CHARACTERIZATION OF HYBRID ENZYMES BETWEEN 6-AMINOHEXANOATE-DIMER HYDROLASE AND ITS ANALOGOUS PROTEIN

6-Aminohexanoate-dimer hydrolase (EII) and its analogous protein (EII'), of Flavobacterium sp. K172 are composed of 392 amino acids, in which 47 are different. The enzyme activity of EII' toward 6-aminohexanoate dimer is approximately 0.5% of that of EII. We have constructed various hybrids of the two genes by exchanging fragments flanked by conserved restriction sites such as PvuII, BglII, SalI, and BamHI (respectively 74, 483, 771, and 1,141 bp downstream of the initiation condon), and purified their gene products to homogeneity. Hyb-12 protein, which was obtained by the replacement of the BglII-SalI region of the EII' with the corresponding region of EII, had 12 times higher specific activity towards the 6-aminohexanoate dimer and its related substrates than EII' protein. Hyb-10, which was composed of the N-terminal -BglII regions of EII' and the BglII- C terminal region of EII, had activity toward these substrates nearly equal to the activity of the EII enzyme. Comparisons of the activity toward 6-aminohexanoate dimer and its analogues has demonstrated that EII, EII', and their hybrid enzymes are highly active only toward the substrates that contain 6-aminohexanoate as the N-terminal residue, while the recognition of the C-terminal residue in the substrate was not stringent. The substrate specificity, pH-activity profile, and heat stability of these enzymes varied slightly.