Redesign of a novel d-allulose 3-epimerase from Staphylococcus aureus for thermostability and efficient biocatalytic production of d-allulose

Abstract

Abstract Background A novel d-allulose 3-epimerase from Staphylococcus aureus (SaDAE) has been screened as a d-allulose 3-epimerase family enzyme based on its high specificity for d-allulose. It usually converts both d-fructose and d-tagatose to respectively d-allulose and d-sorbose. We targeted potential biocatalysts for the large-scale industrial production of rare sugars. Results SaDAE showed a high activity on d-allulose with an affinity of 41.5 mM and catalytic efficiency of 1.1 s−1 mM−1. Four residues, Glu146, Asp179, Gln205, and Glu240, constitute the catalytic tetrad of SaDAE. Glu146 and Glu240 formed unique interactions with substrates based on the structural model analysis. The redesigned SaDAE_V105A showed an improvement of relative activity toward d-fructose of 68%. The conversion rate of SaDAE_V105A reached 38.9% after 6 h. The triple mutant S191D/M193E/S213C showed higher thermostability than the wild-type enzyme, exhibiting a 50% loss of activity after incubation for 60 min at 74.2 °C compared with 67 °C for the wild type. Conclusions We redesigned SaDAE for thermostability and biocatalytic production of d-allulose. The research will aid the development of industrial biocatalysts for d-allulose.

Keywords

• d-Allulose 3-epimerase
• Structural analysis
• TIM-barrel fold
• Site-directed saturation mutagenesis
• Thermostability