Characterization of a thermally stable and organic solvent-adaptative NAD+-dependent formate dehydrogenase from Bacillus sp. F1.

摘要

Aims: To characterize a robust NAD+-dependent formate dehydrogenase firstly obtained from a nonmethylotroph, Bacillus sp. F1. Methods and Results: The Bacillus sp. F1 NAD+-dependent formate dehydrogenase (BacFDH) gene was cloned by TAIL-PCR and heterologous expressed in Escherichia coli. BacFDH was stable at temperatures below 55 degrees C, and the half-life at 60 degrees C was determined as 52.9 min. This enzyme also showed a broad pH stability and retained more than 80% of the activities after incubating in buffers with different pH ranging from 4.5 to 10.5 for 1 h. The activity of BacFDH was significantly enhanced by some metal ions. Moreover, BacFDH exhibited high tolerance to 20% dimethyl sulfoxide, 60% acetone, 10% methanol, 20% ethanol, 60% isopropanol and 20% n-hexane. Like other FDHs, BacFDH displayed strict substrate specificity for formate. Conclusion: We isolated a robust formate dehydrogenase, designated as BacFDH, which showed excellent thermal stability, organic solvent stability and a broad pH stability. Significance and Impact of the Study: The multi-aspect stability makes BacFDH a competitive candidate for coenzyme regeneration in practical applications of chiral chemicals and pharmaceuticals synthesis with a relatively low cost, especially for the catalysis performed in extreme pH conditions and organic solvents.