Isolation of a Bacillus amyloliquefaciens strain producing high-activity alkaline phosphatase and cloning and expression of the phoD gene

Abstract:

[Background] Alkaline phosphatase (ALP), a tool enzyme, is widely used in various fields. Among the ALPs, PhoA has been extensively applied in immunological detection, while the functions of PhoD in immunological detection have been rarely reported. [Objective] This paper aims to screen a bacterial strain producing high-activity PhoD, clone and express the enzyme gene phoD, and study the enzymatic properties of PhoD, which is expected to lay a foundation for the application of PhoD in immunological detection. [Methods] Soil samples rich in organic matter were used to isolate bacteria in organophosphorus plate, and 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP) was employed to detect the enzyme activity of single colonies. The strain with high enzyme activity was selected and the phoD gene was cloned. [Results] S2-4, the strain producing high-activity ALP, was screened out, which was identified as Bacillus amyloliquefaciens by 16S rRNA gene alignment. Its phoD gene was cloned and induced, and the enzymatic properties of purified PhoD are as follows:optimal reaction temperature, pH, and Ca²⁺ concentration of 70℃, 9.8, and 3 mmol/L, respectively, inhibition of Mg²⁺ on PhoD activity, no significant influence of K⁺, Zn²⁺, Mn²⁺, and Fe²⁺ on PhoD activity, Michaelis constant (K_m), maximum reaction rate (V_max), and catalytic constant (k_cat) of 5.94 mmol/L, 31.46 μmol/(L·min), and 103.59 s^-1 at 25℃ in the presence of p-NPP, separately. The k_cat was 1.60 folds that of Escherichia coli alkaline phosphatase (EAP). [Conclusion] S2-4 can synthesize alkaline phosphatase with high activity and its monomer enzyme PhoD has higher catalytic efficiency than EAP, which is more advantageous than EAP as a marker enzyme.

Key words:alkaline phosphatase;screening;Bacillus amyloliquefaciens;phoD;enzymatic properties