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

Abstract

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°C, and the half-life at 60°C was determined as 52·9min. 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 1h. 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. © 2011 The Authors. © 2011 The Society for Applied Microbiology.