DNA molecule, recombinant plasmid and recombinant bacterium for production of D-p-Hydroxyphenylglycine

A technology of p-hydroxyphenylglycine and DNA molecules, applied in the field of DNA molecules, can solve problems such as handling problems, and achieve the effects of environmental friendliness, simple operation and high conversion rate

Inactive Publication Date: 2014-08-20
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since D-hydantoinase can specifically produce D-p-hydroxyphenylglycine, this method has strong specificity and high yield, but the disadvantage is that a large amount of acid still needs to be added in the reaction process, which brings problems to subsequent processing

Method used

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  • DNA molecule, recombinant plasmid and recombinant bacterium for production of D-p-Hydroxyphenylglycine
  • DNA molecule, recombinant plasmid and recombinant bacterium for production of D-p-Hydroxyphenylglycine
  • DNA molecule, recombinant plasmid and recombinant bacterium for production of D-p-Hydroxyphenylglycine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, construction of recombinant plasmid

[0044] 1. Construction of recombinant plasmid pUC-hyd-car

[0045] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

[0046] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair consisting of hyd-for and hyd-rev to obtain a PCR amplification product.

[0047] hyd-for: 5'-TGCTT GTC GAC ATGAGCACTGTCATCAAGGG-3';

[0048] hyd-rev: 5'-ACGAAC TCTAGA TCAGACGCCGCTTGCGGGAAT-3'.

[0049] 3. The PCR amplified product of step 2 was double-digested with restriction enzymes Sal I and Xba I, and the digested product was recovered.

[0050] 4. Digest the vector pUC18 with restriction endonucleases Sal I and Xba I to recover the vector backbone (about 2680 bp).

[0051] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant plasmid pUC-hyd.

[0052] 6. Synthesize the d...

Embodiment 2

[0087] Embodiment 2, the preparation of recombinant bacteria

[0088] The recombinant plasmid pUC-pPL-hyd-car was introduced into Escherichia coli DH5α competent cells to obtain recombinant bacteria DH5α / pUC-pPL-hyd-car.

[0089] The recombinant plasmid pUC-pPAND-hyd-car was introduced into Escherichia coli DH5α competent cells to obtain recombinant bacteria DH5α / pUC-pPAND-hyd-car.

[0090] The recombinant plasmid pUC-pT7A1-hyd-car was introduced into Escherichia coli DH5α competent cells to obtain recombinant bacteria DH5α / pUC-pT7A1-hyd-car.

Embodiment 3

[0091] Embodiment 3, the comparison of different recombinant bacteria expression target protein levels

[0092] Each recombinant bacterium prepared in Example 2 was carried out as follows:

[0093] 1. Pick a single colony of the recombinant bacteria and inoculate it in 15ml of LB liquid medium containing 100μg / ml ampicillin, culture at 37°C with shaking at 200rpm, and obtain OD 600nm =2.8 bacteria solution.

[0094]2. Take 10ml of the bacterial liquid obtained in step 1 and transfer it to 100ml of LB liquid medium containing 100μg / ml ampicillin to obtain OD 600nm =3.0 initial fermentation system, 37 ° C, 200 rpm, shaking culture for 12 hours, to obtain a termination fermentation system.

[0095] 3. Take 2mL to terminate the fermentation system, centrifuge at 10,000rpm for 1min, collect the bacterial precipitate, ultrasonically break (ultrasonic power is 200w, processing time is 4min, stop for 3s every 3s), then centrifuge at 10,000rpm for 1min, and collect the supernatant. ...

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Abstract

The invention discloses a DNA molecule, a recombinant plasmid and a recombinant bacterium for production of D-p-Hydroxyphenylglycine. The DNA molecule provided by the invention comprises a PL promoter, a D-hydantoinase gene and an N-carbamoylase gene, and expression of the D-hydantoinase gene and the N-carbamoylase gene is initiated by the PL promoter. The invention provides the DNA molecule able to stably and massively express D-hydantoinase and N-carbamoylase, and the recombinant plasmid and the recombinant bacterium based on the DNA molecule. At the same time, the invention also provides a method for converting DL-p-hydroxyphenylhydantoin into D-p-Hydroxyphenylglycine by the recombinant bacterium or fermented recombinant bacterium. The method only generates D-p-Hydroxyphenylglycine, has no need of splitting and separating an enantiomer, and the conversion rate is high. The whole production process has the advantages of mild conditions, simple operation, and environmental friendliness.

Description

technical field [0001] The invention relates to DNA molecules, recombinant plasmids and recombinant bacteria for producing D-p-hydroxyphenylglycine. Background technique [0002] D-p-Hydroxyphenylglycine is an important pharmaceutical intermediate used in the production of β-lactam semi-synthetic antibiotics. β-lactam semi-synthetic antibiotics include amoxicillin (amoxicillin), cefadroxil (European) and so on. Compared with traditional penicillin G antibiotics, β-lactam semi-synthetic antibiotics have the advantages of broad antibacterial spectrum, low toxicity, and good oral effect. They are widely used clinically and have a huge market. According to statistics, in 2009, the sales volume of the global antibiotic drug market was 28 billion US dollars, of which β-lactam semi-synthetic antibiotics accounted for about 65%, which was 18.2 billion US dollars. From 2000 to 2009, the production of amoxicillin and cefadroxil alone increased by 614% and 762%, respectively. The la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N1/21C12P13/04C12R1/19
Inventor 蔡真张君丽
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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