Production, purification, characterization, and safety evaluation of constructed recombinant D-psicose 3-epimerase
Issued Date
2024-12-01
Resource Type
eISSN
14752859
Scopus ID
2-s2.0-85200054243
Journal Title
Microbial Cell Factories
Volume
23
Issue
1
Rights Holder(s)
SCOPUS
Bibliographic Citation
Microbial Cell Factories Vol.23 No.1 (2024)
Suggested Citation
Watthanasakphuban N., Srila P., Pinmanee P., Punvittayagul C., Petchyam N., Ninchan B. Production, purification, characterization, and safety evaluation of constructed recombinant D-psicose 3-epimerase. Microbial Cell Factories Vol.23 No.1 (2024). doi:10.1186/s12934-024-02487-x Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/100255
Title
Production, purification, characterization, and safety evaluation of constructed recombinant D-psicose 3-epimerase
Corresponding Author(s)
Other Contributor(s)
Abstract
Background: D-psicose 3-epimerase (DPEase) is a potential catalytic enzyme for D-psicose production. D-psicose, also known as D-allulose, is a low-calorie sweetener that has gained considerable attention as a healthy alternative sweetener due to its notable physicochemical properties. This research focused on an in-depth investigation of the expression of the constructed DPEase gene from Agrobacterium tumefaciens in Escherichia coli for D-psicose synthesis. Experimentally, this research created the recombinant enzyme, explored the optimization of gene expression systems and protein purification strategies, investigated the enzymatic characterization, and then optimized the D-psicose production. Finally, the produced D-psicose syrup underwent acute toxicity evaluation to provide scientific evidence supporting its safety. Results: The optimization of DPEase expression involved the utilization of Mn2+ as a cofactor, fine-tuning isopropyl β-D-1-thiogalactopyranoside induction, and controlling the induction temperature. The purification process was strategically designed by a nickel column and an elution buffer containing 200 mM imidazole, resulting in purified DPEase with a notable 21.03-fold increase in specific activity compared to the crude extract. The optimum D-psicose conversion conditions were at pH 7.5 and 55 °C with a final concentration of 10 mM Mn2+ addition using purified DPEase to achieve the highest D-psicose concentration of 5.60% (w/v) using 25% (w/v) of fructose concentration with a conversion rate of 22.42%. Kinetic parameters of the purified DPEase were Vmax and Km values of 28.01 mM/min and 110 mM, respectively, which demonstrated the high substrate affinity and efficiency of DPEase conversion by the binding site of the fructose-DPEase-Mn2+ structure. Strategies for maintaining stability of DPEase activity were glycerol addition and storage at -20 °C. Based on the results from the acute toxicity study, there was no toxicity to rats, supporting the safety of the mixed D-fructose–D-psicose syrup produced using recombinant DPEase. Conclusions: These findings have direct and practical implications for the industrial-scale production of D-psicose, a valuable rare sugar with a broad range of applications in the food and pharmaceutical industries. This research should advance the understanding of DPEase biocatalysis and offers a roadmap for the successful scale-up production of rare sugars, opening new avenues for their utilization in various industrial processes.