Improving the low-temperature properties of an exo-inulinase via the deletion of a loop fragment located in its catalytic pocket

Abstract

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. 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 t1/2 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. 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. How to cite: He L, Zhang R, Shen J. et al. Improving the low-temperature properties of an exo-inulinase via the deletion of a loop fragment located in its catalytic pocket. Electron J Biotechnol 2022;55. https://doi.org/10.1016/j.ejbt.2021.09.004