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Method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria

An aminocephalosporanic acid acylase and aminocephalosporanic acid technology, which is applied in the biological field, can solve problems such as low enzyme activity, and achieve the effects of simple operation, guaranteed growth and high screening efficiency

Inactive Publication Date: 2012-09-26
广西中医学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is a problem to be solved in the three-enzyme method technology, that is, the enzymatic activity of Gl-7-ACA acylase to AKA-7-ACA is low (this enzyme is to the Michaelis constant Km value of AKA-7-ACA its to GL -4 times of the Km value of -7-ACA), so that the final 7-ACA productive rate is only 76~80%, and there is still some distance from the requirements of industrialized production, because the industrialized dual-enzyme two-step method 7-ACA productive rate≥86 %

Method used

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  • Method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria
  • Method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria
  • Method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Preparation of AKA-7-ACA and its structural analogues:

[0041] Phosphate buffer: Disodium hydrogen phosphate and sodium dihydrogen phosphate are prepared in a buffer solution in a ratio of pH 7.5.

[0042] (1) Preparation of AKA-7-ACA

[0043] 100ml of 2% CPC is dissolved in a phosphate buffer with a concentration of 0.1mol / L and a pH of 7.5, put it into the reactor, and add 3ml of 40U DAAO and 3ml of 2000U CAT. Pure oxygen is introduced during the reaction with constant stirring. The temperature was controlled at 28°C, and the reaction solution was collected by centrifugation after 2h, and the reaction solution was concentrated and crystallized to obtain AKA-7-ACA.

[0044]

[0045] (2) Preparation of Adipoyl-7-ADCA

[0046] 5~10mL adipoyl chloride (adipoyl chloride) was slowly dripped into 5g of 7-ADCA alkaline solution with a pH value of 8~9, adding baking soda solid while stirring, keeping the pH unchanged, and collecting the reaction after 2h by centrifugation The prod...

Embodiment 2

[0071] 1. Preparation of AKA-7-ACA and its structural analogues:

[0072] Phosphate buffer: Disodium hydrogen phosphate and sodium dihydrogen phosphate are prepared in a buffer solution in a ratio of pH 7.5.

[0073] (1) Preparation of AKA-7-ACA

[0074] 100ml of 2% CPC is dissolved in a phosphate buffer with a concentration of 0.1mol / L and a pH of 7.5, put it into the reactor, and add 3ml of 40U DAAO and 3ml of 2000U CAT. Pure oxygen is introduced during the reaction with constant stirring. The temperature was controlled at 27-29°C, and the reaction solution was collected by centrifugation after 2h, and the reaction solution was concentrated and crystallized to obtain AKA-7-ACA.

[0075]

[0076] (2) Preparation of keto-7-ADCA

[0077] 100ml of 2% deacetylated CPC is dissolved in a phosphate buffer with a concentration of 0.1mol / L and a pH of 7.5, put into the reactor, and 2ml of 40U DAAO and 4ml of 2000U CAT are added. Pure oxygen is introduced during the reaction. With constant sti...

Embodiment 3

[0101] 1. Preparation of AKA-7-ACA and its structural analogues:

[0102] Phosphate buffer: Disodium hydrogen phosphate and sodium dihydrogen phosphate are prepared in a buffer solution in a ratio of pH 7.5.

[0103] (1) Preparation of AKA-7-ACA

[0104] 100ml of 2% CPC is dissolved in a phosphate buffer with a concentration of 0.2mol / L and a pH of 7.5, put into the reactor, 4ml of 40U DAAO and 2ml of 2000U CAT are added, and pure oxygen is introduced during the reaction with constant stirring , The temperature was controlled at 27-29°C, the reaction solution was collected by centrifugation after 2.5h, and the reaction solution was concentrated and crystallized to obtain AKA-7-ACA.

[0105]

[0106] (2) Preparation of Adipoyl-7-ADCA

[0107] 5~10mL adipoyl chloride (adipoyl chloride) was slowly dropped into 5g of 7-ADCA alkaline solution with pH 8~9, and baking soda solid was added while stirring, keeping the pH constant, and the reaction product was collected by centrifugation after 3...

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Abstract

The invention discloses a method for screening alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria. The screening process of the method comprises the following steps: (1) preparation of alpha-ketone adipoyl-7-aminocephalosporanic acid and structural analogues thereof; (2) selection and culture of screening system indicator bacteria, enrichment culture of candidate bacteria, and selection and culture of positive and negative control strains; (3) preparation of a three-tier agar plate culture medium screening system; (4) judgement of the objective alpha-ketone adipoyl-7-aminocephalosporanic acid acylase-producing bacteria. According to the method, simple operation is achieved, no expensive and complex experimental instrument is needed, 5000 candidate strains can be screened by one person per day, high screening efficiency is achieved and high throughput screening is obtained. Moreover, the method can be used in breeding of industrial strains.

Description

Technical field [0001] The invention belongs to the field of biological technology, and specifically relates to a method for screening α-ketoadipyl-7-aminocephalosporanic acid acylase producing bacteria. Background technique [0002] 7-aminocephalosporanic acid (7-ACA) is the nucleus of cephalosporin C (Cephalosporin C, CPC), and is the key raw material for the production of more than 50 clinical semi-synthetic cephalosporin antibiotics. At present, the main manufacturers include Austria's Biochemie Company, Italy's Antibioticos Company and the main domestic companies such as Zhongrun Pharmaceutical, Health Yuan, Fukang, Shanxi Weiqida, Harbin Pharmaceutical Group, Lukang Pharmaceutical and United Laboratories. In 2010, the global demand for 7-ACA was more than 4,000 tons, while the actual domestic production of 7-ACA reached about 5,500 tons. Therefore, how to prepare 7-ACA in an environmentally friendly and cost-effective manner, reduce production costs, and improve corporate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12R1/085C12R1/19C12R1/38
Inventor 谭强邓超澄韦海宏陈卫卫邱春会
Owner 广西中医学院
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