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

Yun Feng; Zhongji Pu; Li Zhu; Mianbin Wu; Lirong Yang; Haoran Yu; Jianping Lin

doi:10.1016/j.enzmictec.2022.110054

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

Enzyme Microb Technol. 2022 Sep:159:110054. doi: 10.1016/j.enzmictec.2022.110054. Epub 2022 Apr 26.

Authors

Yun Feng¹, Zhongji Pu², Li Zhu¹, Mianbin Wu¹, Lirong Yang³, Haoran Yu⁴, Jianping Lin⁵

Affiliations

¹ Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
² Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou 311200, China.
³ Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China; Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou 311200, China.
⁴ Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China; Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou 311200, China. Electronic address: yuhaoran@zju.edu.cn.
⁵ Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Electronic address: linjp@zju.edu.cn.

PMID: 35526470
DOI: 10.1016/j.enzmictec.2022.110054

Abstract

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 T₅₀⁵, 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_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.

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

MeSH terms

Clostridium cellulolyticum* / genetics
Enzyme Stability
Fructose
Hydrogen-Ion Concentration
Racemases and Epimerases* / genetics
Temperature

Substances

psicose
Fructose
Racemases and Epimerases