耐有机溶剂果糖苷酶的胞外表达、纯化和结晶

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

摘要:

【目的】克隆表达一种Arthrobacter arilaitensis NJEM01来源的耐有机溶剂β-呋喃果糖苷酶(β-FFase)，纯化并进行结晶条件的研究。【方法】构建pelB信号肽与β-FFase融合表达质粒pET22b-pelB-bff，将其导入Escherichia coli BL21(DE3)中诱导表达，硫酸铵沉淀、DEAE阴离子交换色谱两步纯化重组蛋白，采用坐滴式气相扩散法对β-FFase进行结晶条件筛选和优化。【结果】构建重组质粒pET22b-pelB-bff，通过优化诱导表达条件，IPTG浓度0.1 mmol/L，诱导温度30 °C，诱导时间10 h，比活力高达108 U/mg，实现了果糖苷酶的胞外可溶性表达，纯化获得达到结晶纯度的β-FFase。结晶条件初筛和优化后获得可培养β-FFase晶体的条件为0.15 mol/L氯化钙，0.1 mol/L HEPES pH 6.7，26%聚乙二醇400。晶体衍射分辨率可以达到2.1 ?。【结论】高糖苷合成能力β-FFase表达纯化体系的构建和结晶条件的初步研究，为从结构生物学角度进一步研究果糖苷酶结构与功能的关系，定向进化提高糖苷酶转糖基活性奠定了基础。

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.