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Improved Performance of D-Psicose 3-Epimerase by Immobilisation on Amino-Epoxide Support with Intense Multipoint Attachment.

ABSTRACT: D-allulose is an epimer of D-fructose at the C-3 position. With similar sweetness to sucrose and a low-calorie profile, D-allulose has been considered a promising functional sweetener. D-psicose 3-epimerase (DPEase; EC 5.1.3.30) catalyses the synthesis of D-allulose from D-fructose. Immobilised enzymes are becoming increasingly popular because of their better stability and reusability. However, immobilised DPEase generally exhibits less activity or poses difficulty in separation. This study aimed to obtain immobilised DPEase with high catalytic activity, stability, and ease of separation from the reaction solution. In this study, DPEase was immobilised on an amino-epoxide support, ReliZyme HFA403/M (HFA), in four steps (ion exchange, covalent binding, glutaraldehyde crosslinking, and blocking). Glycine-blocked (four-step immobilisation) and unblocked (three-step immobilisation) immobilised DPEase exhibited activities of 103.5 and 138.8 U/g support, respectively, but contained equal amounts of protein. After incubation at 60 °C for 2 h, the residual activity of free enzyme decreased to 12.5%, but the activities of unblocked and blocked DPEase remained at 40.9% and 52.3%, respectively. Immobilisation also altered the substrate specificity of the enzyme, catalysing L-sorbose to L-tagatose and D-tagatose to D-sorbose. Overall, the immobilised DPEase with intense multipoint attachment, especially glycine-blocked DPEase, showed better properties than the free form, providing a superior potential for D-allulose biosynthesis.

SUBMITTER: Bu Y

PROVIDER: S-EPMC8069956 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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. 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. 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.

Dataset Information

Improved Performance of D-Psicose 3-Epimerase by Immobilisation on Amino-Epoxide Support with Intense Multipoint Attachment.

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