Isolation, Purification, Some Properties And Immobilization Study Of Alkaline Phosphatase From Duck

Alkaline phosphatase(ALP,EC3.1.3.1)is widely existing in microorganism and animal kingdom.In the alkaline conditions,it can catalyze almost all the phosphate monoester hydrolysis, to product inorganic phosphate,and corresponding alcohol phenol or carbohydrate.And also can catalysis the reaction of phosphate group mass metastasis.This enzyme play an important part in the metabolish of phosphate.It is the important metabolic enzymes of duck.And closely related to the growth and development of duck.The alkaline phosphatase has been isolated and purified from the duck liver,by means of homogenation,n-butanol disposal,ammonium sulfate precipitation,ion-exchange chromatography on DEAE-Sepharose Fast Flow column and gel filtration chromatography on Sephacryl S-200.The preparation was shown to be homogenous on polyacrylamide gel electrophoresis.The specific activity was 337.4 uint/mg protein,the purification fold was 58.3.Sephacryl S-200 chromatography and SDS-PAGE showed that the molecular weight of this ALP is 121.0kD,and the molecular weight of subunit was 60.5kD.With phenylphosphoric acid disodium salt being the substrate of alkaline phosphatase,its optimum pH and temperature is 9.0 and 35℃respectively.Na⁺,K⁺ and Li⁺ had not any effect on the enzyme activity.Mg²⁺,Ba²⁺activated the enzyme.Cd²⁺inhibited the enzyme.The Michaelis constant(Km)is 1.28 mmol/L.The alkaline phosphatase was selectively modified by PMSF,NBS,DTT,PCMB,BrAc,IAc,SUAN,formaldehyde,BD and chloramines-t,and changes in the activities of the enzyme have been detected.The results showed that NBS,SUAN,DTT can strongly inhabit the enzyme activities which decreased with the increase of modifier concentration.The formaldehyde and chloramines-t was found to have little inhabition effect on ALP activity.PMSF,PCMB,BrAc,IAc,BD were found without any inhabition effect.All of these suggested that Trp and Lys residues should considered as indispensable groups of ALP,and partial disulfide bonds as essential for the function of the enzyme. Preparation of immobilized enzyme by polyvinyl alcohol and na alginate agglutination.Through single factor and orthogonal experiments L₉(3⁴),four factors were selected,including concentrations of polyvinyl alcohol,CaCl₂ and amount of enzyme,and immobilized time,and employed to test their effects on the yield of immobilized enzyme.In the experiments,we compared the results of properties of the native and immobilized ALP,including the optimum pH and temperature,repeatedly use,stability storage and so on.The result showed that:The effects of polyvinyl alcohol concentration on the yield of immobilized enzyme were the most notable,and amount of enzyme were notable.Both CaCl₂ and immobilized time had inapparent effects on the yield of immobilized enzyme.The optimized conditions of preparing immobilized ALP by polyvinyl alcohol and na alginate agglutination were 15%of polyvinyl alcohol,2.5%CaCl₂,the amount of carrier and enzyme were 1:1 and 1h immobilized time,the optimum pH of immobilized ALP was 10.5 and temperature was 45℃,repeatedly use and stability storage have increased greatly.