Method for reducing decomposition of cephalosporin C

A technology of cephalosporin and aminocephalosporanic acid, which is applied in the field of bioengineering, can solve the problems of low utilization rate of CPC and reduce the decomposition of cephalosporin C, etc., achieve the improvement of substrate utilization rate, reduce the cost of raw materials, and the operation method is fast and efficient Effect

Active Publication Date: 2013-10-16
TSINGHUA UNIV
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  • Description
  • Claims
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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
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Effect test

Embodiment 1

[0031] Decomposition of CPC by E.coli JM105 (Saibaisheng Company), JM109 (DE3) (Dingguo Company) and BL21 (DE3) (Promega Company) cell lysate

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

[0033] In the 10ml LB medium containing 50mg / L kanamycin (the medium consists of: peptone 10g.L -1 , yeast powder 5g.L -1 , sodium chloride 10g.L -1 , pH 7.0) were inoculated with single colonies of Escherichia coli JM105, JM109(DE3) and BL21(DE3), cultured at 37°C and 200rpm for 12h, and made seed bottles. Take 1mL of bacterial solution and transfer to a solution containing 50mg.L -1 Kanamycin 50ml (300ml 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 extract 10g.L -1 , NH 4 Cl 2.5g.L -1 , glycerin 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 st...

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 Escherichia coli E.coli K12 showed that the β-lactamase gene ampC (E.coli K12 gene ID948669) and the acetyl esterase gene aes existed in the genomes of E.coli JM105 and JM109(DE3) (E. coli K12 gene ID 947514). Homologous arm primer pair ampCP1 / ampCP2 were designed, respectively containing 40bp upstream and downstream fragments of ampC gene and 19bp kanamycin resistance gene fragment, which were synthesized by Yingwei Jieji Bioengineering Technology Co., Ltd. (Beijing). The helper plasmid pKD13 was used as a template to amplify the DNA fragment of the kanamycin resistance gene inside and the ampC homology arm outside. A 50 μL system was used for PCR amplification, and the composition was as follows:

[0042]

[0043]

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

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 As shown in A. 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 deg...

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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 converting cephalosporin C into 7-aminocephalosporanic acid catalyzed by 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 part of cephalosporins is its mother 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 preparation process, it has become a development trend to use Cephalosporin C (CPC) acylase to catalyze the cleavage of CPC in one step to produce 7-ACA. [0003] The natural strains produ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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