Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used in this study.
Fig 1.
Multiple amino acid sequence alignment of NPR.
Amino acid sequence alignment of BaNPR from Bacillus amyloliquefaciens K11 with the NPR from Bacillus amyloliquefaciens Y2, NPRE from Bacillus amyloliquefaciens (Bacillus velezensis) (P06832), NPRE from Bacillus subtilis 168 (P68736), NPRE from Bacillus cereus (P05806), THER from Bacillus thermoproteolyticus (P00800), and NPRE from Bacillus caldolyticus (P23384) using the ClustalW program. Two histidine residues in active site are indicated by asterisks.
Table 3.
Production of recombinant BaNPR by Bacillus amyloliquefaciens harboring pUB110-Banpr in shaker flasksa.
Fig 2.
SDS-PAGE analysis of recombinant BaNPR in Bacillus amyloliquefaciens K11.
Lane: M, the molecular mass standards; 1–3 and 5–7, the different transformants harboring pUB110-Banpr; 4, the transformant harboring the empty vector pUB110.
Table 4.
Segregation stability of pUB110-Banpr in recombinant Bacillus amyloliquefaciens 110N-6 by continuous passage cultivationa.
Fig 3.
Optimization of culture conditions for BaNPR production in Bacillus amyloliqufaciens 110N-6.
a Effect of temperature on enzyme production. b Effect of pH on enzyme production. c Effect of working volume on enzyme production. d Effect of inoculum size on enzyme production. e Effect of different amino acids on enzyme production. f Effect of different metal ions on enzyme production. Each value in the panel represents the means ± SD (n = 3).
Fig 4.
Comparison of the production of neutral protease in shake flasks and fermentors.
a The enzyme activity of r-BaNPR and BaNPR produced by Bacillus amyloliquefaciens 110N-6 and Bacillus amyloliquefaciens K11 on flask level under optimized conditions. b The enzyme activity of r-BaNPR and BsNPR produced by B. amyloliquefaciens 110N-6 and Bacillus subtilis 1398 on large-scale batch production in the fermentor. Each value in the panel represents the means ± SD (n = 3).