Construction of hyperthermostable d-allulose 3-epimerase from Arthrobacter globiformis M30 using the sequence information from Arthrobacter psychrolactophilus.

d-Allulose is one of the rare monosaccharides and is considered as a safe ingredient in foods. It can be enzymatically produced from d-fructose by the enzyme d-allulose 3-epimerase. More stable enzymes can operate effectively for longer durations, reducing the need for frequent replacements and thereby lowering costs. In addition, the preparation of the recombinant Arthrobacter globiformis M30 (AgDAE) enzyme requires heat treatment at 60-70 °C to remove host cell debris and potential microbial contaminants. Therefore, to address the need for more thermostable enzymes in d-allulose production, we aimed to create thermostable mutants of AgDAE using the protein engineering method. We cloned d-allulose identified from A. globiformis M30 and, using sequence homology, we constructed thermostable mutants by protein engineering. Each effect of the five mutations used was independent and additive. By integrating positive mutations, we succeeded in the construction of a chimeric enzyme exhibiting hyperthermostability without loss of enzymatic activity. The constructed chimera mutant was highly functional above 95 °C and remained stable under 80 °C. Our approach using structural information for the chimeric construction experiments also suggested that incorporating mutations from other homologous enzymes can impart advantages in enzymes in a simple and effective manner.