Characterization of Q316P mutant of aspartate kinase from Corynebacterium pekinense

Abstract:

[Objective] To obtain a new aspartate kinase (AK) with higher activity and better properties of highly produced aspartic acid family from Corynebacterium pekinense by spatial structure transformation, and attenuate or release the feedback inhibition of AK. [Methods] The Gln(Q)316 of AK was mutated by site-directed mutagenesis, and mutant strain with the highest activity was selected by high throughput screening. The mutant was expressed in Escherichia coli BL21. AK of the wild type and mutant strain was purified by Ni²⁺-NTA column and then the characterized. [Results] The mutant Q316P was constructed, and successfully expressed in E. coli BL21. Compared with the wild type, V_max of Q316P was increased by 8.53 times, and the n values was decreased from 2.15 to 1.29. The optimum temperature was increased from 25 ℃ to 30 ℃, the optimum pH was decreased from 8.0 to 7.5 and the half-time period was increased from 3.8 h to 5.0 h. The inhibition of the substrate inhibitor threonine was not only relieved but also threonine had an active effect on the activity of Q316P. Q316P had better resistance to methanol and K⁺. [Conclusion] Our findings provided a reference for the construction of high-yield aspartic acid family engineering strain.

Key words:Corynebacterium pekinense;aspartate kinase;site-directed mutagenesis;kinetics;characterization