Fermentative method for determining concentration of antibiotic with beta-lactam ring

专利摘要:
Procédé pour la détermination d'antibiotiques à noyau bêta-lactame dans un liquide biologique. Ce procédé comporte les stades suivants: 1) l'incubation du liquide biologique avec la D-alanyl-D-alanine-carboxypeptidase soluble produite par la souche Actinomadura R39; 2) l'incubation du produit obtenu à la fin du stade 1) avec une solution de substrat; 3) la détermination de la quantité de D-alanine formée au stade 2); 4) la comparaison de la détermination du stade 3) avec un étalon; l'enzyme mise en oeuvre étant immobilisée sur un support insoluble dans l'eau séparée du liquide biologique (stade 1) et lavée avant d'être incubée au stade 2). L'invention concerne également une trousse d'essais comprenant les réactifs à mettre en oeuvre pour l'exécution du procédé.
公开号:SU1313353A3
申请号:SU833546754
申请日:1983-01-28
公开日:1987-05-23
发明作者:Дежелаен Жак；Лоффе Альбер；Дюрье Жан-Пьер
申请人:Юцб С.А. (Фирма)；
IPC主号:

专利说明:

113
The invention relates to medicine and can be used to determine the presence of antibiotics in biological fluids and its concentration in fermentation processes,
The aim of the invention is to increase the sensitivity of the method due to the use of the immobilized enzyme.
Example I. Determination of penicillin G concentration in milk „
The poly- (N, N-dimethylacrylamide) resin used as a substrate is obtained by copolymerization carried out in an emution, a mixture of 12 g of S, K-dimethylacrylamide, 1 g of H, H-ethylene bisacrylamide, and 1.4 g of methyl ester N -acryloyl sarcosine 12-13 g of resin, having the form of pearls, are obtained, the particle size of which is 0.1-2 mm.
10 g of resin are introduced into 320 ml of ethylene diamine, and the mixture is stirred overnight at ambient temperature. The resin is then filtered and washed successively with N, H-dimethylformamide and water until the neutral pH is established.
The resin is then washed in methanol and left to eliminate swelling in the presence of diethyl ether.
Get 10 g of resin containing 0.3 mol of ethylenediamine per 1 g
6.7 g of glutaric acid and 12.7 g of N-oxysuccinimide are dissolved in 50 ml of N, N-dimethylacetamide at O C. Then the solution of dicyclohexylcarbodiimide (23.7 g) in dichloromethane (50 ml ); The mixture is left until the temperature of the solution is equal to the ambient temperature, and then after standing overnight, the reaction mixture is filtered and the filtrate is evaporated. The residue is crystallized in diethyl ether. The diester of oxysuccinimide glutaric acid is obtained at a quasi-quantitative yield.
500 mg of a poly- (H51 1-dimethyl-acrylamide) resin containing functional groups derived from ethylene diamine (step q) are suspended in 50 r-yi H, L-dimethylacetamide. Then 2 g of N-OXCM-d-ester of glutaric acid diester are introduced into the mixture (step S). Stir mixture 32
Warm for 24 hours at ambient temperature. The resin is filtered off and washed 5 times in 100 ml of N5N-dimethyl acetate-bank and then in 100 ml.
methanol, the resin is left to eliminate swelling in the presence of diethyl ether.
600 mg of purified enzyme R 39 with a specific activity of 19.8 units / mg
protein (1 unit of enzyme R 39 catalyzes the hydrolysis of 1 mmol of N-alpha, L-epsilon-diacetyl-b-lysyl-B-alanyl-β-D-alanine in 1 m at 377 s during incubation of the enzyme with a solution of 8 mM substrate in the buffer Tris-HCl 0.03 M solution (pH 7.5) containing 3 mM MgCl1) is dissolved in 1 ml of Tris-HCl O. buffer solution, 1 M (pH /, 7) containing 0.2 M NaCl and MgCl j 0.05 M, and dialyzed for 6 h against 200 ml of H. 0.1 M buffer solution (pH 7.7) containing 0.1 M NaCl and MgCl, 0.05 M. Repeat
ryut 4 times.
The 100 mg g1 ol (K, N-dimethylacrylamide) resin obtained in step 6 is allowed to swell to 50 mp K, H-di methyl methyeth – e € yes. Fri; - after about 3 hours, particles reach a point of maximum swelling, and the u-filter is filtered and scrapped five times. put in 100 ml of buffer solution K dreams 0.1 M (pH 7.7), and then wash in 100 ml of buffer O Hepes solution, 1 M
(pH 7.7) 5 containing NaCl O, 1 M and KgCl j 0.05 M.
The resulting wet resin is placed in a flask with a volume of 10 MJ, 70 µg of the R 39 enzyme dissolved in 1.75 ml of a Hepes 0.1 M solution containing 0.1 M NaCl and 0.05 M MgCl are added. Then the flask is rotated at a rate of 100 vol, for 16 h at a temperature surrounding. th Wednesday.
