Engineering a thermostable version of D-allulose 3-epimerase from Rhodopirellula baltica via site-directed mutagenesis based on B-factors analysis.

D-allulose has received increasing attention due to its excellent physiological properties and commercial potential. The D-allulose 3-epimerase from Rhodopirellula baltica (RbDAEase) catalyzes the conversion of D-fructose to D-allulose. However, its poor thermostability has hampered its industrial application. Site-directed mutagenesis based on homologous structures in which the residuals on high flexible regions were substituted according to B-factors analysis, is an effective way to improve the thermostability and robustness of an enzyme. RbDAEase showed substrate specificity toward D-allulose with a Km of 58.57 mM and kcat of 1849.43 min-1. It showed a melting temperature (Tm) of 45.7 °C and half-life (t1/2) of 52.3 min at pH 8.0, 60 °C with 1 mM Mn2+. The Site-directed mutation L144 F strengthened the thermostability to a Δt1/2 of 50.4 min, ΔTm of 12.6 °C, and ΔT5060 of 22 °C. It also improved the conversion rate to 28.6%. Structural analysis reveals that a new hydrophobic interaction was formed by the mutation. Thus, site-directed mutagenesis based on B-factors analysis would be an efficient strategy to enhance the thermostability of designed ketose 3-epimerases.