Stabilization of glutamate dehydrogenase in an aqueous solution

Patent metadata

Field	Value
Publication number	US-2016299140-A1
Application number	US-201415038554-A
Country	US
Kind code	A1
Filing date	Dec 8, 2014
Priority date	Dec 10, 2013
Publication date	Oct 13, 2016
Grant date	—

Abstract

A process for stabilizing glutamate dehydrogenase (GDH) from a bacterium of the Clostridium genus, in an aqueous solution, in order to maintain the antigenic properties thereof, includes the step of mixing the glutamate dehydrogenase and a stabilizing composition which is a carboxylic acid having a carbon-based chain of at least 3 carbon atoms and comprising at least 2 —COOH groups, or a salt thereof. GDH compositions thus stabilized and a method of detecting the presence of bacteria of the Clostridium genus are also disclosed.

First claim

1 . A process for stabilizing glutamate dehydrogenase from a bacterium of the Clostridium genus in an aqueous solution in order to maintain the antigenic properties thereof, comprising the step of mixing said glutamate dehydrogenase and a stabilizing compound which is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two —COOH groups, or a salt thereof.

2 . The process as claimed in claim 1 , wherein the carboxylic acid contains a carbon-based chain of 4, 5 or 6 carbon atoms.

3 . The process as claimed in claim 1 , wherein the stabilizing compound is chosen from the following carboxylic acids and salts thereof:
(i) carboxylic acids containing a carbon-based chain of four carbon atoms having at least two —COOH groups and —CX— groups chosen independently from —CH—, —CH 2 — and —C(H)OH—, and (ii) carboxylic acids containing a carbon-based chain of five carbon atoms having at least two —COOH groups and —CX— groups chosen from —CH 2 — and —C(H)NH 2 , and (iii) carboxylic acids containing a carbon-based chain of six carbon atoms having at least two —COOH groups and “CX” groups chosen from —CH 2 —, —C(H)OH— and —C(O)NH 2 .

4 . The process as claimed in claim 1 , wherein the carboxylic acid contains a double bond in its carbon-based chain and is an E isomer.

5 . The process as claimed in claim 1 , wherein the carboxylic acid contains two —COOH groups.

6 . The process as claimed in claim 1 , wherein the stabilizing compound is chosen from fumaric acid, succinic acid, malic acid, glutaric acid, citric acid, tartaric acid, N-(2-acetamido)iminodiacetic acid, glutamic acid, adipic acid, aspartic acid, pimelic acid and malonic acid, and salts thereof.

7 . The process as claimed in claim 6 , wherein the stabilizing compound is chosen from succinic acid and fumaric acid, and salts thereof.

8 . The process as claimed in claim 1 , wherein the glutamate dehydrogenase is an enzyme from the bacterium of the species Clostridium difficile.

9 . An aqueous composition comprising the glutamate dehydrogenase from a bacterium of the Clostridium genus and a stabilizing compound which is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two —COOH groups, or a salt thereof, it being understood that the stabilizing compound is neither glutamate, nor alpha-ketoglutarate, nor citrate, nor glutarate, nor succinate.

10 . An aqueous composition comprising the glutamate dehydrogenase from a bacterium of the Clostridium genus chosen from the species Clostridium difficile, Clostridium botulinum and Clostridium perfringens , and a stabilizing compound which is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two —COOH groups, or a salt thereof, it being understood that the stabilizing compound is neither glutamate, nor alpha-ketoglutarate, nor citrate.

11 . The composition as claimed in claim 9 , wherein the carboxylic acid contains a carbon-based chain of 4, 5 or 6 carbon atoms.

12 . The composition as claimed in claim 9 , wherein the stabilizing compound is chosen from the following carboxylic acids and salts thereof:
(i) carboxylic acids containing a carbon-based chain of four carbon atoms having at least two —COOH groups and —CX— groups independently chosen from ═CH—, —CH 2 — and —C(H)OH—, and (ii) carboxylic acids containing a carbon-based chain of five carbon atoms having at least two —COOH groups and —CX— groups chosen from —CH 2 — and —C(H)NH 2 , and (iii) carboxylic acids containing a carbon-based chain of six carbon atoms having at least two —COOH groups and “CX” groups chosen from —CH 2 —, —C(H)OH— and —C(O)NH 2 .

13 . The composition as claimed in claim 9 , wherein the carboxylic acid contains a double bond in its carbon-based chain and is an E isomer.

14 . The composition as claimed in claim 9 , wherein the carboxylic acid contains two —COOH groups.

15 . The composition as claimed in claim 9 , wherein the stabilizing compound is chosen from fumaric acid, succinic acid, malic acid, glutaric acid, citric acid, tartaric acid, N-(2-acetamido)iminodiacetic acid, glutamic acid, adipic acid, aspartic acid, pimelic acid and malonic acid, and salts thereof, with the exception of citrate, glutamate, succinate and glutarate.

16 . The composition as claimed in claim 10 , wherein the stabilizing compound is chosen from fumaric acid, succinic acid, malic acid, glutaric acid, citric acid, tartaric acid, N-(2-acetamido)iminodiacetic acid, glutamic acid, adipic acid, aspartic acid, pimelic acid and malonic acid, and salts thereof, with the exception of citrate and glutamate.

17 . The composition as claimed in claim 16 , wherein the stabilizing compound is chosen from succinic acid and fumaric acid, and salts thereof, in particular alkali metal salts.

18 . The composition as claimed in claim 9 , wherein the glutamate dehydrogenase is an enzyme from the bacterium of the Clostridium difficile species.

19 . A kit for detecting the presence of a bacterium of the Clostridium genus in a biological sample that may contain such a bacterium, comprising an aqueous composition as stabilized according to the stabilizing process defined in claim 1 , and also the compounds required for carrying out a process for detecting the presence of a bacterium of the Clostridium genus.

20 - 22 . (canceled)

23 . A process for detecting the presence of a bacterium of the Clostridium genus in a biological sample that may contain such a bacterium, comprising the steps of:
(i) carrying out a process for detecting the presence of a bacterium of the Clostridium genus by detecting or quantifying the presence of glutamate dehydrogenase in said sample using an aqueous composition as stabilized according to the stabilizing process defined in claim 1 , and (ii) a positive result in step (i) makes it possible to conclude that the bacterium is present.

24 . A process for detecting the presence of a toxigenic bacterium of the Clostridium genus which produces at least one toxin, in a biological sample that may contain such a bacterium and at least one such toxin, comprising the steps of:
(i) carrying out a process for detecting the presence of a bacterium of the Clostridium genus by detecting or quantifying the presence of glutamate dehydrogenase in said sample using an aqueous composition as stabilized according to the stabilizing process defined in claim 1 , and (ii) a negative result in step (i) makes it possible to conclude that the bacterium is absent, or (ii′) if the process of step (i) is positive, carrying out a process for detecting or quantifying at least one toxin that may be released by said bacterium of the Clostridium genus, in the same biological sample, or in a new biological sample from the same individual, and concluding that the bacterium is toxigenic and produces said at least one toxin if said at least one toxin is present.

25 . The process as claimed in claim 24 , wherein the bacterium is Clostridium difficile and said at least one toxin comprises toxin A, toxin B or both.

Assignees

• Biomerieux Sa
• Biomerieux Sa

Inventors

• Bettsworth Florence
• Martinez Jérôme

Classifications

• C12Q1/04

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• C12Q1/32

  involving dehydrogenase · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• G01N2333/90616

  with a definite EC number (1.4.1.-) · CPC title

• G01N2333/33

  from Clostridium (G) · CPC title