| China is a large agricultural country,the increase of crop yield and the control of crop diseases and insect pests are inseparable from pesticides.However,the unreasonable application of pesticides has caused many environmental and health problems to a certain extent.In recent years,studies have found that alkaline phosphatase plays an important role in the degradation of organophosphorus pesticides.Alkaline phosphatase(EC 3.1.3.1,ALP)is a nonspecific phosphomonoesterase with optimal activity under alkaline conditions and is widely present in living organisms.It participates in various biochemical reactions and processes.In this experiment,a Lactobacillus strain with high ALP production was selected from 78 strains of lactic acid bacteria.After determining its species,the extraction conditions of the ALP in the target strain were optimized and the ALP was purified to explore its enzymatic properties and its role in degrading organophosphorus pesticides.The main experimental results are as follows:1.Using the traditional culture method,a strain of Lactobacillus with high ALP production was selected from 78 strains of lactic acid bacteria,which was named strain Z23,and its enzyme activity was 2.35±0.12 U/mL.According to morphological and molecular biology identification,strain Z23 was Lactobacillus rhamnosus,the 16Sr RNA gene sequence length was 1473 bp,and the gene registration number was MN582955.2.Through the single factor test and orthogonal test,the extraction conditions of ALP were optimized,and the optimal extraction conditions were determined as follows:cell crushing time 15 min,crushing power 450 W,material-liquid ratio(g/mL)1:6,and extraction pH value 10.0.Under this condition,the enzyme activity was 4.95±0.26 U/mL,which was 2.11 times higher than before optimization.3.Purified enzyme preparation was obtained by ammonium sulfate fractional precipitation,dialysis and desalination concentration,DEAE-52 ion exchange chromatography,Sephadex G-200 gel filtration chromatography,and vacuum freeze drying.The specific enzyme activity was 180.27 U/mg,the purification factor was 48.37,and the enzyme activity recovery was 17.05%.The purity was identified by SDS-PAGE gel electrophoresis,and the molecular weight of ALP was 46.7 kD.The enzymatic properties show that the optimal reaction temperature and pH of ALP were 37? and 9.5 respectively,and the enzyme activity was most stable in the range of 4? and pH 9.0-10.0;Mg2+ and K+are activators,which have an obvious activation effect on ALP.Ba2+ and Cu2+activated ALP when the concentration was 1-3 mmol/L,and showed inhibitory effect when the concentration was higher than 3 mmol/L.Ca2+,Zn2+and EDTA all show strong inhibitory effects on ALP.The Km value of michaelis constant was 3.42 mmol/L,and the maximum reaction rate Vmax value was 1.24 mmol/(L·min).4.The degradation rate of organophosphorus pesticides by ALP was calculated by enzyme inhibition method.The results showed that ALP showed good degradation effect,the whole degradation rate over 85%,and the degradation ability was dichlorvos(95.79 ±0.006%)>methyl parathion(90.69 ± 0.026%)>chlorpyrifos(88.90 ± 0.022%)>trichlorfon(86.07±0.032%)>malathion(85.31 ± 0.019%)>dimethoate(83.18 ± 0.028%),among which the best degradation effect on dichlorvos and methyl parathion,both of which could reach more than 90%.The results of this experiment provide theoretical basis and data support for the screening of ALP-producing strains and the future industrial production and application of ALP. |
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