Screening And Preliminary Application Of Genes For Efficient Bacterial Alkaline Phosphatase

Alkaline phosphatase is widely used as a tool enzyme in many fields,including immunological detection applications.Escherichia coli alkaline phosphatase（EAP）is easy to obtain and operate,and there have been many studies on gene markers.For example,a simple and efficient ELISA method was established after the fusion expression of single-chain antibody and EAP.However,due to the low enzyme activity of EAP,many antigens linked to EAP often fail to meet the detection standard.Therefore,the purpose of this study is to screen a bacterial alkaline phosphatase with high enzyme activity,and to lay a foundation for finding more favorable alkaline phosphatase marker genes for fusion expression.The main research contents are as follows:1.In order to screen bacterial alkaline phosphatase with high enzyme activity,this study used organophosphorus plate to separate bacteria from soil samples with rich organic matter.Enzyme activity of single colony in organophosphorus plate was measured with 4-nitrophenylphosphonate disodium salt（p-NPP）as substrate.The strain with high enzyme activity was selected and its alkaline phosphatase gene was cloned.Finally,two alkaline phosphatases with high enzyme activity were screened:Pho A in Bacillus lysine and Pho D in Bacillus amylatus.The enzymatic properties of purified Pho A and Pho D were studied.The optimum reaction temperature of Pho A was60℃,p H was 10.5,Mg²⁺concentration was 0.5 mmol/L,and Ca²⁺inhibited the activity of Pho A.The optimum reaction temperature of Pho D was 70°C,p H was 9.8,and Ca²⁺concentration was 3 mmol/L.Mg²⁺inhibited the activity of Pho D enzyme.K⁺,Zn²⁺,Mn²⁺and Fe²⁺had no significant effect on Pho D and Pho A activities.Low concentrations of SDS,tratonic X-100,deoxycholate and urea had no significant effect on Pho A,while 0.005%-0.02%SDS and 0.05%-0.1%deoxycholate had a greater effect on Pho D,and the Pho D activity was increased by about one time.Using P-NPP as substrate,the Pho A K_m,V_maxand k_cat values were 1.17 mmol/L,60.86μmol/（L·min）and 339.70 s^-1 at 25°C.The K_m,V_max and k_cat values of Pho D were 5.94 mmol/L,31.46μmol/（L·min）and 103.59 s^-1,respectively.The k_cat values of Pho A and Pho D were 5.2times and 1.6 times of EAP.2.In order to evaluate the application effect of Pho A and Pho D in gene markers,Pho A,Pho D and EAP D153G/D330N mutants（m AP）were fused with the protective antigen Spa A of swine erysipelas,and a double-antigen sandwich ELISA method was established to detect swine erysipelas antibody.Compare the application effect of three alkaline phosphatases.Using Spa A as the coated antigen,square titration test was used to optimize the reaction parameters:the antigen coated amount was 100 ng/well,the serum was diluted 5 times,and the enzyme-conjugated antigen was diluted 2000 times.A double-antigen sandwich ELISA method based on Spa A-m AP was preliminarily established to detect swine erysipelas antibody.The enzyme activity of Pho A and Pho D was not as good as that of m AP.The double-antigen sandwich ELISA method and indirect ELISA method were used to detect 14 negative serums and 17 positive serums at the same time.The coincidence rate of negative and positive serums between the two methods was high.However,due to the insufficient enzyme activity of m AP,the detection value was low,which may lead to false detection of serum with low antibody level.3.Molecular orientation technology has great advantages in modifying enzyme performance.Error-prone PCR was performed for Pho A and Pho D.Finally,two Pho D mutants with elevated enzyme activity were identified by one error-prone PCR round:K224E and R519T.No Pho A mutants with increased enzyme activity were screened.Compared with Pho D,the optimum temperature of K224E and R519T is 70℃.The optimal p H was increased to 10.The optimal Ca²⁺concentration was 1.5 mmol/L and2 mmol/L,respectively.At 37℃,the detection values of K224E and R519T are 1.6times and 1.92 times of Pho D,respectively.At 70℃,the detection values of K224E and R519T are 1.12 times and 1.34 times that of Pho D,respectively.According to the analysis of homology modeling results,R519T may accelerate the release of phosphate group,so the enzyme activity is increased.K224E may indirectly affect the activity of the enzyme by changing the position of the amino acid binding to Ca²⁺.In summary,two alkaline phosphatases with higher enzyme activity than EAP were cloned from environmental microorganisms,Pho A and Pho D,which were isolated from Bacillus amyloliticus and Lysinibacillus fusiformis for the first time.However,due to the low enzyme activity,the application of gene marker antigen could not meet the detection standard.Two Pho D mutants with increased enzyme activity were obtained:K224E and R519T.The results lay the foundation for finding more favorable alkaline phosphatase marker genes for fusion expression.