Optimization,Characteristics And Functions Of Alkaline Phosphatase From Escherichia Coli

Weaning of piglets could increase the risk of getting infected with Gram-negative pathogens,which can furtherly bring about a wide array of virulence factors including the endotoxin lipopolysaccharide(LPS).It is in common practice that the use of antibiotics has been restricted in animal husbandry.Alkaline phosphataseis(AKP)is a non-specific phosphomonoesteras-e,which can affect mammalian intestinal health,including preventing and reducing intestinal inflammation and bacterial translocation,detoxifying local and systemic intestinal of LPS to anti-inflammatory effects.Thus,AKP can be made into an enzyme preparation as a green fodder additive.In this thesis,it was found that the enzyme activity of Pho A from E.coli was increased by about 2.5 times by induction screening.The construction of heterologous expression was used to increase the activity,the test found that compared with E.coli,the activity of Pho A in B.subtilis was increased by 10 times.By genetic modification to remove the signal peptide and site-directed mutation to increase the activity,the test found that the Del Sigpho A is 5 times higher than the Pho A in B.subtilis,the Del Sig D₁₅₃G-D₃₃₀N is 30 times higher than the Del Sigpho A,and the introduction of a stabilizing system into the expression plasmid can significantly improve its stability.50%(v/v)acetone precipitation of AKP protein in the supernatant treated IPEC-J2cells.The effect of AKP on the expression of transporter gene,the Del Sigpho A(p=0.276)and Del Sig D₁₅₃G-D₃₃₀N(p=0.109)has no significant effect on the ASCT2.The effect of AKP on the relative gene expression of tight junction protein,the Del Sigpho A(p=0.002)and Del Sig D₁₅₃G-D₃₃₀N(p<0.001)extremely up-regulated on CLDN 3.The effect of AKP on the expression of cytokine genes,Del Sigpho A showed no effect on IL-6(p=0.863),while Del Sig D₁₅₃G-D₃₃₀N extremely down-regulated on IL-6(p=0.002).Del Sigpho A(p=0.457)and Del Sig D₁₅₃G-D₃₃₀N(p=0.054)showed no significant effect on IL-8.The Del Sigpho A showed no significant effect on TNF-?(p=0.336),and the Del Sig D₁₅₃G-D₃₃₀N has a extremely down-regulation on TNF-?(p=0.005).It can be clearly seen that the Del Sig D₁₅₃G-D₃₃₀N has a beneficial effect on the inhibition of inflammation.By cultivating a large number of AKP engineering bacteria to concentrate enzyme protein as a green feed additive,it can enhance piglet immunity and disease resistance,promote animal growth and development,which provide sustainable development momentum for the pig industry,bring good economic benefits,and improve the quality of life of the people.