The resin is filtered off, washed five times with 10 ml of Hepes 0.1 M buffer solution (pEf 7.7) containing 0.1 M NaCl and 0.05 M MgCl. Then the resin is removed. Vacuum is applied under vacuum B for 30 minutes.
503 mg of resin is obtained. The enzymatic activity of the enzyme R 39, immobilized tea resin, corresponds to 15 μg. Yield or production, immobilization attribute is 21% "
Prepare a substrate solution containing 6 mg N-alpha, L-e (1CH.; 7on - diacetyl-L-lysine-0-al nl-C-al: .ncna or
K-alpha-acetyl-b-lysyl-b-alanyl-b-alanine in 1 ml of water.
A suspension is prepared containing 5 mg of D-amino acid oxidase (specific activity of 15 units, / mg) in 1 ml of an aqueous three molar solution of ammonium sulfate.
Preparing a solution of Flavia-adenine-dinucleotide (FAD) containing 500 µg of FAD in 6 ml of Tris-HCl 0.1 M buffer solution (pH 8.0)
Prepare a solution containing 50 μg peroxidase in 1 ml of water and a solution containing 2.6 mg of o-dianisidine dichlorohydrate in 500 μl of water
The determination is carried out as follows.
Prepare a series of 1 ml samples of milk containing a known concentration of penicillin G, as well as two samples (white and comparative) without penicillin G. To each sample add mg of immobilized ft 39 enzyme and incubate the mixture for 20 min at 37 ° s After incubation, the milk is separated from the fixed enzyme, washed three times in 1 ml of Hepes O buffer, 1 M (pH 7.7), containing 0.1 M NaCl and 0.05 M MgCl. Then, 20 μl of Hepes buffer 0.1 M (pH 7.7) containing 0.1 M NaCl and 0.05 M MgCl j and 10 μl of the substrate solution (dp of the comparative sample and samples containing penicillin G) are added to the enzyme or 20 μl of Hepes buffer solution and 10 μl of water (for a white sample). Incubate the mixture for 30 min at 37 ° C. During the incubation process, add 2 µl of the suspension to the mixture. D-amino acid oxidase, 60 μl of FAD solution. 1.0 μl peroxidase solution and 5 μl o-dianisidine solution. Then the mixture is additionally incubated for 10 minutes at 37 ° C.
The optical density of the resulting colored solutions was measured on a spectrophotometer. This is injected with 1 ml of a methanol-water (1: 1) solution in a cuvette and the optical density is measured at 460 nm.
The optical density value found for the white sample is derived from the values obtained for the comparative sample and all other samples. Then calculate the percentage of residual enzyme activity for each sample, the outcome
from the values of optical density obtained earlier.
The results of determining the concentration of penicillin G in the milk of two series of samples are given in Table 1.
The results obtained are reproduced in the form of a graph, on which axis the concentrations of penicillin in units of units / ml are plotted, and on the ordinate axis - percentage. Residual enzyme activity is observed. "This graph serves as a reference in subsequent determinations of the concentration of the antibiotic in milk.
This method determines the concentration of the antibiotic in the order of 0.0005 units, active / ml of milk (in a known way - 0.01 - 0.001 units of act. / Ml of the test liquid).
Example 2 The concentration of penicillin G in serum or urine is determined. The test was carried out as in Example 1, using 1 ml of serum or urine.
The results of determining the concentration of penicillin G in serum are presented in Table 2.
The results of determining the concentration of penicillin G in the urine are presented in Table 3.
When using serum and urine, the method allows to determine the concentration of penicillin G 0.003 units of act, / ml.
The known method does not allow detection of penicillin in the urine.
PRI and MER 3. The experiment was carried out similarly to examples 1 and 2, but the concentration of cephalosporin C was determined in serum.
The results of determination of the concentration of cephalosporin C in serum are given in table 4.
Example 4. Determination of the concentration of cephalosporin C in serum at high temperature.
The experiment was carried out under conditions similar to Example 3, but the incubation was carried out at a temperature of .47 G and the incubation time was halved. To each serum sample with a volume of 1 ml, 100 µl of Hepes 0.5 M buffer solution (pH 7.7) containing 0.5 M NaCl and MgCl 0525 M are added previously. The results of the determination are shown in Table 5.
From the experience it follows that the method can be carried out with increased
five
temperature, accelerated determination time.

