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

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.