Tyrosine phenol lyase engineering bacteria, construction method and applications 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 large soluble expression, low cost, and high yield Effect

Inactive Publication Date: 2017-11-07
ZHEJIANG LVCHUANG BIOTECHNOLOGY CO LTD
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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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  • Tyrosine phenol lyase engineering bacteria, construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Prepare BL21 (DE3) as 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 pMBP-TPL

[0035] The whole gene fragment of tyrosinase was subcloned to the downstream of the maltose binding protein gene fragment on pSYNO-1 plasmid.

[0036] 4. The expression plasmid pMBP-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 pMBP-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 stan...

example 2

[0043] tyrosine phenol lyase

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

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

[0046] 1) Pick a single colony and inoculate it into a 4ml LB medium test tube, add kanamycin (50mg / 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 the substrate (equal amount of sodium pyruvate and quinone) every half hour, and control the concentration of the two substrates to not be higher than 10g / L;

[0053] 4) When the residual concentration of catechol falls below 1g / L, stop the reaction, and the concentration of L-dopa accumulates to above 100g / L.

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PUM

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Abstract

The invention provides tyrosine phenol lyase engineering bacteria, a construction method and applications thereof, wherein the engineering bacteria are identified as Escherichia coli, are named Escherichia coli MBP-TPL, are preserved in the China Center for Type Culture Collection on April 21, 2017, and have the preservation number of CCTCC NO: M 2017202, wherein the preservation address is Wuhan University, Wuhan, China. According to the present invention, the expression plasmid is constructed by integrally linking the tyrosine phenol lyase gene and the maltose binding protein gene so as to obtain the Escherichia coli MBP-TPL, wherein the expression plasmid can express the maltose binding protein and the tyrosine phenol lyase in the fused manner; and the activity of the fermentation unit bacterial amount of the tyrosine phenol lyase is high, and the soluble expression amount is high.

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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IPC IPC(8): C12N1/21C12N15/70C12P13/22C12R1/19
CPCC12N9/88C12N15/70C12P13/225C12Y401/99002
Inventor 储消和吴黎诚赖秧秧程跃沈建方明山生英涛徐顺青
Owner ZHEJIANG LVCHUANG BIOTECHNOLOGY CO LTD
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