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Acylase for producing 7-ACA (Aminocephalosporanic acid) by splitting CPC (Cephalosporin C) by utilizing a one-step method and polynucleotide encoding same

A technology of acylase and acylase activity, applied in the field of biochemical pharmaceuticals, can solve the problems of low substrate specificity, poor substrate tolerance, low conversion efficiency, etc.

Active Publication Date: 2013-10-30
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved in the present invention is to provide a solution for the problems that the existing CPC acylase that cleaves CPC in vitro to produce 7-ACA has low substrate specificity, poor substrate tolerance and low conversion efficiency. A new CPC acylase that cleaves CPC to produce 7-ACA, the coding gene of the enzyme, a recombinant expression vector and a recombinant expression transformant containing the coding gene, and the production method of the CPC acylase and its synthesis in 7 -Application in ACA

Method used

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  • Acylase for producing 7-ACA (Aminocephalosporanic acid) by splitting CPC (Cephalosporin C) by utilizing a one-step method and polynucleotide encoding same
  • Acylase for producing 7-ACA (Aminocephalosporanic acid) by splitting CPC (Cephalosporin C) by utilizing a one-step method and polynucleotide encoding same
  • Acylase for producing 7-ACA (Aminocephalosporanic acid) by splitting CPC (Cephalosporin C) by utilizing a one-step method and polynucleotide encoding same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Construction of error-prone PCR and product expression plasmids for the starting sequence

[0055] 1.1 Perform error-prone PCR on the starting sequence ecs gene

[0056] ECS stands for CPC Acylase. Wherein, the starting sequence is obtained by connecting segmented PCR products, and its gene sequence is shown in Figure 8 pyg232. When using DNA polymerase to amplify the target gene, by adjusting the reaction conditions, increasing the concentration of magnesium ions or adding manganese ions, using low-fidelity DNA polymerase to change the mutation frequency during the amplification process, so as to increase the mutation frequency at a certain frequency. Randomly introduce mutations into the target gene to obtain random mutants of protein molecules. The PCR reaction system used is as follows:

[0057]

[0058] The sequences of primers P1 and P2 are:

[0059] P 1 : 5'-GCTT GTC GACTCTAGATCAGTGGTG-3' (the underlined part is the Sal I restriction site); ...

Embodiment 2

[0064] Example 2. Primary screening of transformants

[0065] 2.1 Transformant cell culture and protein expression

[0066] Select the engineered bacterium transfer test tube of construction and cultivate overnight, then transfer to the shaking flask that 250ml LB liquid medium is housed with the inoculum of 2% (V / V), and the concentration of kanamycin in the LB medium is 10~50 μ g / ml, cultured at 37°C, 220 rpm for 3 hours;

[0067] Then add IPTG to make the final concentration of IPTG in the medium reach 0.6 mmol, 25°C, 220 rpm, and culture for another 16 hours;

[0068] Centrifuge the bacterial cells at a speed of 8000 rpm in a low-temperature centrifuge.

[0069] 2.2 Primary screening of transformants

[0070] Suspend the collected bacteria with 3ml of 0.1mmol Tris-HCl, take 1ml of concentrated cells and 0.1mg of CPC in a water bath at 25°C, 28°C, 37°C, pH9.6 and pH8.0 respectively for 1h, 13200r / min Centrifuge for 5 minutes to take the supernatant, and measure the abs...

Embodiment 3

[0071] Example 3. Re-screening of highly active transformants

[0072] 3.1 Recultivate the 63 highly active transformants obtained from the primary screening

[0073] 1) 250ml induced expression culture (the induced expression culture obtained in the embodiment 2 step 2.1), centrifuged (8000rpm, 10min) to obtain the thalline, the thalline of the harvest was washed with 5ml P buffer solution (pH8.0, 100 times Diluted 1M disodium hydrogen phosphate and sodium dihydrogen phosphate mixture) suspension, ultrasonic crushing (crushing time 5s / interval time 5s, 500W, ultrasonic crushing 10min).

[0074] 2) Centrifugation and precipitation: after crushing, centrifuge at 4°C at a speed of 8000 rpm (Hitachi Himac CR21G refrigerated high-speed centrifuge, rotor is R12A), and precipitate cell debris.

[0075] 3) Separation and purification: the obtained supernatant (with his tag) was placed in a 100ml beaker, and separated and purified with a nickel column. The specific steps are:

[00...

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Abstract

The invention discloses an acylase for producing 7-ACA (Aminocephalosporanic acid) by splitting CPC (Cephalosporin C) by utilizing a one-step method, polynucleotide encoding the same, a recombinant expression vector containing a nucleotide sequence, a recombinant expression transformant containing the nucleotide sequence, a preparation method of the acylase and an application of a recombinase in synthesis of the 7-ACA. A CPC acylase is a protein of (a) or (b) as follows: (a) a protein which has CPC acylase activity and an amino acid sequence represented by SEQ ID NO.3, wherein 180-site valine is substituted; and (b) a protein which has the CPC acylase activity and an amino acid sequence formed by adding one or multiple amino acids to an N terminal or a C terminal of the amino acid sequence in the (a) and is derivative from (a). According to the CPC acylase disclosed by the invention, both the enzyme activity and the substrate tolerance are improved, so that a strong precondition is provided for manual determinated evolution transformation of the CPC acylase.

Description

technical field [0001] The invention belongs to the field of biochemical pharmacy, and particularly relates to a CPC acylase and its gene for producing 7-ACA by splitting cephalosporin C (CPC) in one step, a recombinant expression vector containing the gene, a recombinant expression transformant, and the CPC Preparation method of acylase and its application in the synthesis of 7-aminocephalosporanic acid. Background technique [0002] 7-Aminocephalosporanic acid (7-ACA), as a key intermediate of semi-synthetic cephalosporin antibiotics, is of great value in the pharmaceutical industry. In industry, chemical method and two-step enzymatic method are often used to produce 7-ACA by using cephalosporin C (Cephalosporin C, CPC). However, the process conditions of the chemical method are relatively harsh, and a variety of toxic substances need to be processed, causing serious environmental pollution. Although a new two-step enzymatic process is being widely adopted, CPC is first ...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12P35/02C12N15/63C12N1/21C12R1/19
Inventor 胡又佳谢丽萍刘艳郑林冲朱宝泉龚桂花
Owner SHANGHAI INST OF PHARMA IND
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