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Inducible expression method in fermentation process of tyrosine phenol-lyase

A technology of inducing expression and tyrosine phenol is applied in the field of inducing expression in the fermentation process of tyrosine phenol lyase, which can solve the problems of low temperature and poor adaptability of inducers, and achieve the effect of reducing toxic effect and reducing toxicity.

Active Publication Date: 2019-01-01
SHANDONG LUKANG PHARMA
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  • Abstract
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Problems solved by technology

[0005] In view of the problems existing in the background technology, the object of the present invention is to solve the problem that the bacteria have poor adaptability to low temperature and inducers, and improve the expression level of tyrosine phenol lyase

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  • Inducible expression method in fermentation process of tyrosine phenol-lyase

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Embodiment 1

[0040] (1) Bacteria preservation and activation

[0041] (1) Strain: Escherichia coli genetically engineered strain (purchased from Hong Kong Biaoxinte Co., Ltd.) that expresses tyrosine phenol lyase (TPL) efficiently. It has kan resistance and can catalyze catechol, Pyruvic acid and ammonia synthesize levodopa.

[0042] (2) Preservation: Preserve at 20% glycerol -80°C (pick a single colony from a fresh plate to a 5mL LB+kan test tube, 37°C, 220rpm, culture for 12-16h; 40% glycerol and LB bacteria suspension volume ratio 1:1 preservation seed ); Store the plate or inclined surface at 4℃, and replace it once every two weeks at most.

[0043] (3) Activation: LB+kan plate or inclined plane with glycerol tube, 37℃, incubate for 12-16h.

[0044] (2) Seed and fermentation culture

[0045] (1) Fermentation process: glycerin tube → LB plate → LB shake flask → TB seed culture → fermentation culture

[0046] (2) Activation: Aseptically use a sterile inoculation needle to pick a single colony fro...

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Abstract

The invention provides an inducible expression method in a fermentation process of tyrosine phenol-lyase, which belongs to the technical field of biological fermentation. The method comprises the following steps: firstly fermenting a culture strain to OD600 of 25 to 30 under the condition of 36 to 38 DEG C, and obtaining to-be-induced fermentation solution; then cooling the to-be-induced fermentation solution in different phases, and adding an induction agent, observing an adaptation state of thallus according to the change of an oxygen dissolving level, obtaining a final induction fermentation solution after the dissolved oxygen amount is stable, and keeping the fermentation state until the fermentation is ended. The thallus is induced by virtue of the method of the invention, so that theharm of the induction agent for the thallus can be maximally reduced, and the problem that the thallus is poor in adaptability to the low temperature and the induction agent can be effectively solved. The thallus OD600 after the fermentation is ended is not less than 100, the wet weight of the thallus is 180 g / L or more, and the expression amount of the tyrosine phenol-lyase in the fermentation solution is greater than 15 g / L.

Description

Technical field [0001] The invention belongs to the technical field of biological fermentation, and specifically relates to a method for inducing expression in the fermentation process of tyrosine phenol lyase. Background technique [0002] Levodopa, also known as 3,4-dihydroxyphenylalanine, is a white crystalline powder, odorless and tasteless, insoluble in ethanol, chloroform and acetone, and easily soluble in dilute acid. As an important biologically active substance, levodopa is the precursor of the neurotransmitter dopamine, which can pass through the blood-brain barrier and enter the cerebral circulation to reach the central nervous system. Under the action of central nervous decarboxylase, it is converted into dopamine, thereby Increase the content of dopamine in brain tissue to achieve the effect of treating Parkinson's disease. Levodopa is the first and most effective drug for the treatment of elderly Parkinson's disease. In addition, levodopa also has the effect of tre...

Claims

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Application Information

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IPC IPC(8): C12N9/88
CPCC12N9/88C12Y401/99002
Inventor 金伟郑长春王博慕东朱维忠卢春玲田晓梅戴晓燕
Owner SHANDONG LUKANG PHARMA
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