Isolation, Purification, Some Properties And Immobilization Study Of Alkaline Phosphatase From The Liver Of Bos Taurus

Alkaline phosphatase was isolated and purified from the cattle(Bos taurus)liver by means of homogeni-zeation,n-butanoldisposal,thermaldenaturation,ultrafilt-ration,DEAE-Sepharose column chromatography and Sephacryl S-200 gel filtration chromatography.Its specificactivity was 19863.50,and the purification fold was 228.74. Its optimum pH and temperature was 10.0 and 45, respectively.Na+,K+and Li+ had no effect on the activity of the enzyme.Mg2+activated the enzyme while Zn2+,Cu2+inhibited its activity.The Km was 1.12mmol/L.The alkaline phosphatase was selectively modified by PCMB,BD,PMSF,DTT,SUAN,NBS,IAc,and changes in the activities of the enzyme have been detected.The results showed that DTT,SUAN,NBS,IAc can strongly inhabit the enzyme activities which decreased with the increase of modifier concentration.While DTT,SUAN,NBS and IAc can selectively modified the disulfide bonds,amino-group, tryptophan and histidine of protein.All of these suggested that amino-group,Try and His should considered as indispensable groups of AKP,and partial disulfide bonds as essential for the function of the enzyme.There were two ways to prepare immobilized enzyme.In the first one, sodium alginate was used as carrier,and in the second one, polyvinyl alcohol and sodium alginate agglutination was used as carrier. Then we optimized the single factor of the two ways and did the orthogonal experiments. through the experiments,we study the properties of immobilized AKP,including the optimum pH and temperature and stability storage.The result showed that:The optimized conditions of preparing immobilized AKP by sodium alginate were 1% of sodium alginate,0.5% CaCl2,the amount of carrier and enzyme were 3:1 and 30 minutes immobilized time.In the orthogonal experiments,the best levels of the factors were that the amount of carrier and enzyme were 3:1,the concentration of CaCl2 was0.2% and 2% of sodium alginate.The highest coefficient of recovery was 80%.The optimum pH of immobilized AKP was 9.5 and temperature was 45℃,stability storage have increased greatly.The optimized conditions of preparing immobilized AKP by polyvinyl alcohol and sodium alginate agglutination were 1% of sodium alginate and 1% of polyvinyl alcohol, 0.5% CaCl2,the amount of carrier and enzyme were 4:1 and 30 minutes immobilized time.In the orthogonal experiments,the best levels of the factors were that the amount of carrier and enzyme were 4:1,the concentration of CaCl2 was 0.2%,1.5% of sodium alginate and 1.5% of polyvinyl alcohol.The highest coefficient of recovery was 95.1%.The optimum pH of immobilized AKP was 10.5 and temperature was 40℃,stability storage have increased greatly.