Alkaline phosphatase
First Claim
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1. An alkaline phosphatase having the following physical and chemical properties:
- (1) the alkaline phosphatase catalyzes the following reaction;
orthophosphoric acid monoester+H2 O→
alcohol+orthophosphoric acid;
(2) activators selected from the group consisting of Mg++ and Co++ ;
(3) stabilizers selected from the group consisting of Mg++ and Co++ ;
(4) a thermal stability wherein the alkaline phosphatase is stable at least for 30 minutes when treated at pH 7.5 and 60°
C.;
(5) a specific activity of at least 2,300 U/mg;
(6) the alkaline phosphatase has no sugar chain; and
(7) a gel filtration molecular weight from about 140,000 to about 150,000 and a SDS-PAGE molecular weight from about 65,000 to about 67,000.
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Abstract
A novel alkaline phosphatase and a method for the production of the alkaline phosphatase are described. A method for detecting a ligand in a sample and a method for analyzing the sequence of nucleic acid in a sample using the alkaline phosphatase are also described.
8 Citations
4 Claims
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1. An alkaline phosphatase having the following physical and chemical properties:
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(1) the alkaline phosphatase catalyzes the following reaction; orthophosphoric acid monoester+H2 O→
alcohol+orthophosphoric acid;(2) activators selected from the group consisting of Mg++ and Co++ ; (3) stabilizers selected from the group consisting of Mg++ and Co++ ; (4) a thermal stability wherein the alkaline phosphatase is stable at least for 30 minutes when treated at pH 7.5 and 60°
C.;(5) a specific activity of at least 2,300 U/mg; (6) the alkaline phosphatase has no sugar chain; and (7) a gel filtration molecular weight from about 140,000 to about 150,000 and a SDS-PAGE molecular weight from about 65,000 to about 67,000.
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2. An alkaline phosphatase having the following physical and chemical properties:
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(1) the alkaline phosphatase catalyzes the following reaction; orthophosphoric acid monoester+H2 O→
alcohol+orthophosphoric acid;(2) activators selected from the group consisting of Mg++ and Co++ ; (3) stabilizers selected from the group consisting of Mg++ and Co++ ; (4) a thermal stability not higher than 60°
C. at pH 7.5 for 30 minutes;(5) an optimum temperature not lower than 60°
C.;(6) a stable pH wherein the pH is from about pH 6 to about 9 at 25°
C. for 16 hours;(7) an optimum pH wherein the pH is from about pH 9 to about 10; (8) a specific activity of at least 2,300 U/mg; (9) no sugar chain in the alkaline phosphatase; (10) a Km value of 0.34 mM (to p-nitrophenyl phosphoric acid); (11) a gel filtration molecular weight from about 140,000 to about 150,000 and a SDS-PAGE molecular weight from about 65,000 to about 67,000; and (12) a substrate specificity wherein the alkaline phosphatase acts on substrates selected from the group consisting of p-nitrophenyl phosphate, 4-methyllumbelliferone phosphate, NADP, DL-α
glycerophosphate, β
-glycerophosphate, phenylphosphate, phosphoethanolamine and glucose-6-phosphate.
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3. An alkaline phosphatase having the following physical and chemical properties:
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(1) the alkaline phosphatase catalyzes the following reaction; orthophosphoric acid monoester+H2 O→
alcohol+orthophosphoric acid;(2) activators selected from the group consisting of Mg++ and Co.sup. 30 +; (3) stabilizers selected-from the group consisting of Mg++ and Co++ ; (4) a thermal stability not higher than 60°
C. at pH 7.5 for 30 minutes;(5) an optimum temperature not lower than 60°
C.;(6) a stable pH wherein the pH is between pH 6-9 at 25°
C. for 16 hours;(7) an optimum pH wherein the pH is between pH 9-10; (8) a specific activity of at least 2,300 U/mg; (9) no sugar chain in the alkaline phosphatase; (10) a Km value of 0.26 mM (to p-nitrophenyl phosphoric acid); (11) a gel filtration molecular weight from about 140,000 to about 150,000 and a SDS-PAGE molecular weight from about 65,000 to about 67,000; and (12) a substrate specificity wherein the alkaline phosphatase acts on a substrate selected from the group consisting of p-nitrophenyl phosphate, 4-methyllumbelliferone phosphate, NADP, DL-α
glycerophosphate, β
-glycerophosphate, phenylphosphate, phosphoethanolamine and glucose-6-phosphate.
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4. An alkaline phosphatase having the following physical and chemical properties:
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(1) the alkaline phosphatase catalyzes the following reaction; orthophosphoric acid monoester+H2 O→
alcohol+orthophosphoric acid;(2) activators selected from the group consisting of Mg++ and Co++ ; (3) stabilizers selected from the group consisting of Mg++ and Co++ ; (4) a thermal stability not higher than 70°
C. at pH 7.5 for 30 minutes;(5) an optimum temperature not lower than 60°
C.;(6) a stable pH wherein the pH is from about pH 6 to about 11; (7) an optimum pH wherein the pH is from about pH 9.5 to about 10; (8) a specific activity of at least 2,300 U/mg; (9) no sugar chain in the alkaline phosphatase; (10) a Km value of 0.28 mM (to p-nitrophenyl phosphoric acid); (11) a gel filtration molecular weight from about 140,000 to about 150,000 and SDS-PAGE molecular weight from about 65,000 to about 67,000; and (12) a substrate specificity wherein the alkaline phosphatase acts on a substrate selected from the group consisting of p-nitrophenyl phosphate, 4-methyllumbelliferone phosphate, NADP, DL-α
glycerophosphate, β
-glycerophosphate, phenylphosphate, phosphoethanolamine glucose-1-phosphate and glucose-6-phosphate.
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Specification