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Escherichia coli expressing thermostable tyrosine phenolase and application thereof

A tyrosine phenol hydrolase and amino acid technology, applied in the field of Escherichia coli, can solve problems such as cytotoxicity

Active Publication Date: 2019-06-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0024] Among the above routes of enzymatic conversion and synthesis of L-DOPA, tyrosine phenol lyase has the highest activity and is the closest to industrial application. The lyase activity has an inhibitory effect and is also toxic to cells. At present, the strategy of substrate flow and feeding is mainly used. Therefore, removing the inhibitory effect and toxicity of catechol is the key to realize the enzymatic preparation of L- The key to the industrialization of DOPA

Method used

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  • Escherichia coli expressing thermostable tyrosine phenolase and application thereof
  • Escherichia coli expressing thermostable tyrosine phenolase and application thereof
  • Escherichia coli expressing thermostable tyrosine phenolase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of recombinant plasmid pET-28(a)-TPL and 25 mutant recombinant plasmids.

[0054] Based on the 3D model TPL-PLP (PDB: 2YHK, ), through the Calculate Mutation Energy (Stability) module of Discovery Studio (DS) around the active center of TPL The amino acids in the range were subjected to virtual mutation to determine that the key amino acids were Gly32, Gly73, Lys155, Gly326, Gly342, Gly189 and Glu313, and then the key amino acids were subjected to virtual saturation mutation through the Calculate Mutation Energy (Stability) module of Discovery Studio (DS), and the mutation The energy table (Table 1) predicted 25 strains expressing thermostable tyrosine phenolase.

[0055] Table 1 Mutation energy and predicted mutation effect

[0056]

[0057]

[0058] The tyrosine phenolase (TPL) gene (nucleotide sequence shown in SEQ ID NO.4) was synthesized by Nanjing Jinweizhi Co., Ltd., using the plasmid pET-28(a)+ as the expression vector, and the e...

Embodiment 2

[0066] Example 2 Recombinant Escherichia coli whole cell transformation method to produce L-DOPA

[0067] Inoculate Escherichia coli strains containing plasmid pET-28(a)-TPL and 25 mutated recombinant plasmids with correct sequencing results into LB plates (add thiokanamycin 50 mg / L), streak, and culture upside down at 37°C A large number of colonies grew in about 12 hours.

[0068] Inoculate a ring of single colonies to LB medium for seed culture, and culture at 220 rpm at 37°C for about 12 hours.

[0069] Inoculate the seed culture solution into the fermentation medium at an inoculum amount of 1%, culture at 37°C, 220rpm for 2h, add inducer 0.4mM IPTG and cool down to 20°C to continue fermentation for 10h, the growth of cells fermented by shake flasks of different strains is similar , OD 600 Both are around 25.

[0070] The prepared fermented broth was centrifuged at 6000 rpm for 10 min to collect wet cells. The prepared wet cells are added to the transformation liquid, ...

Embodiment 3

[0072] The expression situation of the tyrosine phenolase of embodiment 3 recombinant escherichia coli

[0073] Centrifuge the fermentation broth of the strains containing plasmids pET-28(a)-TPL, pET-28(a)-TPL(E313W) and pET-28(a)-TPL(E313M) at 8000rpm for 3min to collect the cells, and use PB buffer (pH 8.5, 50mM KH 2 PO 4 -K2 HPO 4 ) Wash the cells 2-3 times, sonicate until the bacteria liquid is completely broken and become transparent, and centrifuge at 9000rpm for 3min to collect the supernatant. Protein purification was performed using a nickel column Ni-NTA Superflow Cabridge (5 mL) and an AKTA purifier. The purified protein solution was collected, desalted and purified using a desalting column Sephadex-G (2mL) and an AKTA purifier, and the protein concentration was detected by using the Enhanced BCA Protein AssayKit protein quantification kit (purchased from Biyuntian, item number: P0009), and analyzed by SDS- PAGE analysis of intracellular protein expression and p...

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Abstract

The invention discloses escherichia coli expressing thermostable tyrosine phenolase and application thereof, and belongs to the technical field of bioengineering. According to the invention, escherichia coli BL21 is taken as a host, recombinant plasmids pET-28(a)-TPL and 25 tyrosine phenolase mutants are expressed, recombinant escherichia coli containing mutant plasmids for producing L-DOPA is obtained, whole-cell transformation is carried out on the obtained recombinant bacteria to produce the L-DOPA, the yield of the L-DOPA produced by the recombinant bacteria expressing the pET-28(a)-TPL (E313M) reaches 54.9 g / L, compared with a control strain, the yield increases by 142.9%, the half-life period of the expressed tyrosine phenolase is improved to 37.9 min, 17.1 min and 14.6 min respectively under the conditions of 20 DEG C, 40 DEG C and 60 DEG C. The escherichia coli lays the foundation for the L-DOPA production by the escherichia coli modified by metabolic engineering.

Description

technical field [0001] The invention relates to an Escherichia coli expressing a thermostable tyrosine phenolase and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Levodopa (L-DOPA) is a derivative of amino acid and an important intermediate product in the biochemical metabolic pathway from L-tyrosine to catechol or melanin, also known as 3,4-dihydroxyphenyl Alanine, is an important active substance. L-DOPA is a new biochemical drug, which is widely used in the fields of food, medicine and health care products. The derivative of L-DOPA--dopamine is an important neurotransmitter. Since dopamine cannot enter the brain tissue through the blood-brain barrier, Parkinson's disease cannot be treated by supplementing dopamine, and L-DOPA can pass through the blood-brain barrier. Barrier, and decarboxylation in the brain tissue to form dopamine, so that the content of dopamine in the brain tissue increases to achieve the pu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N1/21C12N15/70C12P13/22C12R1/19
Inventor 周景文堵国成陈坚韩红梅曾伟主
Owner JIANGNAN UNIV
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