Engineering the thermostable D-hydantoinases from two thermophilic Bacilli based on their primary structures.

Thermostable enzymes find wide applications to basic studies concerning protein stability as well as to development of industrial and specialty bioprocesses. In the process for the production of optically pure D-amino acids, obtained from the corresponding hydantoin derivatives using a microbial D-hydantoinase,1 limited substrate solubility and enzyme stability were posed as problems. In this context, we have isolated and characterized a thermostable D-hydantoinase from Bacillus stearothermophilus SD1,2 and the gene encoding the enzyme was cloned and its nucleotide sequence was determined.3 However, the low specificity of the enzyme from B. stearothermophilus SD1 toward hydantoin derivatives with an aromatic group at the 5 -position prompted us to isolate an enzyme with improved substrate specificity from B. thermocatenulatus GH2.4 The enzyme of B. thermocatenulatus GH2 was a tetramer and showed high specific activity toward aromatic substrates. On the other hand, previously isolated enzyme was composed of two identical subunits and had a low specific activity for aromatic substrates. In order to get some insights into the difference in the biochemical characteristics of the two enzymes, the gene coding for the enzyme from B. thermocatenulatus GH2 was cloned and expressed in E. coli, and its nucleotide sequence was determined. Based on the primary structures of the two enzymes, hybrid and truncated mutant enzymes were constructed, and their catalytic properties were compared with wild-type enzymes. Details are reported here.