摘要
Alkaline phosphatase(AKP),from the succus entericus of silkworm,was purified using 10%-50%ammonium sulfate fractions,ion exchange chromatography of DEAE-Sepharose,and size exclusion chromatography of Sephacryl S-200.The purification fold was 464 times and specified activity was 3936 U/mg.Optimum pH value of the phosphatase was 10.5,and was stable between pH 7.5 and 11.The optimum temperature of the phosphatase was 40℃ and it was unstable over 50℃.Km value of the phosphatase was 1.25 mmol/L.In a given condition,the phosphatase was selectively modified by PCMB,NBS,PMSF,TNBS,SUAN,DTT,BrAc,and IAc,the results indicate that PMSF,SUA,BrAc,IAc,and TNBS could obviously inhibit the activity of the phosphatase,and the degree of inhibition depended on the concentration of these reagents.There was little effect on the activity of phosphatase after treatment by PMSF,DTT,and NBT.We primarily conclude that mercapto and imidazole are essential for AKP from silkworm.Also,Lys residue and disulfide bands are necessary to protect the catalysis of the AKP.
