Screening of a novel and thermostable nicotinate dehydrogenase-producing strain Bacillus paramycoides for efficient synthesis of 6-hydroxynicotinic acid

Abstract

6-Hydroxynicotinic acid emerged as a pivotal intermediate for fine chemicals. The current study aims to screen novel nicotinate dehydrogenase-producing strains. Based on the high-throughput UV coloration method, a novel nicotinate dehydrogenase-producing strain named Bacillus paramycoides was first and successfully screened from soil and gram staining as 16S rRNA gene sequencing was conducted to further confirm its accuracy. The fermentation medium formula is determined as seignette salt (5 g/L), tryptone (10 g/L), K2HPO4 (2 g/L), and nicotinic acid (4 g/L) after a single factor and orthogonal test. The activity of nicotinate dehydrogenase produced by B. paramycoides after optimization reached 1.23 U/mL, which was the highest ever reported. In addition, the enzyme characterization results revealed that the optimum reaction temperature and pH were 40 °C and 7.0, respectively. Furthermore, B. paramycoides nicotinate dehydrogenase exhibited good thermostability with more than 50% of the highest enzyme activity retained at 50 °C after 8 h incubation, which was better than ever reported. The pH stability results suggested that nicotinate dehydrogenase possessed a narrow pH ranging from 6.0 to 8.0. In addition, shaker speed demonstrated a positive relation with enzyme activity since shaker speed could affect mass transfer and dissolved oxygen concentration. Finally, it could completely transform nicotinic acid to 6-HNA within 3 h when the substrate loading is 15 g/L. In conclusion, these results indicated that B. paramycoides nicotinate dehydrogenase could serve as a potential candidate for bioproduction of 6-HNA.