METHOD FOR THE PURIFICATION OF UREASE AND GLUTAMATE DEHYDROGENASE

Abstract: A urea assay mixture, particularly for use in the determi­ nation of urea in physiological fluids such as blood, which comprises urease and glutamate dehydrogenase enzymes from the same micro-organism source together with cofac­ tors. The application also discloses a process for the production of the mixture, a method for the purification together of the enzymes urease and glutamate dehydroge­ nase and a method for testing for urea in a fluid using the assay mixture.

Description: Urea assay mixture and Process for the production thereof This invention relates to a urea assay mixture particularly for determining urea in physiological fluids such as blood, to a process for the production of the mixture, to a method for the purification of enzymes which can be used in the process for the production of the mixture and to a method of testing physiological fluids and particularly blood serum using the mixture. A test for urea in clinical blood samples which is widely used in hospital pathological laboratories relies upon the two enzymes glutamate dehgdrogenase (ODE) and urease. The test is carried out using an assay mixture containing the two enzymes and cofactors WAD(P)H (i.e. the reduced forts of nicotinamide adenine dinucleotide (phosphate) - the cofactor of the GIE enzyme) and o- ketoglutarate in a suitable buffered medium. In assay mixtures which are commercially available at the present time the two enzymes have been derived from different sources, i.e. urease from Jack bean meal and GDH from bovine liver, and have been separately purified from those sources. According to the present invention we provide a urea assay mixture comprising urease and glutamate dehydrogenase (G13H) enzymes from the same micro-organism source and cofactors, for the reactions whereby these enzymes convert urea into glutamate. Further according to the present invention we provide a process for the production of a urea assay mixture which com- prises the steps of purifying the enzymes urease and glutamate dehydrogenase (GDH) from the same micro-organism source and admixing the purified enzymes with cofactors, for the reactions whereby the enzymes convert urea into glutamate. Further according to the invention we provide a method for the purification together of the enzymes urease and glutamate dehydrogenase (GDH) from the same micro-organism source which comprises a step (A) in which a solution containing the enzymes is contacted with ammonium sulphate under conditions such that a precipitate containing both enzymes is produced followed by a step (3) in which the precipitate is re-dissolved, the resulting solution is applied to an ion exchange material, suitably a dextran, and a liquid fraction containing the enzymes is collected. Further according to the present invention we provide a method for testing for urea in a fluid wherein a sample of urea containing fluid is contacted with an assay mixture comprising urease and glutamate dehydrogenase (ode) enzymes from the same micro-organism source and cofactors, for the reaction whereby these enzymes convert urea into glutamate, in an appropriate buffered medium. Fu:+vher according to the present invention we provide an enzyme component of a urea assay mixture which comprises urease and glutamate dehydrogenase (ODE) enzymes extracted from the sase micro-organism source. Further according to the present invention we provide a process for the production of an enzyme component of a urea assay mixture which comprises the step of purifying the enzymes urease and glutamate dehydrogenase (GDE) from the same micro-organism source. The assay mixture is useful for determining urea in physiological fluids and in particular for determining blood urea. The reaction mechanism by which the assay mixture converts urea from e.g, blood into glutamate involves two reactions. In a first reaction, which is catalysed by the enzyme urease, urea is converted into carbon dioxide and ammonia. In a second reaction, which is catalysed by the GDE enzyme, the ammonia produced by the first reaction and q-Retoglutarate are converted into glutamate. During this second reaction cofactor MAD(P)H is converted into NAD(P) (i.e. the cofactor in its oxidised form). This conversion of NAD(P)H to NAI)(P) provides a means by which the extent of the reaction and hence the amount of urea in a blood sample can be determined by 1W spectrophotometry. NAD(P)H absorbs at 340 nm in the UV