After immobilisation, the hydantoinase activity was retained at 86% of the unimmobilised level and 15% of this activity was retained even after 4 weeks, as compared with the unimmobilised hydantoinase activity which was completely lost after 2 weeks. The N-carbamoylase activity in the biocatalyst was significantly enhanced in terms of both activity and retention of activity during storage; after immobilisation the NCAAH activity was increased to 4x the non-immobilised level, and 33% of this high activity was retained after 4 weeks of storage, compared with complete loss of activity after 2 weeks in the non-immobilised case. The biocatalyst was re-used in 18 biocatalytic reaction cycles before activity levels declined by 50% in the case of NCAAH activity, and in 28 cycles in the case of the hydantoinase activity. D 2006 Elsevier Inc. All rights reserved.