Fusion of the peptide derived from the acidic tail of alpha-synuclein improves the thermostability and soluble expression of recombinant Agrobacterium sp. d-allulose 3-epimerase

The Engineering, Expression, and Immobilization of Epimerases for D-allulose Production

“…ATCC 31,749 (AsDAEase) was reported to have a relatively high specific activity and yield in E. coli [ 28 ]. Although the protein could reach a high yield, it faced rapid heat inactivation when exposed to temperatures of or above 60 °C, typically within 30 min [ 24 ]. In an effort to improve its thermostability and solubility, Tseng et al adopted the strategy of implementing a fusion tag derived from the C-terminal acidic tail of α-synuclein (ATS), which is both thermostable and possesses chaperone activity.…”

“…In an effort to improve its thermostability and solubility, Tseng et al adopted the strategy of implementing a fusion tag derived from the C-terminal acidic tail of α-synuclein (ATS), which is both thermostable and possesses chaperone activity. In Tseng’s study, a 22-amino-acid-long ATS peptide sequence (residue 119–140) was fused to both wild-type and thermostable AsDAEase mutants before expression in E. coli BL21(DE3) [ 24 ]. A total of four different combinations of enzymes were characterized: the wild type, wild type fused with ATS, mutant (I33L/S213C), and mutant fused with ATS (I33L/S213C-ATS fusion, known as LCATS).…”

“…Compared to the wild type, the LCATS demonstrated a significantly improved thermostability up to 65 °C, along with 19-fold increase in its half-life. Its catalytic efficiency was also slightly higher than that of the wild type at 64.1 ± 12.0 compared to 41.2 ± 12.0 min −1 mM −1 [ 24 ].…”

“…In this review, we discussed how new epimerases can be discovered [ 15 , 16 , 17 ], as well as methods to improve their stability and activity via protein engineering [ 21 , 22 , 24 , 25 , 28 ], the application of B. subtilis [ 11 , 25 , 29 ], and the potential of immobilization techniques to enhance performance [ 34 , 35 , 36 , 37 ]. These advancements have the potential to significantly reduce production time and costs associated with D -allulose biosynthesis.…”

Computer-Aided Targeted Mutagenesis of Thermoclostridium caenicola d-Allulose 3-Epimerase for Improved Thermostability

“…Recently, a heat-tolerant DAEase (DaeM) from a thermal spring metagenome in India has been reported with half-life values of 165 and 54 h at 60 and 70 °C, respectively; however, its specific activity for catalyzing the epimerization of d -fructose into d -allulose is only 7 U/mg . On the other hand, several methods of molecular modification have been used for engineering thermostable versions of DAEase, such as gene fusion, , directed evolution, ,, and rational/semirational design. − Identifying potential mutation hotspots that affect thermostability plays a critical role in the rational/semirational design. Extensive research has shown that the difference in the free energy of folding between a wild-type protein and its mutant (ΔΔ G fold ) indicates the mutational effect .…”