Production, Purification, Characterization, and Safety Evaluation of Constructed Recombinant D-Psicose 3-Epimerase

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 garnered considerable attention for healthy alternative sweetener due to its remarkable 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, with a strong emphasis on scalability. Experimentally, this research created the recombinant enzyme, explored the optimization of gene expression systems and protein purification strategies, and then investigated the enzymatic characterization to establish a robust and scalable process for D-psicose production. Additionally, the optimization of D-psicose production using the recombinant DPEase was also studied. Finally, the produced D-psicose syrup was 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 (IPTG) induction, and controlling induction temperature. The purification process was strategically designed to facilitate industrial-scale operations. A nickel column and an elution buffer containing 200 mM imidazole were employed, resulting in purified DPEase with a remarkable 21.03-fold increase in specific activity compared to the crude extract. The optimum D-psicose conversion conditions of purified DPEase were performed at pH 7.5 and 55°C under Mn²⁺ addition as the cofactor. Kinetic parameters of purified DPEase, V_max and K_m, were determined, demonstrating the high substrate affinity and efficiency of DPEase, which are crucial factors for achieving cost-effective for D-psicose production that was confirmed by binding site of fructose-DPEase-Mn²⁺ structure. Stability studies were conducted to ensure the enzyme's robustness during storage, which the glycerol supplementation and storage at -20°C were identified as effective strategies for maintaining its stability. D-psicose production using purified DPEase was investigated across a range of fructose concentrations, with a focus on optimizing yields for industrial applications. Moreover, the results from acute toxicity study showed no toxicity to rats, supporting the safety of 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. Our research advances 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.