Construction of hybrid genes of 6-aminohexanoic acid-oligomer hydrolase and its analogous enzyme. Estimation of the intramolecular regions important for the enzyme evolution.
Hybrids were constructed of the genes for two homologous enzymes, 6-aminohexanoic acid-oligomer hydrolase (EII, one of the nylon oligomer degradation enzymes), and its probable evolutionary antecedent (EII'). The structural genes of EII (nylB) and EII' (nylB') have 88% similarity in their nucleotide sequences, an open frame encoding a peptide of 392 amino acids, conserved restriction sites, and in vitro recombination between these genes at the corresponding restriction sites generated genes directing various hybrid enzymes. In a comparison of the EII, EII', and the hybrid enzymes, we concluded that one or more of the four amino acid alterations that occurred in the intramolecular region (between amino acids 162-257) of EII' is essential to the adaptation of the enzyme to nylon oligomer degradation, and that its effect is enhanced 20-fold by one or more further alterations in the 258-380 region. Our results also suggest that this technique is useful for improving enzyme characteristics.
D000581:Amidohydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005796:Genes; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000235:genetics*; D009693:Nucleic Acid Hybridization
D007527:Isoenzymes; D000581:Amidohydrolases; C026250:6-aminohexanoate-dimer hydrolase