Improving low-temperature activity and thermostability of exo-inulinase InuAGN25 on the basis of increasing rigidity of the terminus and flexibility of the catalytic domain

Abstract

Enzymes displaying high activity at low temperatures and good thermostability are attracting attention in many studies. However, improving low-temperature activity along with the thermostability of enzymes remains challenging. In this study, the mutant Mut8S, including eight sites (N61E, K156R, P236E, T243K, D268E, T277D, Q390K, and R409D) mutated from the exo-inulinase InuAGN25, was designed on the basis of increasing the number of salt bridges through comparison between the low-temperature-active InuAGN25 and thermophilic exo-inulinases. The recombinant Mut8S, which was expressed in Escherichia coli , was digested by human rhinovirus 3 C protease to remove the amino acid fusion sequence at N-terminus, producing RfsMut8S. Compared with wild-type RfsMInuAGN25, the mutant RfsMut8S showed (1) lower root mean square deviation values, (2) lower root mean square fluctuation (RMSF) values of residues in six regions of the N and C termini but higher RMSF values in five regions of the catalytic pocket, (3) higher activity at 0–40°C, and (4) better thermostability at 50°C. This study proposes a way to increase low-temperature activity along with a thermostability improvement of exo-inulinase on the basis of increasing the rigidity of the terminus and the flexibility of the catalytic domain. These findings may prove useful in formulating rational designs for increasing the thermal performance of enzymes.

Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance

“…On the contrary, an increase in intraprotein interactions resulted in the thermostability enhancement of the exo-inulinase from Sphingobacterium sp. GN25 ( He et al, 2020 ; Zhang et al, 2020 ). Therefore, the close numbers of salt bridges and cation-π interactions should be responsible for similar thermal characteristics of InuAMN8 and its mutant MutG169Δ4.…”

“…Exo-inulinases are classified in family 32 of glycoside hydrolases (GH32), which usually consist of two domains, with the N-terminal catalytic domain belonging to Glyco_hydro_32N (PF00251) and the C-terminal domain belonging to Glyco_hydro_32C (PF08244) ( Mistry et al, 2021 ). To date, studies on thermal properties and substrate recognition, as well as related mechanisms of exo-inulinases, have been reported ( Nagem et al, 2004 ; Arjomand et al, 2016 ; Zhou S. H. et al, 2016 ; Singh et al, 2018b ; Germec and Turhan, 2019 , 2020 ; Ma et al, 2019 , 2020 ; He et al, 2020 , 2022 ; Zhang et al, 2020 ; Wang et al, 2021 ). Among these studies, deletion of loops at the N-terminal tail and catalytic domain influences the thermal performance of exo-inulinases ( Arjomand et al, 2016 ; He et al, 2020 , 2022 ).…”

Purification and partial characterization of a thermostable peroxidase isoenzyme from Bambara groundnut (Vigna subterranea L. Verdc) seedlings

“…However, the fact that thermostable enzymes are generally less flexible at room temperature and reach their maximum catalytic potential only at temperatures over 50 °C or 70 °C presents an inherent challenge with them [7]. This is a major disadvantage for applications such as enzyme immunoassays, which are done at ambient temperature.…”