Bifunctional enzymes find a wide application as a monitoring facility and a potential biocatalyst in molecular biology and biotechnology. Recombination of natural enzymes to a bifunctional fusion offers valuable tools, but the functional and structural instability of artificial fusion enzymes remains to be solved. Based on structural traits of microbial D-hydantoinase, we attempted to construct a bifunctional N-carbamylase/D-hydantoinase fusion enzyme that would be useful for the synthesis of nonnatural D-amino acids in a concerted fashion. The bifunctional ability of D-hydantoinase, as a fusion partner, was noticeable, but the resulting fusion enzyme was subjected to serious proteolysis in vivo, as generally encountered in the expression of large the multidomain polypeptide in E. coil. In an effort to improve the structural instability imposed by artificial linear fusion, directed evolution of the fusion enzyme was performed using DNA shuffling with a consensus primer to maintain a crucial domain for the enzyme activity. The evolved fusion enzyme, F11,was selected after repeated rounds, and this enzyme was found to show sixfold increased performance in the production of D-amino acid compared with the parent fusion enzyme, which was mainly due to the enhanced structural stability of the evolved fusion enzyme. This result is an example showing that directed evolution of the linearly fused polypeptide may broaden the opportunity to generate a fusion enzyme with greater potential. (C) 2000 John Wiley & Sons, Inc.