Trosine phenol lyase engineering bacteria as well as construction method thereof 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 less discharge of three wastes. Effect

Active Publication Date: 2016-08-24
ZHEJIANG UNIV OF TECH +2
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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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  • Trosine phenol lyase engineering bacteria as well as construction method thereof and application 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 pFPL

[0035] The whole gene fragment of tyrosinase was subcloned into pET24a plasmid, restriction sites BamHI, XhoI.

[0036] 4. The expression plasmid pFPL 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 pFPL 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;

[0039] 3) Gently transfer the compete...

Embodiment 2

[0043] 1. Prepare BL21 (DE3) as competent cells

[0044] Use the TAKARA Competent Cell Preparation Kit and operate according to the instructions to prepare BL21 (DE3) competent cells.

[0045] 2. Whole gene synthesis of tyrosine phenol lyase gene fragment

[0046] According to the sequence provided by Gene ID: X66978.1, the whole gene fragment of tyrosine phenol lyase was synthesized.

[0047] 3. Construction of tyrosinase expression plasmid pFPL

[0048] The whole gene fragment of tyrosinase was subcloned into pET24a plasmid, and restriction sites BamHI and XhoI were cut.

[0049] 4. The expression plasmid pFPL is introduced into competent cells

[0050]6) Immediately insert the competent state into an ice-water bath for 3 minutes after taking it out from -70°C;

[0051] 7) Add 1 microliter of plasmid pFPL 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;

[0052] 8) Gently trans...

Embodiment 3

[0064] The difference from Example 2 is that pKJE7 is selected as the chaperonin expression plasmid.

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Abstract

The invention relates to trosine phenol lyase engineering bacteria as well as a construction method thereof and application thereof. A tyrosine phenol lyase gene is constructed to express plasmids; chaperonin is introduced to express the plasmids to obtain the trosine phenol lyase engineering bacteria which are identified as escherichia coli FPLF8 (Escherichia coli HPLF8) preserved in China Center for Type Culture Collection on February 19, 2016, with a preservation number being CCTCCNO:M 2016065. The engineering bacteria can be synthesized into levodopa by fermentation and conversion, and are efficiently expressed; the expressed product is stable and high in activity, and can be synthesized into levodopa by conversion in the presence of related substrates; and the process is simple, the cost is low, the yield is high, emission of three wastes is a little, and the application value for industrial production is achieved.

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): C12N1/21C12N15/60C12N15/70C12P13/22C12R1/19
CPCC12N9/88C12P13/222
Inventor 储消和吴黎诚余炜方明山徐顺清周卫国张拥军
Owner ZHEJIANG UNIV OF TECH
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