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

Catalytic pocket
Deletion
Enzyme
Exo-inulinase
Glycoside hydrolase
Intraprotein interactions
Inulase
Inulin
Loop fragment
Low-temperature
Mutagenesis
Protein engineering
Thermostability

How to Cite

1.
He L, Zhang R, Shen J, Miao Y, Zeng C, Tang X, Wu Q, Zhou J, Huang Z. Improving the low-temperature properties of an exo-inulinase via the deletion of a loop fragment located in its catalytic pocket. Electron. J. Biotechnol. [Internet]. 2022 Jan. 4 [cited 2024 Nov. 11];55. Available from: https://www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/2021.09.004

Abstract

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 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.

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.

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