Determination of free phosphate release from AP substrate
Tris-HCl (50mmol L-1, pH6.0-8.0) buffer was used to prepare 10mM of ATP, ADP, and AMP solution (ATP, Biotopped; ADP and AMP, Shanghai source leaf biology) and also to dilute recIAP (1000U ml-1) to 0.5, 5.0, 50.0U ml-1. 950μl of recIAP and 50μl of ATP, ADP, and AMP solutions were mixed together thoroughly, producing a final concentration of the solution is 0.5 mM, and incubated at 37°C for 60 min. In the above measurement, recIAP that was inactivated at 65 ° C for 60 minutes was used as a blank control. An inorganic phosphorus test box (C006-1-1, Nanjing Jiancheng Biotechnology Research Institute) was used to measure the content of free phosphate (Pi) released through the recIAP’s effect on ATP, ADP, and AMP (0.5mM) at different pHs, ranging between 6.5-8.0. The released Pi interacts with molybdic acid to generate phosphomolybdic acid, which is then reduced to molybdenum blue and exhibits a unique absorption spectrum with a maximum peak at 660nm.