whilst NED(P) does not absorb at this wavelength. Hence the urea concentration in e.g. blood is determined from measurements of the decline in the height of the peak at 340 nm in the UV spectrum of a sample under test. The source micro-organism for the enzyme component of the assay mixture may be any micro-organism which contains both enzymes and from which they may be purified. The micro-organism may be one which contains both enzymes naturally or it may be one into which DNA material comprising the gene or genes specifying either or both enzymes has been introduced by a genetic manipul ation technique. Preferably the source micro-organism is a bacterium. Useful bacterial strains are urease-containing strains of the species Methylophilus methylotrophus (formerly names Pseudomonas methylotroDha) which have been genetically modified to include in them the GDH enzyme. Cultures of Methylonhilus methylotrophus strains which could be genetically modified in this way to make them suitable for use as source micro-organisms for the enzyme component of the present invention are described in our BE Patent No. 1370892 and are deposited as follows: NCIB: 10508 - 10515 and 10592 - 10596, all inclusive; NRRL: B 5352 - 3 5364 inclusive; FRI: 1215 - 1227 inclusive. Especially useful are GDH-containing strains derived from one of the above-mentioned Methylophilus methylotronhus strains, strain NCLB 10515, by genetic manipulation, in particular the modified GDH-containing strain NCIB 11585 and variants and mutants thereof. Genetically modified GDE-containing strains of Methylohilus metQylotrophus, in particular strain NCIB 11585 are described in our co-pending European Patent Application No. 81300538. Two advantages of strain NCIB 11585 are (a) the urease and GDH enzymes are readily co-purified and (b) the GDH enzyme is constitutive and the alternative pathway using glutamine synthetase and glutsmine glutamate aminotransferase cannot occur. Note: NGIB is the National Collection of Industrial bacteria, Torrey Research Station, Aberdeen, Scotland, BE; SWIRL is the collection maintained by the US Department of Agriculture, Peoria, Illinois; ml is the Fermentation Research Institute, Japan. When the source micro-organism is a strain of Methylonhilus methylotro1xiis such as NCLB 11585 it is suitably produced by a process such as that described in our UK Patent No. 1370892. In such a process a culture of the micro-organism is produced in a fermented. The process of the present invention for the production of an enzyme component or a blood assay mixture may be carried out as an addition to the process for producing the source micro-organism or the production of the source micro-organism and the purification of the enzyme component from the micro-organism may be performed separately, for instance at different locations Moreover the production of the enzyme component and its admixture with the other components of the assay mixture may be performed together as a two-stage process at the same location or separately at different locations. The extent to which the assay mixture is made up ready for use before being supplied to the user can vary considerably. For instance the enzyme component alone could be supplied, the user then adding everything else. Alternatively the user could be supplied with a mixture fully made up and containing enzymes, cofactors and such other ingredients as stabilisers, which are desirably included, in a buffered medium, i.e. a liquid mixture. Preferably however the user is supplied with an assay mixture which contains the dry ingredients - enzymes, cofactors, buffering materials and other desired ingredients and which on addition to a solvent, usually water, will give the mixture in a suitable buffered medium. The cofactors to be included in he assay mixture are suitably oC-keto glutarate and MAD(P)H. Suitable buffering materials include tris(hydroxymethyl) amino methane, triethanolamine, phosphate, imidazol e, N-2 hydrcxyethylpiperazine-N-2 ethane sulphonic acid (Hepes). Other ingredients include stabilisers, for example albumin (from bovine serum), dithiothreitol or mercapto ethanol. The amounts of the various components of the mixture In typical assay mixtures are in the following ranges: urease : 0.5 to 10 units/ml 2 : 2 to 40 units/ml t-ketoglutarate : 0.2 to 10 mM NAD(P)H : 0.05 to 0.5 mM Hepes buffer : 10 to 100 mM - giving a pH in the range 7 - 8. Such a mixture is suitably provided as dried mixture to which water is added to give