Abstract
Random mutations were introduced into a cryptic enzyme (Hyb-24) having low 6-aminohexanoate-dimer (Ald) hydrolyzing activity (0.023 µmole/min/mg purified protein) using PCR. One of the mutant enzyme produced by a plasmid pS1M77 had 4 amino acid replacements in the sequence of Hyb-24 protein, and possessed 9 times higher specific activity toward Ald than the parental Hyb-24 protein. DNA shuffling experiments using the five mutants including pS1M77 suggested that a single amino acid alteration from Asp to Tyr at position 370 (D370Y) in the Hyb-24 is crucial for increasing of the enzyme activity. Moreover, since the corresponding amino acid at position 370 was Asp in the functional 6-aminohexanoate-dimer hydrolase (EII: having 200 times higher specific activity toward Ald), D370Y mutation was introduced to the wild-type EII enzyme. By this amino acid replacement, the specific activity increased to 2.3 times of the value of the parental EII. This result demonstrates that identification of amino acid alteration suitable for increasing the activity in the cryptic enzyme is also effective for improving the functional EII enzyme.
