Purification And Properties Of Alkaline Phosphatase From Pearl Oyster Pinctada Fucata

Pine tada martens! i dunker alkaline phosphatase (EC3. 1. 3. 1) is a metalioenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoester. in this present paper, we purified theerizyme from the mantle of Pinctadaiuar~ensii Dunker~ The relative~activity of the enzyme was8723 unit /mg. The kinetics characters of the enzyme have been studied. The optimutntemperature for the enzyme to catalyze the hydrolysis of p-nitrophenylphosphate (pNPP) is at 450C. If the p11 value of the assay system was equal to 9. 72, the enzyme would become the mostactive. The activation energy of the enzyme is 20. 14kj/mol. The Michaelis-Menten constant (K..) of the hydrolysis pNPP catalyzed by the enzyme is 2.86mmol /L and the maximum velocity (Vs) is 9. 09 pinol. L~ miwi1, the initial velocity (V~) of the hydrolysis is 6. 1 1.unol U'o mm'.The product 11P04>and the product-analog W04~ could competitively inhibit the activity of the enzyme, and the inhibitory constants are 0.894 mmol/L and 1.203 mmol/L, respectively. Thepositivemonovalent alkali ions (Li, Na~, KD have no effects on the activity of the enzyme. positive bivalent ions have different effects on the enzyme: Mg>, Ca>, Cci, Mn> could active the enzyme, but Zn>, Cu:, Cd> could inhibi t the enzyme.The eonformational changes of the Pinctada Fucata alkaline phosphatase have been studied in Gu-HCJ, Urea, SDS solutions with different concentrations, respectively. The results showed that increasing the Gu-HC1, Urea or SOS concentrations caused the alkaline phosphátase activity decrease to different degrees.o6mol/L Gu-UCI, 6mo1/L Urea or 50 mtuol/L SOS has been commixed with the enzyme and incubated at 40C for 24 hours, the activity of theenzyme was less than 3%, 28. 1% or 4. 7% of the original activity, respectively. The conformational changes caused by Gu-HC1L, Urea or SDS have been studied by using ultraviolet contrast spectra. The kinetics of inactivation by Gu-I-ICl at low concentrations has been studied using the kinetic method of the sub~trate reaction. Gu-HCJ can irreversibly change the conformation of the enzyme, the inhibition rate constant K1~O. 303 min'~moL1.L.EDTA can combine the metal ions from the molecular of alkaline phosphatase. Commixed EDTA in different concentrations with the enzyme, incubated at 40C for 24 hours, the activity was less than 1% of the original activity. The inactivation type of EDTA has been studied, the results showed that EDTA could competitively inhibit the activity of the enzyme.Bromoacetic acid (BrAc), dithoibisnitrobenzaic acid (DT\B), N-bromosuccinimide (NBS), acetyl acetone, formaldehyde have been used to modify the amino acid residues, respectively. The results showed that the active site of the enzyme have one histidine residue and one tyrosine residue.Dithiothreitol (DTT) can break the bisulfide bonds. When the solution contained different concentrations of DTT, the activity of the enzyme would be inhibited obviously. The left activity was only 7% of the original activity, when the concentration of DTT was up to 0.b25mmol/L in the solution. The inhibition type of DTT on the enzyme was competitive.Some chemical reagents, such as PVP, ATP and so on, were used to treat the little pieces of the mentle. Studied the activity changes of the akaline phosphatase on the little pieces of the mentle. The rsults showed that all the reagents could rise the activity of the enzyme, and if all of the reagents mixed together,the activity rised the highest.