the concentrations indicated. A preferred mixture has the following composition: urease - 1.6 units/ml; GIR - 10 units/ml; U-ketoglutarate 1.2 mM; AD(P)H - 0.18 zit and Hepes buffer 50 mum to pH 7.5. In performing the method of testing of the invention, the assay mixture in a suitable buffered medium is prepared and a fluid sample eOg. a sample of blood serum is added to it in a conventional manner. Preferably 0.5 to 5 of the sample (or equivalent larger volume of diluted material) are added to 0.5 to 5 ml of buffered medium. Blood serum which will be a common material to be tested is formed by removing cells from blood. After addition of the sample e.g. of serum to the buffered medium spectrophotometeric measurements are made at 340 nm and the urea content of the test sample is determined. Urea in other fluids such as urine or plasma may be similarly determined. The source micro-organism for the enzyme component of the assay mixture is preferably grown continuously under carbon limitation and preferably with urea as a nitrogen source. In ideal circumstances cultivation under nitrogen limitation is preferred but we have found that with 013 11585 at least this results in some loss of GDH activity. Hence for practical purposes at present growth under carbon limitation is preferred. In the production of the enzyme component of the invention from the source micro-organism cells, suspended in water or buffer, are suitably broken, e.g. mechanically using a bead mill, pressare cell or ultrasonics, and the material thus produced centrifuged to remove insoluble matter and to give a soluble fraction containing urease and GDH enzymes This soluble fraction is treated according to steps (A) and (3) of the purification method of the invention. Preferably in step (A) ammonium sulphate is added to the solution of enzymes at dOC to give ) v saturation with ammonium sulphate. This causes a precipitate of protein not containing the urease and GIN enzymes to form. This precipitate is removed by centrifugation. The ammonium sulphate concentration in the solution is then preferably raised to 5 which causes precipitation of both urease and GDH. The precipitate containing urease and GDH can be re-dissolved, de-salted, e.g. by dialysis, diafiltration or gel chromatography, to remove ammoniun sulphate, and treated according to step (3) of the purification method, Preferably in step (3) the enzyme-contsining solution in suitable buffer is applied to a column containing an ion-exchange material, e.g. a column containing a cellulose or other polymer such as dimethyl amino ettyi-Sepharose (Sepharose is a trade name for a product of Phaacia Chemical Co). The enzymes are retained on the column which is then washed with buffer containg an increasing concentration of sodium chloride solution. The urease and GDH enzymes are eluted from the column in the same or closely adJacent fractions at a concentration of sodium chloride between 0.25 and 0.4 M and are collected together. The purification method of the invention provides a simple and effective means of purifying the urease and GDH enzymes and is the preferred purifiation method for the process for the production of the enzyme component or the assay mixture. However other purification methods may be used and the enzymes may be purified either separately or together in the production process. Preferably the enzymes are purified together. The invention is illustrated by the following Example:- EXAMPLE MethyloDhilus methylotronhus strain MCLB 11585 was grown continuously under carbon limitation with -2rea as nitrogen source. Cells obtained from the culture were broken mechanically and the material thus produced was centr fuged to remove insoluble matter and to give a crude extract containing urease and GIN enzymes. The crude extract was dissolved in water and ammonium sulphate was added at 40C to give 30% saturation with -amtnonium sulphate. This caused precipitation of protein not containing urease and GIN enzymes. After centrifuging to remove this precipitate further ammonium sulphate was added to raise the ammonium sulphate concentration to 50% and to cause precipitation of both urease and GIN. The precipitate containing urease and GDH was re-dissolved, desalted and applied to a column containing diethyl amino ethyl (DEAE) Sepharose in 10 mM phosphate buffer pH 7.5. The enzymes were retained -lpon the column which was washed with an increasing concentration of sodium chloride (0 - 0.5 M) in the same buffer and the