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High-throughput screening method of beta-lactam antibiotic synthetase

A screening method and antibiotic technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of low detection efficiency and narrow application range, and achieve the effect of low cost, simple operation and accurate detection

Active Publication Date: 2014-03-26
NORTH CHINA PHARMA HEBEI HUAMIN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a high-throughput screening method for lactam antibiotic synthetase to solve the problems of low detection efficiency and narrow application range of existing methods

Method used

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  • High-throughput screening method of beta-lactam antibiotic synthetase
  • High-throughput screening method of beta-lactam antibiotic synthetase
  • High-throughput screening method of beta-lactam antibiotic synthetase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The reagent preparation method used in this embodiment is as follows:

[0031] ①LB medium: 1% Trptone; 0.5% Yeast Etract; 1% NaCl;

[0032] ②Lysis Buffer: 10mM-50mM Tris-Hcl (pH 7.2); 1-5% Glycerol; 10mM-50mM NaCl;

[0033] ③KPB solution: configure 0.02-0.2M KH 2 PO 4 and 0.02-0.2M K 2 HPO 4 , adjust the pH value to 5.0-7.0;

[0034] ④PDAB chromogenic solution: Weigh 0.4g-1.8g PDAB, add 20mL methanol and 44mL pure water to make solution I; add 16mL glacial acetic acid to 60mL pure water, add 0.08g NaOH to dissolve to prepare solution II; mix solution I and solution II, avoid Store at light 4°C;

[0035] ⑤ Synthetic solution: Weigh 30-100mg 6-APA, 7-ADCA or 7-ACCA β-lactam antibiotic nucleus and 40-120mg HPGME·HCL or PGME·HCL and other synthetic side chains and dissolve them in 10mL KPB solution.

[0036] In this embodiment, Clone1-12 in the library are screened according to the following groupings:

[0037]

[0038] The screening method is as follows:

[003...

Embodiment 2

[0047] Embodiment 1 While carrying out enzyme label determination, take the reaction solution obtained in step 2.1), and use the HPLC method to detect the concentration of mother nuclei in the reaction solution. The detection conditions of different mother nuclei are as follows:

[0048] (1) 6-APA concentration HPLC detection method:

[0049] Chromatographic conditions: high performance liquid chromatography Agelent LC 1200, SB-C18 column, 150*4.6*5, plus SB-C18 pre-column. The column temperature is 35°C. Mobile phase A: 30% acetonitrile, mobile phase B: 50Mm NaH 2 PO 4 , PH=5, 0-2min, A:B=2:98; 2-10min, A:B=10:90. Flow rate is 1mL / min, detection wavelength: 210nm. Injection: 20 μL.

[0050] Sample preparation: take 500 μL of the reaction solution, add water to dilute it into a 25mL volumetric flask, then take 500 μL into a 25mL volumetric flask, add mobile phase B to dilute to the mark, filter through a 0.45μm filter membrane, take the filtrate and inject a sample, and de...

Embodiment 3

[0058] (1) Comparison of absorbance data and concentration data obtained in Example 1 and Example 2 with 6-APA as the reaction core:

[0059] Example 1 Screening with 6-APA as the reaction mother nucleogenesis enzyme results (6-APA absorbance value change) are shown in Table 1 and figure 1 :

[0060] Table 1: Changes in absorbance value of 6-APA

[0061]

[0062] Example 2 simultaneously measured the 6-APA concentration (μg / ml) obtained by HPLC method, the results are shown in Table 2 and figure 2 :

[0063] Clone1 Clone2 Clone3 Clone4 CK 30min 11.28 10.10 19.70 18.47 20.54 60min 11.12 9.95 19.30 12.12 20.37 120min 12.64 11.78 19.49 16.23 20.42

[0064] Depend on figure 1 and figure 2 As can be seen from the change trend, the change trend of the absorbance value of the enzyme label detection 6-APA is consistent with the concentration change detected by HPLC, thus showing that the inventive method can well detect ...

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Abstract

The invention discloses a high-throughput screening method of beta-lactam antibiotic synthetase. The method comprises the steps as follows: a synthetic liquid is added in plate holes of an ELISA (enzyme linked immunosorbent assay) plate, then a to-be-tested enzyme sample is added, the mixture reacts in a microplate oscillation reactor at the temperature of 10-30 DEG C for 0.1-3 hours, in the reaction process, the ELISA plate is taken out every 1-30 minutes, a PDAB (paradimethylaminobenzaldehyde) color development solution is added for color development, and a light absorption value in the position of 415 nm is measured through ELISA; according to variation trend of the light absorption values and a minimum value of the light absorption values, enzyme with high activity and good synthesis capacity is screened out; the synthetic liquid is prepared by dissolving mother nucleuses and side chains of beta-lactam antibiotics into a KPB solution; and the to-be-tested enzyme sample is beta-lactam antibiotic synthetase. The high-throughput screening method of beta-lactam antibiotic synthetase can be used for screening enzymatic synthesis enzyme of lactam antibiotics such as amoxicillin, cefalexin, cefaclor, cephadroxil, ampicillin and the like, has the characteristics of simplicity in operation, low cost, accurate detection and the like, and is suitable for large-scale high-throughput screening of synthesis enzyme.

Description

technical field [0001] The invention relates to high-throughput screening technology, in particular to a high-throughput screening method for β-lactam antibiotic synthetase. technical background [0002] At present, enzymes for the synthesis of lactam antibiotics are mainly concentrated in the industrial production of 7-ACA, 6-APA, amoxicillin, ampicillin, etc., among which the most widely used is penicillin acylase (EC 3.5.1.11), the earliest For the production of 6-APA core. In recent years, with the development of biotechnology and the breakthrough of enzyme molecular transformation technology, various enzyme molecules for the enzymatic synthesis of lactam antibiotics have appeared one after another. DSM has realized the production of amoxicillin, cephalexin, cefaclor, etc. Industrialization of antibiotics; domestic United Laboratories has also realized the enzymatic synthesis of amoxicillin. [0003] The discovery of new enzymes can be screened through the method of na...

Claims

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Application Information

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IPC IPC(8): C12Q1/34
Inventor 刘力强贾娟娟牛昆范俊辉石晨光王召业吕娜武芳王欢杨丽萍邸胜苗刘东
Owner NORTH CHINA PHARMA HEBEI HUAMIN PHARMA
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