Purification And Characterization Of Alkaline Phosphatase From Ruditapes Philippinarum

Alkaline phosphatase （ALP） is an important regulator enzyme in livingorganisms which could catalyze the hydrolytic and transfer reactions of phosphategroups. ALP extensively participates in the absorption of glucose, lipid, inorganicphosphorus and calcium in fishes. And it plays an important role in the maintenanceof the calcium-phosphorus ration and the formation of the shellfishes’shells. The ALPafter purification has now been widely applied in enzyme immunoassay and geneengineering areas. The ALP now used is mainly separated from colon bacillus or calfintestine and it has some limitations. For example, the ALP from colon bacillusdoesn’t have enzyme activity at the temperature of25℃as the optimum temperatureof colon bacillus is80℃and the ALP from calf intestine is difficult to be heatinactivated in the process of cloning and amplification of DNA. Therefore, new-styleALP from new sources is now widely studied.In consideration of the unique living environment of marine organisms, they arenow gradually become the important source of new-style active materials. In thisresearch, the ALP is separated from Ruditapes philippinarum which is one kind ofmarine mollusk and the purification technique is optimized. The enzymatic propertiesof ALP is also studied. The results are as follows:1. The ALP in the visceral mass of Ruditapes philippinarum has the highestenzymatic activity followed successively by gill, foot and pallium. The ALP invisceral mass has the highest enzymatic activity in spring and actually the differencesamong four seasons are not significant.2. The ALP is separated from the visceral mass of Ruditapes philippinarum bysalt precipitation with ammonium sulphate, DEAE-Sepharose Fast Flow ion-exchangechromatography and gel filtration chromatography with Sephadex G-75gel column.The purified factor is14.42and the specific activity is131.08U/mg. The result ofSDS-PAGE is a single band and the molecular weight of subunit is about44kDa. 3. The optimum temperature of ALP from Ruditapes philippinarum is45℃andthe optimum pH is9.0. It has high enzymatic activity at the temperature of25℃and italso has good stability in the pH range of7-11. By use of the Lineweaver Burkmethod with the zymolyte of PNPP, the dynamical parameter of ALP could beobtained as follows: K_m=0.098mmol/L，V_max=1.01μmol/（mL·min）.4. The effects of metallic ions on the enzymatic activity of ALP is studied and theresult indicates that metallic ions including Na⁺, K⁺, Ca²⁺and Mg²⁺could activate theALP in various degrees. Ca²⁺and Mg²⁺could magnificently activate the ALP. Mn²⁺and Fe³⁺could activate the ALP when they at low concentrations, but they showinhibited effects on the ALP with the increasing of the concentrations. In addition,Ba²⁺and Cu²⁺could also inhibit the activity of ALP.5. The effects of organic solvent on the enzymatic activity of ALP is studied andthe result shows that methanol, ethanol and isopropyl alcohol all have strong inhibitedeffects on the activity of ALP and the inhibited effects become stronger with increaseof the concentrations of the organic solvents. When the organic solvents are at theconcentration of40%, the enzymatic activity of each group treated with differentorganic solvent is5.23%,3.13%and2.09%, respectively. The result indicates that theinhibited effects of organic solvent on the enzymatic activity of ALP become strongerwith increase of the nonpolarity.6. Chemical modification is carried out on the functional groups of ALP fromRuditapes philippinarum and the result show that histidine and disulfide bond areessential functional groups while serine, lysine, tryptophan and sulfhydryl are not.