enzymes eluted between 0.25 and 0.4 M, the eluted fractions containing the two enzymes being collected together. The specific activities of urease and GIN in the material at various stages of this purification, i.e. crude extract, precipitate after 30 - 504/0 ammonium sulphate treatment and after elution from nFAF Sepharose, are given in Table 1 together with the specific activities of the enzymes in a commercial reagent. In Table 1 specific activity is measured in p mol/minute/mg protein. Table 1 EMI8.1 <tb> <SEP> urease <SEP> urease <SEP> specific <SEP> GDH <SEP> specific <tb> <SEP> Material <SEP> activity <SEP> activity <tb> Crude <SEP> extract. <SEP> 0.25 <SEP> 0.45 <tb> <SEP> Precipitate <SEP> after <SEP> (SH4)2S04 <SEP> <tb> <SEP> -eatment. <SEP> 0.96 <SEP> 1.99 <tb> <SEP> Product <SEP> from <SEP> IIEAE <SEP> treatment <SEP> 4.97 <SEP> 14.9 <tb> <SEP> Commercial <SEP> reagent <SEP> 3.21 <SEP> 3.86 <tb> A series of standard urea solutions containing respectively 10, 20, 30, 40 and 50 mg urea per 100 ml solution were made up. The concentrations of urea in these test solutions were then measured using in turn an assay mixture containing the enzyme mixture purified as described above and a commercially available assay mixture. The procedure was to put the assay mixture into a recording spectrophotometer and record absorbance at 340 nm against time obtaining a constant reading. The standard urea solution under test was then added and the reduction in absorbance, due to oxidation of NAD(P)H to NED(P) as the reaction proceeds, was observed. This takes 2 to 5 minutes. The total change in absorbance is proportional to the amount of urea in the standard solution under test and hence the concentration of urea in the sample can be calculated by conventional means. This was done for each of the standard urea solutions using both the assay mixture of the invention and a commercially available assay mixtures The results are given in Table 2. The assay mixture of the invention comprised 200 pl of an enzyme extract prepared as described above and containing 194 g protein, 1.2 mum < -ketoglutarate, 0.18 mM NXD(P)H and 50 mM Hexes buffer, being buffered to pH 7.5. Table 2 EMI9.1 <tb> <SEP> Commercial <SEP> reagent <SEP> Assay <SEP> mixture <SEP> of <tb> <SEP> Conc. <SEP> (1.5 <SEP> mg <SEP> protein) <SEP> invention <tb> <SEP> Urea <tb> <SEP> added <SEP> Concentration <SEP> of <SEP> Response <SEP> Concentration <SEP> of <SEP> ; <SEP> Response <tb> Mg/100 <SEP> ml <SEP> urea <SEP> indicated <SEP> Time <SEP> urea <SEP> indicated <SEP> Time <tb> <SEP> in <SEP> assay <SEP> by <SEP> assay <tb> <SEP> 10 <SEP> 9.7 <SEP> mg/100 <SEP> ml <SEP> 2.0 <SEP> miii <SEP> 10.1 <SEP> mg/l00 <SEP> ml <SEP> 3.0 <SEP> min <tb> <SEP> 20 <SEP> 21 <SEP> mg/100 <SEP> ml <SEP> 3.0 <SEP> miii <SEP> 21 <SEP> mg/l0O <SEP> ml <SEP> 3.0 <SEP> min <tb> <SEP> 30 <SEP> 32 <SEP> mg/100 <SEP> ml <SEP> 3.5 <SEP> min <SEP> 32 <SEP> mg/100 <SEP> ml <SEP> 3.5 <SEP> min <SEP> <tb> <SEP> 40 <SEP> 41 <SEP> mg/500 <SEP> ml <SEP> 3.5 <SEP> min <SEP> 41 <SEP> mg/100 <SEP> ml <SEP> 4.0 <SEP> min; ; <SEP> <tb> <SEP> 50 <SEP> 52 <SEP> mg/100 <SEP> ml <SEP> 4.0 <SEP> min <SEP> 51 <SEP> mg/100 <SEP> ml <SEP> 4.0 <SEP> min <SEP> <tb> As can be seen from Table 2, the results obtained using the assay mixture of the invention are comparable with those obtained using an available commercial reagent. alternatively the concentration of urea may be calculated from the maximum rate of the reaction which is achieved 5 - 10 seconds after adding the urea-containing sample to the reaction mixture described above0 The concentration of urea in an unknown sample is determined by reference to a curve constructed from reaction rates obtained with standard urea solutions. Such a standard curve and the results of tests with standard serum samples are shown in the Figure. The reaction rate is proportional to the mass of urea in the range 0 - 8 pg urea/ml assay volume. The results obtained with standard serum samples were in good agreement with the standard curve. This method has the advantage that, since the maximum reaction rate is achieved only 5 - 10 seconds after mixing, the measurement may be completed in 30 seconds or less so that the procedure is particularly suitable for use with centrifugal reaction rate analysers. In the Figure the different types of points are shown as follows: e : points obtained using urea standards in distilled water; o : points obtained using standard sera produced by the Wellcome Foundation.

