索诺拉沙漠芽孢杆菌s262过氧化氢酶的异源表达及酶学性质

Heterologous expression and enzymatic properties of catalase from Bacillus sonorensis s262

摘要:

【背景】索诺拉沙漠芽孢杆菌(Bacillus sonorensis) s262所产过氧化氢酶具有降解黄曲霉毒素B₁ (Aflatoxin B₁, AFB₁)的能力。【目的】在大肠杆菌(Escherichia coli)中表达索诺拉沙漠芽孢杆菌(Bacillus sonorensis) s262过氧化氢酶，对表达条件进行优化并测定酶学性质。【方法】将过氧化氢酶基因katA连接到pET28a(+)载体中，构建重组表达质粒pET28a-katA，转化至Escherichia coli BL21(DE3)感受态细胞中，通过优化IPTG浓度、诱导温度和诱导时间确定重组酶的最佳表达条件。使用Ni-NTA Sefinose^TM Resin分离纯化过氧化氢酶。通过测定过氧化氢酶最适反应温度及热稳定性、最适pH及pH稳定性和金属离子对酶活力的影响对重组酶酶学性质进行分析，通过双倒数作图法对重组酶进行动力学分析。高效液相色谱法检测重组过氧化氢酶对AFB₁的降解率。【结果】重组表达质粒pET28a-katA经双酶切(BamH Ⅰ和Hind Ⅲ)及测序证明构建成功。纯化的重组酶分子量大小在55 kDa，在E. coli中的最佳表达条件为：IPTG浓度0.8 mmol/L，诱导温度25 ℃，诱导时间10 h。酶最适催化温度50 ℃，最适pH 7.0时，其动力学参数V_max为(760.17±19.61) mmol/(min·L)，K_m为(63.73±3.87) mmol/L。0.1 mmol/L Fe²⁺、Zn²⁺和Cu²⁺对酶有促进作用，酶活分别提高40%、8%和12%，但该浓度下K⁺对酶起抑制作用，使酶活降低39%。重组酶对AFB₁的降解率为61.44%。【结论】成功表达并纯化了索诺拉沙漠芽孢杆菌源过氧化氢酶，为过氧化氢酶的工业生产及应用奠定了基础。

Abstract:

[Background] The catalase produced by Bacillus sonorensis s262 has the ability to degrade aflatoxin B₁ (AFB₁).[Objective] To express Bacillus sonorensis s262 peroxidase in Escherichia coli, optimize the expression conditions, and determine the enzymatic properties. [Methods] The catalase gene katA was ligated into the pET28a(+) vector to construct the recombinant expression plasmid pET28a-katA, which was then transformed into E. coli BL21(DE3) competent cells. Furthermore, the expression conditions including IPTG concentration, the induction temperature, and the induction time of the recombinant enzyme were optimized. Ni-NTA Sefinose^TM resin was used to purify the recombinant enzyme. The optimal reaction temperature and thermal stability, the optimal pH and pH stability, and the effects of metal ions on the enzyme activity were determined, and the kinetics of the recombinant enzyme was analyzed by double-reciprocal plotting. The degradation rate of AFB₁ by the recombinant catalase was measured by high performance liquid chromatography. [Results] The recombinant expression plasmid pET28a-katA proved to be successfully constructed by double digestion (BamH Ⅰ and Hind Ⅲ) and sequencing. The molecular weight of the purified recombinant enzyme ranged from 55 kDa. The expression conditions in E. coli were optimized as follows: induction with 0.8 mmol/L IPTG at 25 ℃ for 10 h. The kinetic parameters V_max and K_m were (760.17±19.61) mmol/(min·L) and (63.73±3.87) mmol/L, respectively, at the optimal catalytic temperature of 50 °C and optimal pH 7.0. In addition, 0.1 mmol/L Fe²⁺, Zn²⁺, and Cu²⁺ increased the enzyme activity by 40%, 8%, and 12%, respectively, while 0.1 mmol/L K⁺ decreased the enzyme activity by 39%. The degradation rate of AFB₁ by the recombinant enzyme was 61.44%. [Conclusion] We successfully expressed and purified the catalase of B. sonorensis in E. coli, laying a foundation for the industrial production and application of catalase.