Revision as of 14:43, 29 September 2024

T7 promoter-RBS-AJC7/I129T-6xHis-T7 termonator

AJC 7 single-point mutation expression plasmid

Construction

Primers were designed for the I129T point mutation, and the plasmid containing the pET-28a(+) vector was amplified using PCR. Following the mutation, the PCR products were verified by nucleic acid gel electrophoresis to confirm the presence of the desired bands. The plasmid containing the correct bands was subsequently transformed into E. coli BL21 (DE3) competent cells.

Fig.1 Point mutation localisation and primer design

Fig. 2 Nucleic acid gel plot of colony PCR

Indicator

The mutant and wild-type strains were subjected to activation and amplification culture, followed by a series of protein purification procedures to extract the target proteins, as outlined in [Experimental]. The volume of the purified enzyme solution needed for the 500 μL reaction system was determined based on the protein concentration detailed in [Experimental]. The final concentration of fructose in the reaction mixture was 100 g/L, and it included 10 μL of Ni²⁺ as a catalyst. The reaction was conducted at 70°C for 5 hours, and the resulting products were subsequently analyzed using High-Performance Liquid Chromatography (HPLC).

Result

The results indicated that the concentration of products obtained from the AJC7 enzyme after the double mutation of S125D/I129T was higher compared to the wild-type AJC7-S125D enzyme activity, when both were subjected to a reaction at 70℃ for 5 hours. However, the observed enhancement was not notable.

Fig.3 Fig.3 The concentrations of tagose produced in the system after WT, S125D, T181A, H342L, I129T, and L140P reacted with 100 g/L substrate fructose for 5 h