Enhancing the thermostability of D-allulose 3-epimerase from Clostridium cellulolyticum H10 via a dual-enzyme screening system.

Enzyme Microb Technol

Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address:

Published: September 2022

D-Allulose 3-epimerase (DAE) is promising to be used for the production of the rare sugar D-allulose in industry. However, the poor thermostability and low catalytic efficiency limited its large-scale industrial applications. A dual-enzyme screening method was developed to measure the activity of the D-allulose 3-epimerase from Clostridium cellulolyticum H10 by employing a xylose isomerase, enabling high-throughput screening of mutants with higher thermostability. After two rounds of directed evolution, the H56R, Q277R, H56R/Q277R and H56R/Q277R/S293R variants were obtained with 1.9, 1.8, 3.5 and 7.1 °C improvement in T, the temperature at which the enzyme activity becomes half of the original after the 5 min treatment and 3.1-, 4.2-, 4.4- and 9.47- fold improvement in the half life at 60 °C, respectively, compared with the wild-type enzyme. Among them, triple mutant H56R/Q277R/S293R showed significant improvement in k/K compared to the wild type enzyme. Molecular dynamics simulations provided the insights into improving the thermostability by three arginine mutations. The research will aid the development of industrial biocatalysts for the production of D‑allulose.

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http://dx.doi.org/10.1016/j.enzmictec.2022.110054DOI Listing

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