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Engineered bacterium to improve stability of tyrosine phenol-lyase and construction method and application thereof

A tyrosine phenol and construction method technology, applied in the field of genetic engineering, can solve the problems of low L-DOPA yield, difficult to control reaction conditions, many by-products, etc., and achieve high yield, low cost, and improved stability. Effect

Pending Publication Date: 2019-03-08
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, the reaction conditions are difficult to control, the stability is poor, there are many by-products, and the yield of L-DOPA is low

Method used

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  • Engineered bacterium to improve stability of tyrosine phenol-lyase and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Prepare BL21(DE3) into competent cells

[0031] Use the TAKARA Competent Cell Preparation Kit and operate according to the instructions to prepare BL21(DE3) competent cells.

[0032] 2. Whole gene synthesis of tyrosine phenol lyase gene fragment

[0033] According to the sequence provided by Gene ID: X66978.1, the whole gene fragment of tyrosine phenol lyase was synthesized.

[0034] 3. Construction of tyrosinase expression plasmid pTrx-TPL

[0035] The whole tyrosinase gene fragment was subcloned into the downstream of the thioredoxin gene fragment on the pET32a plasmid.

[0036] 4. The expression plasmid pTrx-TPL is introduced into competent cells

[0037] 1) Immediately insert the competent state into an ice-water bath for 3 minutes after taking it out from -70°C;

[0038] 2) Add 1 microliter of plasmid pTrx-TPL to the competent state in the ultra-clean bench, flick and mix well, immediately insert into the ice water bath for 25 minutes, and let stand;

[003...

example 2

[0043] tyrosine phenol lyase

[0044] LB medium: tryptone 10g / L, yeast extract 0.5g / L, sodium chloride 10g / L, pure water.

[0045] Fermentation medium: tryptone 12g / L, yeast extract 24g / L, glycerin 5g / L, potassium dihydrogen phosphate 2.31g / L, dipotassium hydrogen phosphate trihydrate 16.43g / L, pure water.

[0046] 1) Pick a single colony and inoculate it into a 4ml LB medium test tube, add ampicillin (100mg / L), culture at 37°C, 220rpm for 12h, and obtain first-grade seeds;

[0047]2) The primary seeds were inoculated into 100ml of fermentation medium shake flask, 37°C, 220rpm, cultured for 4h, added IPTG to a final concentration of 1mM, 25°C, 220rpm, cultured for 12h;

[0048] 3) Centrifuge the bacterial liquid in step (2) to collect the bacterial cells, and place in a -20°C refrigerator.

example 3

[0049] Example 3: Conversion of tyrosine phenol lyase to produce L-dopa

[0050] 1) 1L substrate solution: 14g / L sodium pyruvate, 10g / L catechol, 40g / L ammonium chloride, 2g / L sodium sulfite, 1g / L EDTA, adjust pH to 8.0;

[0051] 2) To 35g of bacterial cells, add 1L of substrate solution, stir well, seal and shake at 25°C;

[0052] 3) Add a substrate (an equivalent amount of sodium pyruvate and quinone) every half hour, and control the substrate concentration of the two substrates to not be higher than 10g / L;

[0053] 4) When the residual catechol concentration is below 1g / L, stop the reaction, and the accumulated L-dopa concentration is above 120g / L.

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Abstract

The invention relates to an engineered bacterium and a construction method thereof, in particular to an engineered bacterium to improve stability of tyrosine phenol-lyase and a construction method andapplication thereof, and belongs to the technical field of genetic engineering. The engineered bacterium is identified as Escherichia coli Trx-TPL, collected in China Center for Type Culture Collection at Wuhan university in Wuhan, China on 13th, 11, 2017; its collection number is CCTCC NO: M2017684. Two tyrosine phenol-lyase genes are linked with a thioredoxin gene to construct expression plasmids; stability, particularly thermal stability, of expressed enzymes can be effectively improved; conversion synthetic time of levodopa can be extended at the premise of providing a related substrate;substrate concentration is improved; the process is simple; the cost is low; the yield is high; the engineered bacterium herein is worthy of application in industrial production.

Description

technical field [0001] The invention relates to an engineering bacterium and a construction method thereof, in particular to a tyrosine phenol lyase engineering bacterium, a construction method and application thereof, belonging to the field of genetic engineering. Background technique [0002] The chemical name of levodopa (3,4-dihydroxyphenyl-L-ananine, referred to as L-DOPA) is 3,4-dihydroxyphenylalanine, and its structural formula is: [0003] [0004] As an important biologically active substance, L-DOPA is an important intermediate product in the biochemical metabolic pathway from L-tyrosine to catechol or melanin. [0005] In the 1960s, many foreign scholars began to devote themselves to the research of microbial enzymatic synthesis of L-DOPA. In order to increase the yield of L-DOPA and the conversion rate of the substrate, researchers have conducted a lot of research on the process of microbial enzymatic synthesis of L-DOPA. [0006] Tyrosine phenol lyase (Tyro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/62C12P13/22C12R1/19
CPCC07K2319/35C12N9/88C12N15/62C12N15/70C12P13/225C12Y401/99002
Inventor 储消和吴黎诚沈建生英涛陈万河方明山程跃赖秧秧
Owner ZHEJIANG UNIV OF TECH
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