Microorganism and inoculum preparation
A gene-modified strain (B. subtilis168/pHT01-aprN1 )
was used to express NK. The medium for
flask and fed-batch culture was as
follows: yeast extract, 60 g L-1; glucose, 40 g
L-1; glycerol, 20 mL L-1;
NH4Cl, 3 g L-1;
K2HPO4·3H2O, 1 g
L-1; MgSO4·7H2O, 2 g
L-1; MnSO4·H2O, 0.02 g
L-1; FeSO4·7H2O, 0.05
g L-1; CoCl2·6H2O,
0.01 g L-1; ZnCl2, 0.01 g
L-1; 2,6-pyridinedicarboxylic acid, 0.167 g
L-1. Glucose and
MgSO4·7H2O were autoclaved separately
and aseptically added to the medium. Nutrient solution contained 100 g
L-1 yeast extract and 250 mL L-1glycerol was used as feed medium. To avoid plasmid loss and prevent
microbial contamination, chloramphenicol (5 μg mL-1)
was added to all media after being dissolved in anhydrous ethanol and
filtered through a 0.22 μm membrane.