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. 2020 Dec;11(1):1233-1244.
doi: 10.1080/21655979.2020.1837476.

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

Rui Zhang  1   2   3 , Limei He  1   2   3 , Jidong Shen  1   2   3 , Ying Miao  1   2   3 , Xianghua Tang  1   2   3 , Qian Wu  1   2   3 , Junpei Zhou  1   2   3 , Zunxi Huang  1   2   3
Affiliations

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

Rui Zhang et al. Bioengineered. 2020 Dec.

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.

Keywords: Enzyme; biochemical property; mechanism; mutagenesis; structure.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Amino acid sequence alignment of InuAGN25 with GH 32 thermophilic exo-inulinases
Figure 2.
Figure 2.
The salt bridge network (green) formed within the N-terminal tail of Mut8S
Figure 3.
Figure 3.
MD analysis of wild-type RfsMInuAGN25 and its mutant RfsMut8S at 323 K
Figure 4.
Figure 4.
SDS-PAGE analysis
Figure 5.
Figure 5.
Effects of pH and temperature on purified wild-type RfsMInuAGN25 and its mutant RfsMut8S
Figure 6.
Figure 6.
Arrhenius plots for the determination of Ea for inulin hydrolysis by wild-type RfsMInuAGN25 and its mutant RfsMut8S
Figure 7.
Figure 7.
TLC analysis. G, 1.0% (w/v) glucose; F, 1.0% (w/v) fructose; S and CK, inulin with the active and inactivated enzymes, respectively
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