Patent US4614714A successfully refreshed from Google Patents.
US4614714A
Expired / Lapsed
US
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Use of novel L-glutamic acid oxidase
Assignee: Yamasa Shoyu KK
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Filing: 1983-06-28
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Granted: 1986-09-30
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Expires: 2003-06-28
Abstract
Abstract The present invention consists of an analytical method for assay of L-glutamic acid in a sample by the use of an L-glutamic acid oxidase which is an L-amino acid oxidase catalyzing the oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide, and having a very high substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine, and also having a high stability, a reagent for analysis to practice the analytical method, a kit for analysis comprising the reagent, and a biosensor employing the enzyme.
Inventors
- 👤 Hitoshi Kusakabe
- 👤 Hiroshi Yamauchi
- 👤 Yuichiro Midorikawa
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 11:03
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0012; C12N9/0014; C12N9/0022; C12Q; C12Q1/00; C12Q1/001; C12Q1/005; C12Q1/26; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/817
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
198 words
A method for analysis of L-glutamic acid, which comprises acting L-glutamic acid oxidase with L-glutamic acid in a sample to be analyzed in the presence of oxygen and water, and detecting the consumption of oxygen or the formation of hydrogen peroxide, ammonia or α-ketoglutaric acid accompanying the reaction, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of flavin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 2
Independent Claim
198 words
A method for analysis of L-glutamic acid, which comprises acting L-glutamic acid oxidase with L-glutamic acid in a sample to be analyzed in the presence of oxygen and water, and detecting the consumption of oxygen or the formation of hydrogen peroxide, ammonia or α-ketoglutaric acid accompanying the reaction, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of flavin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 3
Independent Claim
209 words
A method for analysis of L-glutamic acid, which comprises reacting L-glutamic acid oxidase with L-glutamic acid at pH 5-6 in the presence of oxygen and water in a sample to be analyzed containing L-asparatic acid together with L-glutamic acid, and detecting the consumption of oxygen or the formation of hydrogen peroxide, ammonia or a α-ketoglutaric acid accompanying the reaction, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of falvin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 4
Independent Claim
209 words
A method for analysis of L-glutamic acid, which comprises reacting L-glutamic acid oxidase with L-glutamic acid at pH 5-6 in the presence of oxygen and water in a sample to be analyzed containing L-asparatic acid together with L-glutamic acid, and detecting the consumption of oxygen or the formation of hydrogen peroxide, ammonia or a α-ketoglutaric acid accompanying the reaction, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of falvin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 5
Dependent Claim
172 words
A reagent for analysis of L-glutamic acid, comprising an L-glutamic acid oxidase and a buffering agent suitable for use in enzymatic reaction, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of flavin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 6
Dependent Claim
172 words
A reagent for analysis of L-glutamic acid, comprising an L-glutamic acid oxidase and a buffering agent suitable for use in enzymatic reaction, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of flavin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 7
Dependent Claim
166 words
A reagent for analysis of L-glutamic acid, comprising an L-glutamic acid oxidase immobilized on a carrier, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of flavin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 8
Dependent Claim
166 words
A reagent for analysis of L-glutamic acid, comprising an L-glutamic acid oxidase immobilized on a carrier, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of flavin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 9
Dependent Claim
169 words
A kit for analysis of L-glutamic acid, comprising an L-glutamic acid oxidase and a detecting reagent for enyzmatic reaction, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of flavin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 10
Dependent Claim
169 words
A kit for analysis of L-glutamic acid, comprising an L-glutamic acid oxidase and a detecting reagent for enyzmatic reaction, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of flavin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 11
Dependent Claim
22 words
A kit according to claim 5 wherein the detecting reagent is a reagent for detection of hydrogen peroxide, ammonia or α-ketoglutaric acid.
Claim 12
Dependent Claim
28 words
A kit according to claim 5 wherein the detecting reagent is a single color forming agent or a combined reagent of a color forming agent and a coupler.
Claim 13
Dependent Claim
197 words
A biosensor comprising a receptor portion for a substance to be detected comprising an L-glutamic acid oxidase, and a transducer portion which detects chemical or physical change in a sample for analysis by the action of said L-glutamic acid oxidase and transduces it to an electrical signal, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of falvin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 14
Dependent Claim
197 words
A biosensor comprising a receptor portion for a substance to be detected comprising an L-glutamic acid oxidase, and a transducer portion which detects chemical or physical change in a sample for analysis by the action of said L-glutamic acid oxidase and transduces it to an electrical signal, wherein said L-glutamic acid oxidase is an L-amino acid oxidase having activity of oxidative deamination of the α-amino group of L-glutamic acid in the presence of water and oxygen to form α-ketoglutaric acid, ammonia and hydrogen peroxide; having a substrate specificity for L-glutamic acid substantially without acting on L-glutamine and L-histidine; and having the following properties: (a) stable in the pH range from 5.5 to 10.5 under maintenance conditions of 37° C. and 60 minutes; (b) stable in the temperature range of up to 65° C. under maintenance conditions of pH 5.5 and 15 minutes; (c) optimum activity in the pH range from 7 to 8.5; (d) is not inactivated by cupric chloride in a concentration of 1.0 mM at pH 7.4; (e) contains 2 mols of falvin adenine dinucleotide per mol of the oxidase as a coenzyme; and (f) a molecular weight of 135,000±10,000 as estimated by gel filtration.
Claim 15
Dependent Claim
28 words
A biosensor according to claim 8 wherein the transducer portion is an electrode which detects a chemical change in the sample and transduces it to an electrical signal.
Claim 16
Dependent Claim
15 words
A biosensor according to claim 8 wherein the receptor portion comprises immobilized L-glutamic acid oxidase.
References Cited (Backward Citations)
JPS5743685A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-09-29 | 1990-03-31 | 1990-09-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-09-29 | 1994-03-31 | 1994-09-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-09-29 | 1998-03-31 | 1998-09-30 | $7700.00 | Window Closed / Lapsed |