Escherichia coli for expressing tyrosine phenol lyase active inclusion body and application of escherichia coli

A technology of tyrosine phenol and recombinant Escherichia coli, which is applied in the field of bioengineering, can solve problems such as environmental pollution, complicated process, and high cost, and achieve good application prospects, simple construction method, and easy operation

Active Publication Date: 2020-10-02
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

[0004] L-DOPA mainly exists in plants such as broad beans and edamame in nature. The separation process with the chiral isomer D-DOPA is eliminated in the extraction method, and the extraction yield of L-DOPA has also been improved accordingly. , but due to the limited source of raw materials and low output, the production cost is high, and it is difficult to extract L-DOPA from plants for large-scale production, which is far from meeting market demand
Although the chemical synthesis method is widely used in the commercial production of L-DOPA, a large amount of lead and other metals are required as catalysts in the chemical synthesis process, and the process is complicated, the conversion efficiency and enantioselectivity of the product are low, and there are costs. High, serious environmental pollution and other issues
The enzymatic conversion method using tyrosinase, p-hydroxyphenylacetate 3-hydroxylase and tyrosine phenol lyase as the main biocatalyst can effectively overcome the environmental problems caused by chemical methods. The above enzymatic conversion synthesis L- Among the routes of DOPA, tyrosine phenol lyase has the highest activity, which is the closest to industrial application, but when the concentration of catechol, one of the reaction substrates, is higher than 0.1M, it has an inhibitory effect on the activity of tyrosine phenol lyase. It also has certain toxicity to cells. At present, the strategy of substrate flow and feeding is mainly used. Therefore, the key to realize the industrialization of L-DOPA prepared by enzymatic method of tyrosine phenol lyase is to relieve the inhibitory effect and toxicity of catechol

Method used

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  • Escherichia coli for expressing tyrosine phenol lyase active inclusion body and application of escherichia coli
  • Escherichia coli for expressing tyrosine phenol lyase active inclusion body and application of escherichia coli
  • Escherichia coli for expressing tyrosine phenol lyase active inclusion body and application of escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of recombinant plasmid pET-28(a)-TPL-peptide fused with functional short peptide.

[0058] Using the primer pET-28-TPL-F / R shown in Table 2, the plasmid pET-28 (a)-TPL (construction method reference publication number is CN109897845A patent application) is carried out linearization by polymerase chain reaction (PCR). PCR amplification, PCR conditions: 95°C pre-denaturation for 3min; 98°C denaturation for 30s; 55°C annealing for 15s; 72°C extension for 6min, 30 cycles of reaction; final extension at 72°C for 5min, PCR products were subjected to 1% agarose gel electrophoresis Detection and recovery by DNA purification kit. Nine short peptides (shown in Table 1) ELK16, DLK6, L6KD, Linker-ELP10, ELP20, L6K2, EAK16, 18A and GFIL16 were synthesized by Wuxi Tianlin Biotechnology Co. Primers TPL-ELK16-F / R, TPL-DLK6-F / R, TPL-L6KD-F / R, TPL-ELP10-F / R, TPL-ELP20-F / R, TPL-L6K2-F / R, TPL -EAK16-F / R, TPL-18A-F / R and TPL-GFIL16-F / R amplify the synthesized short ...

Embodiment 2

[0060] The expression situation of the tyrosine phenol lyase of embodiment 2 recombinant escherichia coli

[0061] The recombinant Escherichia coli TPL-ELK16, TPL-DLK6, TPL-L6KD, TPL-ELP10, TPL-ELP20, TPL-L6K2, TPL-EAK16, TPL-18A and TPL-GFIL16 constructed in Example 1 were respectively placed in TB medium After culturing for 3 hours, 0.2 mM IPTG as an inducer was added, and the temperature was lowered to 20° C., and the fermentation was continued for 10 hours. The fermentation broth was centrifuged at 8000rpm for 3min to collect the thalli, and PB buffer (pH 8.5, 50mM KH 2 PO 4 -K 2 HPO 4 ) Wash the cells 2-3 times, sonicate until the bacterial liquid is completely broken and become transparent, centrifuge at 9000rpm for 3min, and collect the supernatant and precipitate respectively. The intracellular supernatant fragment was purified by nickel column Ni-NTA Superflow Cabridge (5mL) and AKTA purifier, and the purified protein solution was collected and desalted and purifi...

Embodiment 3

[0063] TPL chemical properties and thermostability expressed by different strains of embodiment 3

[0064] The catalytic activity of the enzyme expressed by the recombinant bacteria constructed in Example 2 was measured under different pH environments, and the TPL enzyme activity was measured by the racemization reaction of tyrosine, and an enzyme activity unit was defined as decomposing and producing 1 μmol of sodium pyruvate per minute The amount of reaction substrate solution prepared by using 50mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate (pH 6-8) and 50mM glycine-sodium hydroxide (pH 8-10) buffer solution, 100 μmol of tyrosine solution, to 900 μL Add 100 μL of pure enzyme solution (containing 30 μmol cofactor PLP) to the reaction substrate to activate the enzyme to catalyze the reaction. The reaction pH range is 6-10, the reaction temperature is 20°C, and it is protected from light. After 10 minutes of reaction, add 100 μL of 2M to the reaction solution...

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Abstract

The invention discloses escherichia coli for expressing tyrosine phenol lyase active inclusion body and an application of the escherichia coli, and belongs to the technical field of bioengineering. Escherichia coli BL21 is used as a host to express a recombinant plasmid pET-28 (a)-TPL and 9 functional oligopeptides fused with the carboxyl terminal of tyrosine phenol lyase separately, recombinant escherichia coli for producing L-DOPA is obtained, and the L-DOPA is produced through whole-cell transformation of the obtained recombinant bacteria, wherein the L-DOPA yields of the whole cell reactions of the strains TPL-ELP10, TPL-EAK16, TPL-18A and TPL-GFIL16 are 37.8 + / -1.5 g / L, 53.8 + / -2.1 g / L, 37.5 + / -0.5 g / L and 29.1 + / -1.9 g / L respectively; and compared with a control strain, the yield isimproved by 111%, 201%, 109% and 62.6% respectively.

Description

technical field [0001] The invention relates to an Escherichia coli expressing active inclusion body of tyrosine phenol lyase and 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 achi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/22C12R1/19
CPCC12N9/88C12Y401/99002C12N15/70C12P13/225
Inventor 周景文陈坚韩红梅曾伟主堵国成
Owner JIANGNAN UNIV
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