权利要求:
Claims (1)
[1]
Invention Formula
Enzymatic method for determining the concentration of an antibiotic with a beta-lactam ring in a biological fluid by incubating the test sample with B-alanyl-B-alanine-carboxypeptine produced by Actino-madura R 39, followed by incubating the obtained antibiotic complex with the enzyme with a solution substrate N-alpha, N-epsilon-diacetyl-B-lysyl-B-alanyl-B-alanine or N-alpha-acetyl-b-lysyl-B-apanyl-B-ala13353 5
nina, followed by determining the amount of B-alanine formed, the concentration of which determines the residual enzymatic activity,
5 and determining the amount of the antibiotic from the difference in the concentration of the enzyme compared to the control, characterized in that, in order to increase the sensitivity of the method,
JO incubation of the sample is carried out with an enzyme immobilized covalently on a poly (H, K-dimethylacrylamide) resin of a network structure, before incubating the substrate solution
15 the complex of the antibiotic with the enzyme is separated and washed with a buffer solution with a pH of 7j7
Table 1
Yellow yellow
Zhepto Brown Brown 11
GB is yellow
Light yellow
Yellow
Tan brown
100 Brown
S.V e tl o-yellow ThiH
Table 2
Table3
TableA
O. Light yellow
8 44 Yellow brown
100 Brown
-Light yellow
10 2 1
Oh oh
0.040 0.035 0.095
Oh, 180 0.035
Compiled by G.Smirnova Editor N. Lazarenko. Tehred M. Khodanych Proofreader E. Roshko
Order 1984/59 Circulation 500 Subscription
VNIIGSH State Committee of the USSR
for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5
Production and printing company, Uzhgorod, Projecto st., 4
Table
ABOUT
ABOUT
41
Light yellow
Tan brown
100 brown
- Light yellow

类似技术:

---

公开号 | 公开日 | 专利标题

---

US3907644A|1975-09-23|Creatinine amidohydrolase composition and process for the determination of creatinine

---

US4371612A|1983-02-01|Immobilization of biological material with an acrylonitrile polymer

---

CA1149712A|1983-07-12|Method and reagent for the determination of glycerine

---

SU1313353A3|1987-05-23|Fermentative method for determining concentration of antibiotic with beta-lactam ring

---

EP0550450B1|2003-05-14|Use of crosslinked crystals as a novel form of enzyme immobilization

---

CA2007885A1|1990-07-17|Beta-lactamase assay employing chromogenic precipitating substrates

---

US4543325A|1985-09-24|Process and reagent for the determination of creatinine

---

SU515471A3|1976-05-25|Method for quantitative determination of hydrolytic enzymes

---

CA1245587A|1988-11-29|Stabilization of enzymes useful in the production ofglucosone and other enzymatic processes

---

EP0019937A1|1980-12-10|A method for producing alcohol oxidase, alcohol oxidase, enzyme preparation and a method for determining the concentration of a compound in an alcohol-containing sample

