Revision as of 13:17, 28 September 2024

AJC7/S125D/ I129T

AJC7 two-point mutant

Construction

The mutation site of I129T is located close to S125D, prompting the redesign of the I129T mutation primer using pET-28a(+)-AJC7-S125D as a template. This allowed for the successful construction of the S125D/I129T double mutant. After the mutation process, the PCR product was verified using nucleic acid gel electrophoresis. Subsequently, the plasmid displaying the correct band was transformed into competent E. coli BL21 (DE3) cells.

Fig. 1 point-mutation-localisation-and-primer-design

Fig. 2 nucleic-acid-gel-plot-of-colony-pcr

Indicator

The two-point 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 described in [Experimental]. The volume of the purified enzyme solution required for the 500 μL reaction system was determined based on the protein concentration indicated in [Experimental]. The final concentration of fructose in the system was set at 100 g/L, with the addition of 10 μL of Ni²⁺ as a catalyst. The reaction was conducted at 70°C for 5 hours, after which the products were analyzed using High-Performance Liquid Chromatography (HPLC).

Result

The results indicated that the concentration of products obtained from the AJC7 enzyme after the two-point mutation of S125D/I129T showed an increase compared to the enzyme activity of the wild-type AJC7-S125D under the reaction conditions of 70℃ for 5 hours; however, the enhancement observed was not significant.

Fig. 3 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P after reaction with 100g/L fructose substrate for 5 h, respectively