Expression, purification, and crystallization of an organic solvent-tolerant β-fructofuranosidase

Abstract:

[Objective] This study aims to clone, express and purify an organic solvent-tolerant β-fructofuranosidase (β-FFase). [Methods] The recombination plasmid pET22b-pelB-bff by fusing the signal peptide gene pelB with bff was expressed in Escherichia coli BL21(DE3), encoding β-FFase from Arthrobacter arilaitensis NJEM01. Ammonium sulfate precipitation and DEAE anion exchange chromatography were used to purify β-FFase. Crystallization condition was determined using the sitting drop diffusion technique. [Results] Plasmid pET22b-pelB-bff was constructed. Under the optimal induction condition as 0.1 mmol/L IPTG at 30 °C for 10 h, β-FFase with the specific activity of 108 U/mg was expressed in the culture medium, which was further purified to the crystalline purity. The single crystal of β-FFase could be diffracted up to 2.1 ? resolution at the optimal culture condition consisting of 0.15 mol/L calcium chloride dehydrate, 0.1 mol/L HEPES (pH 6.7) and 26% (V/V) polyethylene glycol 400. [Conclusion] β-FFase has been successfully expressed, purified and crystallized with an efficient transfructosylation activity, which lays a foundation for the investigation on the structure-function relationship and improvement of β-FFase catalytic efficiency by directed evolution of gene manipulation in the future.