Identification of the open reading frame for the Pseudomonas putida D-hydantoinase gene and expression of the gene in Escherichia coli.

A DNA fragment containing the gene for D-hydantoinase was cloned from Pseudomonas putida CCRC 12857 into Escherichia coli. The cloned gene contained an open reading frame (ORF) of 1485 nucleotides encoding a protein of 53.4 kDa in which the carboxyl terminal end is longer than that previously deduced from strain DSM 84. This ORF was verified by amino acid sequencing of amino and carboxyl termini, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid sequence comparison. Deletion analysis revealed that 32 amino acids from the carboxyl terminal end were essential for D-hydantoinase activity. Tagging of six consecutive histidyl residues to the amino terminus or to the carboxyl terminus of the enzyme did not significantly affect D-hydantoinase activity. Under the control of T5lac promoter and lactose induction, the D-hydantoinase activity of transformed E. coli reached 200 U l-1 which is about 20-fold higher than that of gene donor strain.