LmAPX基因在大肠杆菌和酵母菌中的表达研究

目的：研究枸杞抗坏血酸过氧化物酶基因（ascorbate peroxidase，LmAPX）在原核中的表达和酶学特性以及在酵母菌中的抗氧化能力，为进一步研究逆境诱导的抗氧化胁迫的作用机理奠定理论基础。方法：将LmAPX转入大肠杆菌BL21中进行异源表达，采用 Ni²⁺亲和层析，纯化重组蛋白，并对不同温度和pH值下的酶活进行研究，Lineweaver-Burk双倒数作图法测定该酶的Km和Vmax值。将LmAPX转入酵母菌株W303中进行H₂O₂和NaCl氧化胁迫处理。结果：该酶的最适温度和最适pH值分别为40℃和6.5。当抗坏血酸（Ascorbic acid，AsA）浓度过量时，对H₂O₂的Km和Vmax分别是0.17±0.02 mmol/L和11.78±1.88 mmol/min·mg；当H₂O₂浓度过量时，对AsA的Km和Vmax分别是2.19±0.40 mmol/L和58.82±3.51 mmol/min·mg。含有LmAPX基因的酵母菌株，在半乳糖的诱导下在8 mmol/L H₂O₂和100 mmol/L NaCl的培养基上的生长都明显优于对照组。结论：LmAPX蛋白具有很好的抗氧化性和耐盐性。

Expression Studies of Ascorbate Peroxidase from Lycium chinense Mill. in E.coli and Yeast

Objective:The aim of the study is to investigate the expression and enzymatic properties in E.coli and oxidation resistance in yeast of ascorbate peroxidase (LmAPX) cloned from Lycium chinense Mill., which will provide the theory foundation of antioxidant stress for future studying.Method: LmAPX was transformed into heterologous expression system E.coli BL21 and wild type yeast strain W303. The recombinant protein was purified by Ni²⁺ affinity chromatography and the enzyme activities at different temperature and pH were detected. The enzyme kinetics constant Km and Vmax were calculated by double-reciprocal plot. Results: The results showed that the optimal pH and temperature for this enzyme were 6.5 and 40℃, respectively.At fixed concentration of AsA, the Km and Vmax values of the enzyme for H₂O₂ were 0.17±0.02 mmol/L and 11.78±1.88 mmol/min·mg. And at fixed concentration of H₂O₂, the Km and Vmax values for AsA were 2.19±0.40 mmol/L and 58.82±3.51 mmol/min·mg. Tansgenic yeast stain transformed with LmAPX gene showed higher stress resistance than the control in the medium containing 8 mmol/L H₂O₂ or 100 mmol/L NaCl under the inducement of β-galactose. Conclusion: LmAPX enhanced the tolerance to oxidative stresses.