Mutational analysis and stability characterization of a novel esterase of lipolytic enzyme family VI from Shewanella sp

- A novel esterase belonging to lipolytic enzyme family VI, Esth, has been cloned.
- Mutated Esth has the highest catalytic efficiency (Kcat/Km) in its family.
- Mutated Esth displays cold-adapted and salt-tolerant features.
- Mutational analysis indicates that replacement of Ala with Gly at 129 improves the thermosatabilty.

Esterases are widely used in different industries. Here, a novel esterase, Esth, with low identity with previously reported esterases, was identified and characterized. The results showed that Esth was a cold-adapted esterase and retained 50% of its maximum activity at 0 °C. Besides, Esth showed great activity and stability in high concentrations of NaCl. When treated with some organic solvents, detergents and metal ions, Esth showed high activity as well. The kcat/Km value of Esth was 29.5 s−1 mM−1, suggesting that it has higher catalytic efficiency than all the previously reported esterases from the same family, lipolytic enzyme family VI. The structural modeling showed that changing Ala129 into Gly would form a new hydrogen bond between ser125 and Gly129 and make theα-helix longer, which might influence on the thermostability of enzymes (Kumar, 2000). To confirm this, the mutant EsthA129G was obtained by site-directed mutagenesis. The result indicated that EsthA129G retained over 70% of the activity versus 12% for Esth after incubation at 55 °C for 120 min, showed a nearly six fold increase when compared with wild type. Overall, Esth shows a potential application prospect in extreme conditions and the mutation research can provide some structural information about thermostable enzymes.