Claims: 1. A urea assay mixture comprising urease and glutamate dehydrogenase (sub) enzymes from the same micro-organism source and cofactors, for the reactions whereby these enzymes convert urea into glutamate. 2. A urea assay mixture according to claim 1 wherein the micro-organism source is a urease-containing strain of the species Methylohilus met4slotroDhus which has been genetically modified to include in it the GIR enzyme. 3. A urea assay mixture according to claim 2 wherein the micro-organism source is a strain of Meth,yloDhilus methylotroDhus selected from the group NCIB 10508 - 10515 and 10592 - 10596 which has been genetically modified to include in it the GDH enzyme. 4. A urea assay mixture according to claim 3 wherein the micro-organism source is MethYloDhilus methylotroDhus strain NClB 11585. 5. A urea assay mixture according to any one of the preced- ing claims containing enzymes, cofactors, buffering materials and other desired ingredients in a dry mixture. 6. A urea assay mixture according to any one of the preceding claims containing as a buffering material tris (hydroxymethyl) amino methane, triethanolamine, imidazole, N-2 hydroxyethylpiperazine-N-2 ethane sulphonic acid (repels) and/or a phosphate. 7. A urea assay mixture according to any one of the preceding claims containing as a stabiliser albumin (from bovine serum), dithiothreitol and/or mercapto ethanol. 8. A process for the production of a urea assay mixture which oomprises the steps of purifying the enzymes urease and glutamate dehydrogenase(GDE) from the same micro-organism source and admixing the purified enzymes with cofactors, for the reactions whereby the enzymes convert urea into glutamate. 9. A process for the production of a urea assay mixture according to claim 8 wherein the micro-organism source is a urease-containing strain of the species Methylophilus methylotronhuss which has been genetically modified to include in it the GDH enzyme. 10, A method for the purification together of the enzymes urease and glutamate dehydrogenase (ode) from the same micro-organism source which comprises a step () in which a solution containing the enzymes is contacted with ammonium sulphate under conditions such that a pr-- cipitate containing both enzymes is produced followed by a step (3) in the precipitate is re-dissolved, the resulting solution is applied to an ion exchange material, and a liquid fraction containing the enzymes is collected. 11. A purification method according to claim 10 wherein in step (A) ammonium sulphate is added to the enzyme-containing solution to cause a first precipitate of protein not containing the enzymes to forn, the first precipitate is removed and thereafter the ammonium sulphate concentration in the isolation is raised to cause a second precipitate of protein containing urease and GDH enzymes to form. 12. A purification method according to claim 10 or claim 11 wherein in step (3) the ion exchange material is a dextran. 13. A method for testing for urea in a fluid wnerein a sample of urea-containing fluid is contacted with an assay mixture comprising urease and glutamate dehydrogenase (ODE) enzymes from the same microorganism source and cofactors, for the reaction whereby these enzymes convert urea into glutamate, in an appropriate buffered medium. 14. A method for testing for urea according to claim 13 wherein the fluid is a physiological fluid. 15. A method for testing for urea according to claim 14 wherein the phyiological fluid is blood. 16. A method for testing for urea according to any one of claims 13 to 15 wherein 0.5 to 5 1 of the sample (or equivalent larger volume of diluted material) is added to 0.5 to 5 mls of buffered medium containing the assay mixture and spectrophotometric measurements are made at 340 nm to determine the urea content of the sample. 17. An enzyme component of a urea assay mixture which comprises urease and glutamate dehydrogenase (ODE) enzymes extracted from the same micro-organism source. 18. An enzyme component according to claim 17 wherein the micro-organism source is a urease-containing strain of the species MethvloDhi¯us 3nethylotronhus which has been genetically modified to include in it the GDH enzyme. 19. An enzyme component according to claim 18 wherein the micro-organism source is a strain of Methylonhilus methylotroDhus selected from the group NOIB 10508 - 10515 and 10592 - 10596 which has been genetically modified to include in it the GDH enzyme. 20. An enzyme component according to claim 19 wherein the micro-organism source is Methyloxhilus mettylotronhu;s strain NCIB 11585. 21. A process for the production of an enzyme component of a urea assay mixture which comDrises the step of purifying the enzymes urease and glutamate dehydrogenase (GDH) from the same micro-organism source. 22. A process for the production of an enzyme component according to claim 21 wherein the micro-organism source is a urease-containing strain of the species MethvloDhilus meth,ylotroDhus which has been genetically modified to include in it the GDH enzyme. 23. A process for the production of an enzyme component according to claim 21 or claim 22 wherein cells of the micro-organism source are broken and the material thus produced is centrifuged to remove insoluble matter and to give a soluble fraction containing urease and GDH enzymes. 24. A urea assay mixture comprising urease and glutamate dehydrogenase enzymes from the same micro-organism source substantially as described and as shown in the Example. 25. A process for the production of a urea assay mixture substantially as described and as shown in the Example. 26. A method for the purification together of the enzymes urease and glutamate dehydrogenase substantinlly as described and as shown in the Example. 27. A method for testing for urea in a fluid substantially as described and as shown in the Example.

Inventors: COLLINS, STEPHEN HUGH, SENIOR, PETER JAMES
Applicants: IMPERIAL CHEMICAL INDUSTRIES PLC
Application year: 1982
Main classification: C12Q1
IPC codes: C12N9, C12Q1, C12N15, G01N33