---

EP0169920B1|1989-02-01|Process for thermally stable alpha-amylase production

---

US4845029A|1989-07-04|Method for the determination of sarcosine creatine or creatinine

---

FR2509324A1|1983-01-14|SUBSTRATE FOR DOSING A-GLUTAMYL TRANSPEPTIDASE AND DOSING METHOD USING THE SAME

---

US5273885A|1993-12-28|Conjugates of monophenyl thyroid analogs useful in assays

---

US5246830A|1993-09-21|Enzymatic process for the determination of β-lactam antibiotics

---

Bódalo‐Santoyo et al.1999|Production of optically pure L‐valine in fluidized and packed bed reactors with immobilized L‐aminoacylase

---

Kiba et al.1995|Determination of branched-chain L-amino acids by flow-injection analysis with co-immobilized leucine dehydrogenase/NADH oxidase and chemiluminescence detection

---

Sundhagul et al.1966|Effect of tryptophan on growth and morphology of Hansenula schneggii cells

---

SU1059002A1|1983-12-07|Method for immobilizing l-lysine decarboxylase

---

JP3795972B2|2006-07-12|Thermostable D-amino acid oxidase, process for producing the same, and microorganism

---

Hinkson1962|CRYSTALLINE YEAST L |-LACTATE DEHYDROGENASE, SOME OF ITS CHEMICAL, PHYSICAL AND ENZYMIC PROPERTIES

---

JP4068722B2|2008-03-26|D-amino acid oxidase derived from the genus Gibberella

---

Katchalski‐Katzir1989|Preparation, properties and uses of polymer‐enzyme conjugates

---

SU1333712A1|1987-08-30|Method of producing reagent for quantitative determination of glucose

---

SU799318A1|1982-03-30|Method for preparing gamma-aminobutyric acid

同族专利:

---

公开号 | 公开日

---

CA1185881A|1985-04-23|

---

IE830185L|1983-08-01|

---

JPH0376919B2|1991-12-06|

---

PL144340B1|1988-05-31|

---

IE53882B1|1989-03-29|

---

EP0085667A3|1983-08-17|

---

DK161205B|1991-06-10|

---

DK161205C|1991-11-25|

---

DK29883D0|1983-01-26|

---

EP0085667A2|1983-08-10|

---

AT19654T|1986-05-15|

---

AU559670B2|1987-03-19|

---

DE3363319D1|1986-06-12|

---

DK29883A|1983-08-02|

---

JPS58138397A|1983-08-17|

---

AU1096483A|1983-08-11|

---

US4546076A|1985-10-08|

---

EP0085667B1|1986-05-07|

---

PL240339A1|1983-08-15|

---

BG42679A3|1988-01-15|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

IL46178A|1974-12-03|1978-10-31|Rehovot Res Prod|Method for the performance of enzymatic reactions|

---

JPS5650833B2|1977-11-05|1981-12-01|US4806478A|1984-10-17|1989-02-21|Minnesota Mining And Manufacturing Company|Dry enzyme formulations containing D-amino acid oxidase|

---

US4686182A|1984-10-17|1987-08-11|Minnesota Mining And Manufacturing Company|Method and kit for detecting antibiotics in a liquid sample|

---

GB9009692D0|1990-04-30|1990-06-20|Ucb Bioproducts|An enzymatic method of determining beta-lactam ring antibiotics|

---

US5263345A|1992-09-24|1993-11-23|Nikola Zagorac|Anti-theft device for a motor vehicle|

---

EP1023603B1|1997-10-07|2005-06-01|Ucb S.A.|Test device and method for determining analytes in a liquid dairy product|

---

US6143513A|1999-06-23|2000-11-07|Biacore Ab|Method and kit for detecting betalactam-containing compounds|

---

CA2466497A1|2001-11-08|2003-05-15|Tibotec Pharmaceuticals Ltd.|Protease assay for therapeutic drug monitoring|

---

EP2766735B1|2011-10-14|2017-02-01|Universite De Liege|Method for measuring beta-lactam antibiotics|

法律状态:

优先权:

---

申请号 | 申请日 | 专利标题

---

GB8202790|1982-02-01|

---