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Application of vitreoscilla hemoglobin in improving expression quantity of cephalosporin C acylase

A technology of cephalosporin and Vibrio vitreous, applied in application, enzyme, bacteria and other directions, can solve the problems of difficult reaction control, unfriendly chemical deacylation method, harsh reaction conditions, etc.

Inactive Publication Date: 2020-10-30
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical deacylation method has the disadvantages of being unfriendly to the environment and harsh reaction conditions, and has been eliminated from the market
The two-step enzymatic method has been basically used in industrial production, but the conversion rate of the two-step enzymatic method is low, and the reaction is not easy to control
[0004] Because the activity of cephalosporin C acylase existing in nature is low and unstable, it is difficult for industrial application, so obtaining a cephalosporin C acylase with high conversion efficiency becomes the key to solve the problem

Method used

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  • Application of vitreoscilla hemoglobin in improving expression quantity of cephalosporin C acylase
  • Application of vitreoscilla hemoglobin in improving expression quantity of cephalosporin C acylase
  • Application of vitreoscilla hemoglobin in improving expression quantity of cephalosporin C acylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] [Example 1] Experimental material

[0036] Strains and plasmids

[0037] Escherichia coli E.coli BL21(DE3) and Escherichia coli DH5α were purchased from Shenzhen Kangti Life Technology Co., Ltd. Plasmids pETDuet-1, pACYCDuet-1, pRSFDuet-1 were purchased from Addgene.

[0038] experimental method:

[0039] 1. P1 phage transduction recipient bacteria

[0040] (1) Lysis of the donor bacterium JM1087 (JM1087 genotype: BW25113 ptsG::kan, phenotype: Kan+, the ptsG gene of the donor bacterium has been knocked out, and the corresponding position is replaced by the kanamycin resistance gene, which with FRT sequences at both ends)

[0041] ①Cultivate donor bacteria JM1089 in 5 mL of LB until OD600=0.1-0.2, add 50 μL of 1M CaCl2, and mix well.

[0042] ② Add 10-100 μL of wild-type P1 phage, and incubate on a shaker at 37°C for at least 3 hours until the cells are lysed.

[0043] ③ Add 1 to 3 drops of chloroform to each ml of culture medium and shake to kill the cells.

[00...

Embodiment 2

[0104] [Example 2] Experimental method

[0105] Cloning and Sequence Analysis of Cephalosporin C Acylase Gene and Vitiligo hyaline Hemoglobin Gene

[0106] The cephalosporin C acylase gene is re-synthesized according to the codon recognition characteristics of Escherichia coli to optimize the GC content, as shown in SEQ ID NO.9, which is more conducive to its expression in Escherichia coli.

[0107] Strain recovery

[0108] 1. Take out the strains stored at -80°C and dissolve them on ice.

[0109] 2. Use an inoculation loop to inoculate the thawed preserved bacterial strains into a shaking tube containing 5 mL of liquid LB containing corresponding antibiotics in a clean bench. 37°C, 200rpm, cultured overnight.

[0110] 3. In the ultra-clean bench, take a circle of overnight cultured bacteria and streak it onto a solid LB plate with corresponding antibiotics. Incubate overnight at 37°C.

[0111] 4. Pick a single colony on the plate for subsequent experimental operations. ...

Embodiment 3

[0162] [Example 3] Experimental results

[0163] 1. Amplification and sequence comparison of cephalosporin C acylase

[0164] In order to obtain the cephalosporin C acylase (CA) gene, Escherichia coli BL21 (DE3) strain was activated according to the strain recovery steps described in Example 2, and the plasmid pET28α-CA was extracted. Using primers SEQ ID NO.1 and SEQ ID NO.2 (CA-1F / CA-1R) as primers to amplify the cephalosporin C acylase gene sequence, the agarose electrophoresis results of the amplified products are shown in the appendix figure 1 . The size of the PCR amplification product CA is about 2500bp, which is in line with the expected result. After the PCR product of the cephalosporin C acylase gene that met the expected results was sent to Aiji Sequencing Company for testing, the sequencing results were consistent with the target sequence results.

[0165] 2. Amplification and sequence comparison of the hemoglobin gene of Vibrella hyalineus

[0166] In order to...

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Abstract

The invention relates to an application of vitreoscilla hemoglobin in improving expression quantity of cephalosporin C acylase, and discloses a method for improving cephalosporin C acylase expressionquantity, which comprises the step of co-expression of a cephalosporin C acylase gene and a vitreoscilla hemoglobin gene in escherichia coli.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of Vitreosella hyaline hemoglobin in increasing the expression of cephalosporin C acylase. Background technique [0002] Cephalosporins are semi-synthetic antibiotics widely used in the world. In clinical use, the intermediate produced by most cephalosporin antibiotics is 7-aminocephalosporanic acid. 7-Aminocephalosporanic acid is mainly derived from the deacylation of cephalosporin C, and the main deacylation methods include chemical method, two-step enzymatic method and one-step enzymatic method. The disadvantages of chemical deacylation, such as unfriendly environment and harsh reaction conditions, have been eliminated from the market. The two-step enzymatic method has been basically used in industrial production, but the conversion rate of the two-step enzymatic method is low, and the reaction is not easy to control. One-step enzymatic method can dir...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/31C12N15/55C12N15/70C12N1/21C12R1/19
CPCC07K14/195C12N9/80C12N15/70C12Y305/01093
Inventor 余少文李星谢宁
Owner SHENZHEN UNIV
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