Abstract:

 Aspartate kinase (AK) is the first critical enzyme in the biosynthesis pathway of vital amino acids, in which
AK is strictly regulated by metabolites. In the present study, AK from Corynebacterium pekinense was mutated using sitedirected
mutagenesis combined with high-throughput screening and G377F with high activity was successfully constructed.
Then, the purified AK from G377F was characterized. The results showed that the optimum reaction pH of G377F was 9.0,
which was slightly higher than that of the wild-type strain (WT). The optimum reaction temperature of G377F was 25 ℃,
which was consistent with that of WT. The half-life period of G377F increased from 2.6 h (WT) to 5.3 h. The pH tolerance
of G377F maintained more than 50% of its original vitality in 5 h, which was identical to that of WT (3 h). In addition, AK
from G377F had a good tolerance to metal ions and organic solvents. To some extent, the inhibition by Lys could be released and
the co-presence of Lys and Met had an active effect on the G377F. Kinetic studies showed that the Vmax of AK from G377F was
10.3 times higher than that of WT. The n value was 1.0, which was significantly lower than that of WT, indicating that the positive
cooperativity of AK from G377F decreased, which showed that AK from G377F tended to be a Michaelis enzyme.

Key words: Corynebacterium pekinense E31, aspartate kinase, site-directed mutagenesis, enzymatic activity