Purification, immobilization, evolution, and characterization of D-allulose 3-epimerase from Antarctic Microbacterium

D-allulose is a rare, low-calorie sugar substitute with multiple physiological functions and is widely used in food and pharmaceutical industries. D-allulose is primarily produced by the catalytic conversion of d-fructose via D-allulose 3-epimerase (DAEase). In this study, a DAEase gene, dpema4, was isolated from an Antarctic bacterium and expressed in Escherichia coli. The recombinant DAEase was a homotetramer with an optimal temperature of 60 °C and pH of 7.5. Its catalytic activity was not strictly dependent on metal ions, making it a safer alternative to the other reported DAEases. The recombinant DAEase showed exhibited the highest activity towards D-allulose, and the bioconversion rate was 29 %. For immobilization, the cellulose-binding domain (CBD) was fused to DAEase, and the fusion protein was immobilized on microcrystalline cellulose. The immobilized DAEase showed highly improved pH stability and maintained approximately 44 % of catalytic activity after 10 continuous reaction cycles. The single-point mutant A248H showed high thermal stability and catalytic activity at 60 °C, and the bioconversion rate of d-fructose reached 32 %. In summary, the recombinant DAEase can serve as a good candidate enzyme for the production of D-allulose, and the establishment of a one-step purification and immobilization of DAEase can facilitate its industrial application.