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

A technology of tyrosine phenol and construction method, which is applied in the field of genetic engineering, can solve the problems of low yield of L-DOPA, difficulty in controlling reaction conditions, and many by-products, and achieve high yield, low cost, and improved product quality. 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, the by-products are many, and the productive rate 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
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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 pCBD-TPL

[0035] The whole tyrosinase gene fragment was subcloned into the downstream of the cellulose binding domain gene fragment on the pET35b plasmid.

[0036] 4. The expression plasmid pCBD-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 pCBD-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 s...

Embodiment 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, glycerol 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 kanamycin (50mg / L), 37°C, 220rpm, and cultivate for 12h to obtain first-grade seeds;

[0047] 2) Inoculate primary seeds into 100ml of fermentation medium shake flasks, incubate at 37°C, 220rpm for 4h, add IPTG to a final concentration of 1mM, incubate at 25°C, 220rpm, for 12h;

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

Embodiment 3

[0049] Example 3: Extraction and immobilization of tyrosine phenol lyase

[0050] 1) Add 3 times the volume of water to the bacteria, and ultrasonically disrupt the cells for 20 minutes;

[0051] 2) High-speed centrifugation to obtain supernatant enzyme liquid;

[0052] 3) Add 50%-200% cellulose to the enzyme solution, shake and mix at 20-30°C for 1-12 hours;

[0053] 4) Filtrate, collect the immobilized crude enzyme, wash with potassium phosphate buffer (20-100mM, pH7-8.5) several times, and filter to obtain the immobilized enzyme;

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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 CBD-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: M2017685. Two tyrosine phenol-lyase genes are linked with a cellulose binding domain gene to construct expression plasmids; target enzymes can be extracted effectively and immobilized; levodopa can be catalytically produced at the premise of providing a related substrate; purification yield is greatly increased; product quality 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 an engineering bacterium for improving the stability of tyrosine phenol lyase, a construction method and application thereof, and belongs to the technical 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 substrates, researchers have conducted a lot of research on the process of microbial enzymatic synthesis of L-DO...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/60C12N15/70C12P13/22C12R1/19
CPCC12N9/80C12P13/225C12Y401/99002
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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