4.5 Article

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

Journal

ENZYME AND MICROBIAL TECHNOLOGY
Volume 159, Issue -, Pages -

Publisher

ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2022.110054

Keywords

D-allulose; D-allulose 3-epimerase; High-throughput screening; Thermostability; Molecular dynamics simulations

Funding

  1. National Key Research and Development Program [2019YFA0905404]

Ask authors/readers for more resources

A dual-enzyme screening method was developed to obtain several variants of D-allulose 3-epimerase, among which the triple mutant showed significant improvement, aiding the development of industrial biocatalysts for D-allulose production.
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 ? improvement in T-50(5) , 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 ?, respectively, compared with the wild-type enzyme. Among them, triple mutant H56R/Q277R/S293R showed significant improvement in k(cat)/K-m 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.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.5
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available