Revision as of 15:29, 27 September 2024

T7 promoter-RBS-TET-6xHis-T7 termonator

TET structural gene expression plasmid

Construction

The screened sequence AJC7, which has potential tagatose-4-epimerase activity, was sent to a company for synthesis, and the sequence was subsequently ligated into the pET-28a(+) vector. (Figure 1).

 Fig. 1 Mapping of pET-28a(+)-AJC7 plasmid

Indicator

The pET-28a(+)-AJC7 construct was transformed into E. coli BL21 (DE3) and incubated in an inverted culture at a constant temperature of 37°C for 14 hours. Individual colonies were selected from the transformed growth, and colony PCR was performed. Single colonies with the correct bacterial plasmid were then transferred to LB medium containing kanamycin (LB Kan) for activation and amplification culturing, followed by protein purification as outlined in [Experiment]. The final concentration of fructose in the reaction system was set at 10 g/L, supplemented with 10 µL of Ni²⁺ as a catalyst. The volume of pure enzyme solution required was determined based on the protein concentration, as indicated in [Experimental]. The reaction was conducted at 70℃ for 5 hours, and the products were analyzed using high-performance liquid chromatography (HPLC) (Figure 2).

 Fig. 2 Concentration of tagatose produced by AJC7 and UxaE under identical reaction conditions.

Result

From the results obtained in the liquid phase, it is evident that the concentration of tagatose produced by AJC7 is approximately four times higher than that produced by UxaE under the same reaction conditions. This indicates that AJC7 demonstrates significantly greater potential activity as a tagatose-4-epimerase, leading to its selection for subsequent studies.

Sequence and Features