Enzymes that exhibit superior catalytic activity, stability and substrate specificity are highly desirable for industrial applications. These goals prompted the designed substrate specificity of Bacillus stearothermophilus D-hydantoinase toward the target substrate hydroxyphenylhydantoin (HPH). Positions crucial to substrate specificity were selected using structural and mechanistic information on the structural loops at the active site. The size and hydrophobicity of the involved amino acids were rationally changed, and the substrate specificities of the designed D-Hyd mutants were investigated. As a result, M631/F159S exhibited about 200-fold higher specificity for HPH than the wild-type enzyme. Systematic mutational analysis and computational modeling also supported the rationale used in the design. (C) 2008 Elsevier Inc. All rights reserved.