高酶活枯草芽孢杆菌碱性磷酸酶phoA基因克隆表达及降解内毒素的应用

The alkaline phosphatase gene phoA from a Bacillus subtilis strain with high enzyme activity: cloning, expression, and application in degrading endotoxin

摘要:

【背景】 碱性磷酸酶有降解内毒素活性，在胃肠道中有预防和减弱肠道炎症的作用，不同的枯草芽孢杆菌能分泌不同酶活性水平的碱性磷酸酶，可能是该菌保健作用的一个值得重视的因素。【目的】 筛选并分离出一株产高酶活碱性磷酸酶的枯草芽孢杆菌(Bacillus subtilis)，对其phoA基因进行克隆并原核表达，研究其酶学性质并对内毒素进行降解研究。【方法】 以有机质丰富的土壤环境微生物为样本，在有机磷筛选培养基中分离枯草芽孢杆菌，以4-硝基苯磷酸二钠盐(4-nitrophenyl phosphate disodium salt hexahydrate, p-NPP)为底物进行酶活测定，筛选产高酶活碱性磷酸酶的细菌，克隆表达其phoA基因并研究重组PhoA的酶学特性，最后用小鼠试验证明该酶对降低内毒素LPS的毒性作用。【结果】 筛选到14株产高酶活碱性磷酸酶枯草芽孢杆菌，并克隆一株最高酶活的枯草芽孢杆菌35-16-1的phoA基因，在大肠杆菌(Escherichia coli) BL21(DE3)中实现可溶性表达。研究纯化后PhoA的酶学性质，PhoA的最适反应温度为70 ℃；最适反应pH为9.8；Mg²⁺对PhoA的酶活性呈激活作用，当Mg²⁺为14 mmol/L时，激活作用最大；Ca²⁺对PhoA的酶活性呈抑制作用；K⁺、Zn²⁺、Mn²⁺和Fe²⁺对PhoA的酶活性无明显影响；以p-NPP为底物，在37 ℃下测得PhoA的V_max为179.86 mmol/(L·min)，K_m为2.38 mmol/L，k_cat为246.83 s^-1。最适条件下测得酶比活为6 550.00 U/mg。在重组PhoA处理后，LPS对小鼠的毒性明显降低。【结论】 枯草芽孢杆菌35-16-1的phoA基因能重组表达出高酶活性的碱性磷酸酶，对内毒素具有降解功能。

Abstract:

[Background] Alkaline phosphatase (ALP) with the ability of degrading endotoxin (lipopolysaccharide, LPS) can prevent and weaken intestinal inflammation. Different strains of Bacillus subtilis can secrete ALP with different activities, which may be an important factor in the health effects of B. subtilis. [Objective] To study the enzymatic properties and endotoxin-degrading performance of the recombinant protein by cloning the ALP gene phoA from a B. subtilis strain with high ALP activity and expressing this gene in Escherichia coli. [Methods] We used the organophosphorus agar medium to isolate B. subtilis strains from the soil samples with rich organic matter. The ALP activity was assayed with 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP) as the substrate. The strains producing ALP with high activity were screened and phoA was cloned and expressed. The enzymatic characteristics of recombinant PhoA were studied, and the LPS-attenuating effect of PhoA was evaluated in mice. [Results] Fourteen strains of B. subtilis with high ALP activities were screened out, and phoA was cloned from the strain 35-16-1 with the highest ALP activity and expressed solubly in E. coli BL21(DE3). The enzymatic properties of PhoA after purification were studied. PhoA showcased the optimum reaction performance at 70 ℃ and pH 9.8. Mg²⁺ activated the activity of PhoA, with the maximum activating effect at 14 mmol/L. Ca²⁺ inhibited the activity of PhoA. K⁺, Zn²⁺, Mn²⁺, and Fe²⁺ had no significant effect on PhoA activity. With p-NPP as the substrate, PhoA presented the V_max of 179.86 mmol/(L·min), K_m of 2.38 mmol/L, and k_cat of 246.83 s^‒1 at 37 ℃. The specific activity of 6 550.00 U/mg under optimal conditions. The toxicity of LPS treated with recombinant PhoA was significantly reduced in mice. [Conclusion] The phoA gene of B. subtilis 35-16-1 can be expressed with high ALP activity in E. coli and the recombinant protein can degrade endotoxin.