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Method for reducing decomposition of cephalosporin C

A cephalosporin and host bacteria technology, applied in the field of bioengineering, can solve the problems of reducing the decomposition of cephalosporin C and low utilization rate of CPC, and achieve the effects of improving the utilization rate of substrates, simplifying the production process and reducing the cost of raw materials

Active Publication Date: 2012-07-04
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The object of the present invention is to provide a method for reducing the decomposition of cephalosporin C, and solve the problem of low utilization rate of CPC in the process of generating 7-ACA from industrial enzyme catalyzed CPC

Method used

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  • Method for reducing decomposition of cephalosporin C
  • Method for reducing decomposition of cephalosporin C
  • Method for reducing decomposition of cephalosporin C

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Decomposition of CPC by E.coli JM105 (Cyberson Corporation), JM109(DE3) (Dingguo Corporation) and BL21(DE3) (Promega Corporation) Cell Lysate

[0032] (1) Preparation of cell lysate and crude enzyme solution:

[0033] In 10ml LB medium containing 50mg / L kanamycin (medium composition is: peptone 10g.L -1 , yeast powder 5g.L -1 , sodium chloride 10g.L -1 , pH 7.0) were inoculated with single colonies of E. coli JM105, JM109 (DE3) and BL21 (DE3) respectively, cultured at 37°C and 200rpm for 12h to make seed flasks. Take 1mL of bacterial solution and transfer it to 50mg.L -1 kanamycin in 50 ml (300 ml shake flask) fermentation medium. Among them, the inducible medium of Escherichia coli JM109 (DE3) and BL21 (DE3) is: corn steep liquor 50g.L -1 , Yeast paste 10g.L -1 , NH 4 Cl 2.5g.L -1 , glycerol 5g.L -1 , KH 2 PO 4 2.3g.L -1 , K 2 HPO 4 .3H 2 O 20.4g.L -1 , lactose 3g.L -1 , pH 7.5. The constitutive medium of Escherichia coli JM105 is: corn steep liquor ...

Embodiment 2

[0039] Knockout of β-lactamase and acetylesterase in E. coli JM105 and JM109(DE3)

[0040] (1) Knockout of β-lactamase gene ampC:

[0041] The genome sequence analysis of E.coli K12 showed that the β-lactamase gene ampC (E.coli K12 gene ID948669) and the acetylesterase gene aes coexisted in the genomes of E.coli JM105 and JM109(DE3) (E. coli K12 gene ID 947514). Homologous arm primer pair ampCP1 / ampCP2 was designed, containing 40bp upstream and downstream fragment of ampC gene and 19bp kanamycin resistance gene fragment, respectively, and was synthesized by Yingweijieji Bioengineering Technology Co., Ltd. (Beijing). Using the helper plasmid pKD13 as a template, a DNA fragment containing the kanamycin resistance gene inside and the ampC homology arm outside was amplified. PCR amplification adopts 50μL system, the composition is as follows:

[0042]

[0043]

[0044] Among them, rTaq DNA polymerase and dNTPs were purchased from Takara Company (Dalian).

[0045] The ups...

Embodiment 3

[0056] Decomposition of CPC by Cell Lysate of β-lactamase and Acetylesterase Knockout Engineering Bacteria

[0057] According to the method described in Example 1, cultivate the following three kinds of β-lactamase and acetyl esterase knockout type engineering bacteria E.coli JM105 (ΔampC), E.coli JM105 (Δaes), E.coli JM105 ( ΔampC, Δaes), and E.coli JM105 was used as the control. Determination of cell growth curves, such as Figure 4 A shown. The results showed that knockout of β-lactamase and acetylesterase had no significant effect on the growth of recombinant bacterial cells. According to the method as described in Example 1, the cell lysate of each bacterial strain was prepared, and the cell lysate of the bacterial strain before natural degradation and double enzyme knockout was used as a control to investigate the effect of each cell lysate CPC decomposition, the results are as follows Figure 4 Shown in B. The E.coli JM105 cell lysate without gene knockout degrades ...

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Abstract

The invention belongs to the technical field of bioengineering and relates to a method for reducing decomposition of cephalosporin C (CPC). The method comprises the following steps: knocking out beta-lactamase genes and acetyl esterase genes of host bacteria by using a Red recombinant system; transforming cephalosporin C acylase genes into the host bacteria; expressing the cephalosporin C acylaseby using the host bacteria; preparing cephalosporin C acylase crude enzyme solution or immobilized cephalosporin C acylase crude enzyme; and adding the cephalosporin C acylase crude enzyme solution or immobilized cephalosporin C acylase crude enzyme into cephalosporin C substrate working liquid and catalyzing to generate 7-aminocephalosporin acid (ACA). CPC acylase is prepared by the method. In the process of catalyzing the CPC to generate 7-ACA by using the harvested crude enzyme, the utilization ratio of the CPC is increased to over 98 percent.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for reducing the decomposition of substrate CPC in the process of catalyzing the conversion of cephalosporin C to 7-aminocephalosporanic acid using cephalosporin C acylase expressed by genetically engineered bacteria. Background technique [0002] Cephalosporins belong to β-lactam broad-spectrum antibiotics, which play a bactericidal effect by interfering with the synthesis of bacterial cell walls and accelerating the destruction of cell walls. The antibacterial site of cephalosporins is the parent nucleus 7-aminocephalosporanic acid (7-ACA), which is an important intermediate of various semi-synthetic cephalosporin antibiotics. Due to the simple, efficient and low pollution of the preparation process, the one-step catalytic cracking of CPC with Cephalosporin C (CPC) acylase to produce 7-ACA has become a development trend. [0003] Natural strains pro...

Claims

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Application Information

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IPC IPC(8): C12P35/02C12R1/19
Inventor 于慧敏王颖张婧罗晖沈忠耀
Owner TSINGHUA UNIV
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