Dataset Information

Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704.

ABSTRACT: The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²? as optimum cofactor for activity. In addition, Mn²? improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T m) and the urea midtransition concentration (C m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²?-bound enzyme showed higher T m and C m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s?¹, and 64.5 mM?¹ s?¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.

SUBMITTER: Zhang W

PROVIDER: S-EPMC3639893 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704.

Zhang Wenli W Fang Dan D Xing Qingchao Q Zhou Leon L Jiang Bo B Mu Wanmeng W

PloS one 20130430 4

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, t ...[more]

PMID: 23646168

Similar Datasets

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

Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704.

Publications

Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets