Background: Engineering thermal adaptations of enzymes is a popular field of study. Enzymes active at low temperature have been used in many industries; however, reports seldom describe improvements in enzyme activity at low temperatures using protein engineering.

Results: Multiple amino acid sequence alignment of glycoside hydrolase (GH) family 32 showed an unconserved region located in the catalytic pocket. The exo-inulinase InuAGN25 showed the highest frequency of charged amino acid residues (47.4%) in this region among these GH 32 members. Notably, five consecutive charged amino acid residues (¹³⁷EEDRK¹⁴¹) were modeled as a loop fragment in this region of InuAGN25. Deletion of the loop fragment broke two salt bridges, one cation–π interaction, and the α-helix–loop–310-helix structure at the N-terminal tail. The mutant exo-inulinase RfsMutE137Δ5 without the loop fragment was expressed in Escherichia coli, digested using human rhinovirus 3C protease for removal of the fused sequence at the N-terminus, and purified using immobilized metal affinity chromatography. Compared to the wild-type enzyme, the optimum temperature and t_1/2 at 50°C of purified RfsMutE137Δ5 decreased by 10°C and 31.7 min, respectively, and the activities at 20°C and 30°C increased by 11% and 18%, respectively.

Conclusions: In this study, we engineered the loop to obtain the mutant exo-inulinase that showed an improved performance at low temperatures. These findings suggest that the loop may be a useful target in formulating rational designs for engineering thermal adaptations of GH 32 exo-inulinases.