Enhancing the thermostability of Pantoea dispersa sucrose isomerase using semi-rational design strategy

Abstract

Isomaltulose stands out for its low glycemic index and caries-resistant properties, and shows great potential for applications in the food and medical fields. Belonging to the glycoside hydrolase family GH13, sucrose isomerase is capable of converting sucrose to isomaltulose. The sucrose isomerase from Pantoea dispersa UQ68J (PdSI) is favored for its high conversion rates. However, poor thermostability limits its application in industrial production. To enhance the thermostability of PdSI, we combined sequence analysis and computer-aided design to identify and exclude key sites that might affect catalytic activity, and then screened 14 candidate mutants for point mutation validation. During the study, single-point mutants M62E, V105I, N109H, D232P, V447E and S481M demonstrated improved thermostability in preliminary experiments. Among them, the mutant V447E performed particularly well, with a 1.38-fold increase in half-life at 40 °C compared to the wild type, and showed an increase in the optimal temperature from 30 °C to 35 °C. Further combined mutation studies revealed that mutant V447E/D232P showed better thermostability. Compared with the wild type, mutant V447E/D232P increased the optimal temperature by 5 °C, and its half-life at 40 °C was prolonged by 1.52-fold. The results of molecular dynamics simulations further confirmed the low root-mean-square fluctuations of V447E and V447E/D232P compared with the wild type, indicating a significant enhancement in structural stability. This study offers a reference for improving the thermostability modification of sucrose isomerase and promotes the industrial application of isomaltulose.

Keywords

Sucrose isomerase / Thermostability / Semi-rational design / Molecular dynamics simulations

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Funding

National Natural Science Foundation of China(32272264)

Jiangsu Provincial Key Research and Development Program(BE2023686)

Fundamental Research Funds for Central Universities of the Central South University(JUSRP122012)

RIGHTS & PERMISSIONS

Jiangnan University