Improving the enzyme property of D-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense through rational design

Zhangliang Zhu; Lei Li; Wei Zhang; Chao Li; Shuhong Mao; Fuping Lu; Hui-Min Qin

doi:10.1016/j.enzmictec.2021.109850

Improving the enzyme property of D-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense through rational design

Enzyme Microb Technol. 2021 Sep:149:109850. doi: 10.1016/j.enzmictec.2021.109850. Epub 2021 Jun 12.

Authors

Zhangliang Zhu¹, Lei Li¹, Wei Zhang¹, Chao Li¹, Shuhong Mao², Fuping Lu³, Hui-Min Qin⁴

Affiliations

¹ Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, PR China.
² Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, PR China. Electronic address: shuhongmao@tust.edu.cn.
³ Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, PR China. Electronic address: lfp@tust.edu.cn.
⁴ Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, PR China. Electronic address: huiminqin@tust.edu.cn.

PMID: 34311887
DOI: 10.1016/j.enzmictec.2021.109850

Abstract

The rare sugar d-allulose is an attractive sucrose substitute due to its sweetness and ultra-low caloric value. It can be produced from D-fructose using d-allulose 3-epimerase (DAE) as the biocatalyst. However, most of the reported DAEs show low catalytic efficiency and poor thermostability, which limited their further use in food industrial. Here, a putative d-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense (HcDAE) was characterized, showing optimal activity at pH 8.0 and 70 °C in the presence of Mg²⁺. Saturation mutagenesis of Y7, C66, and I108, the putative residues responsible for substrate recognition at the O-4, -5, and -6 atoms of D-fructose was performed, and it yielded the triple mutant Y7H/C66L/I108A with improved activity toward D-fructose (345 % of wild-type enzyme). The combined mutant Y7H/C66L/I108A/R156C/K260C exhibited a half-half (t_1/2) of 5.2 h at 70 °C and an increase of the T_m value by 6.5 °C due to the introduction of disulfide bridges between intersubunit with increased interface interactions. The results indicate that mutants could be used as industrial biocatalysts for d-allulose production.

Keywords: D-allulose; D-allulose 3-epimerase; Saturation mutagenesis; Thermostability.

MeSH terms

Firmicutes
Fructose*
Hydrogen-Ion Concentration
Racemases and Epimerases*

Substances

psicose
Fructose
Racemases and Epimerases

Supplementary concepts

Halanaerobium congolense