Gene Cloning, Expression, Characteristics And Application Of Phosphatases From Bacillus Amyloliquefaciens YP6

Phosphatase can hydrolyze the phosphate ester bonds to release phosphate groups from phosphorus-containing substances,thus it has broad application prospects in molecules,medicine,and industry.The research on the ability of solubilize phosphate in microorganisms is mainly focused on organic acids.However,the ability of phosphatase in Bacillus amyloliquefaciens to release phosphorus from phosphate rock and shell powder,and degrade kinds of organophosphorus pesticides have not been comprehensively reported.This study started from the selection of phosphatase-producing microorganisms from the rhizosphere of phosphate rock plants.The strain with the best phosphatase activity was selected as the experimental object.The phosphatase gene was cloned,the recombinant vector was constructed,the heterologous expression of the protein was performed,and the applications were done.The main findings are as follows:?1?118 strains were obtained from the rhizosphere of phosphate rock plants,a strain named YP6 with high phosphatase activity was obtained and identified from by the method of Benzene Disodium Phosphate Colorimetry.At last,YP6 was identified as B.amyloliquefaciens YP6 by analyzing the 16S rRNA results.?2?Five phosphatase genes were obtained,which were ap1?684 bp?,ap2?1752 bp?,ap3?1380 bp?,ap4?1740 bp?and ap5?489 bp?.Four phosphatase enzymes were finally obtained,and the AP3 was alkaline phosphatase,AP2,AP4 and AP5 were acid phosphatases.The molecular weights of AP2?AP3?AP4?AP5 were 66.0,49.6,64.5 and 18.6 kDa,respectively.?3?When use pNPP as substrate.The optimum pH of AP2,AP3,AP4 and AP5 were 5.5,10.3,5.0 and 5.0,respectively,and the optimum reaction temperatures were 30,40,30 and35°C,respectively.Used the DPP and pNPP as the substrates,the kinetic parameters were as follows:for AP2,K_m was 3.8 and 260.4 mmol·L^-1,and V_m was 361 and 213?mol·min^-1·mg^-1.For AP3,the K_m values were 101.2 and 12.2 mmol·L^-1,and the V_m values were 3068 and 4033?mol·min^-1·mg^-1.For AP4,the K_m values were 12.5 and 389.4 mmol·L^-1,and the V_m values were 633 and 288?mol·min^-1·mg^-1.For AP5,K_m was 11.8 and 333.3 mmol·L^-1,and V_m values were 1087 and 490?mol·min^-1·mg^-1.?4?Under the optimal conditions,the degradation rates of AP3 to chlorpyrifos,dichlorvos,trichlorfon,phoxim and triazophos within 1 h were 0.38,0.53,0.23,1.72 and 2.36mg·m L^-1·h^-1.?5?AP2?AP3?AP4?AP5 acted on phosphate rock powder,which can significantly increase free phosphorus in the system.And the four enzymes also have a certain phosphorus-solubilization effect on oyster meal,oyster meal and lotus root meal.The phosphorus-solubilizing effect indicating that phosphatase is also one of the reasons for microbial release the soluble phosphorus.