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Conversion and extraction method for producing levodopa by enzyme method

A technology of levodopa and extraction method, which is applied in the field of bioengineering, can solve the problems of large amount of acid and alkali, easy oxidation of levodopa, etc., to reduce the amount of acid and alkali, avoid denaturation and synthesis of by-products in the reaction system, Avoid the effect of oxidation

Pending Publication Date: 2020-07-14
山东惠仕莱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The tyrosine phenol lyase method to prepare levodopa mainly faces the problems that excessive catechol inhibits the activity of TPL enzyme, levodopa is easily oxidized during the conversion process, and the adsorption rate of activated carbon decolorization to levodopa is as high as 20%. Crystallization causes problems such as excessive acid and alkali dosage

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: the preparation of escherichia coli fermented liquid

[0035] Incline medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar powder 15g / L. pH to 7.0, the final concentration of kanamycin is 50mg / L.

[0036] Seed medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH to 7.0, final concentration of kanamycin 50mg / L.

[0037] Fermentation medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH to 7.0, final concentration of kanamycin 50mg / L.

[0038] One ring of freshly cultured E. coli was inoculated into the seed medium, and cultured at 37° C. and 180 rpm for 16 hours. A 20L automatic fermenter was used for fermentation and cultivation, and the liquid seeds were inoculated into the fermentation medium with an inoculation amount of 5%, and cultivated to OD at 37°C and 300rpm. 600 When =6, add IPTG (filter-sterilized) with a final concentration of 0.2mM as an inducer, induce enzyme production at 25°C, and culture for 24h. The wet weight of th...

Embodiment 2

[0039] Embodiment 2: the preparation of tyrosine phenol lyase

[0040] The fermentation broth was centrifuged at 8000rpm for 10min to collect Escherichia coli, and the bacteria were resuspended in 0.2M phosphate buffer (pH7.2) with 10% of the volume of the fermentation broth, and the wall was broken by a high-pressure homogenizer at a pressure of 800bar for 15min; The wall liquid passes through the ceramic membrane with a pore size of 200nm. Add 80% of the volume of the feed liquid to clean the ceramic membrane and collect the filtrate; pass the filtrate through the ultrafiltration membrane with a molecular weight cut-off of 100KD, add 80% of the feed liquid volume to clean the ultrafiltration membrane membrane to collect the retentate; the retentate was treated with a macroporous adsorption decolorizing resin (SP207) at a flow rate of 2BV, and the effluent was collected to obtain tyrosine phenol lyase with an enzyme activity of 55.6U / mL.

Embodiment 3

[0041] Embodiment 3: the preparation of tyrosine phenol lyase

[0042]The fermentation broth was centrifuged at 6000rpm for 20min to collect Escherichia coli, and the bacteria were resuspended in 0.2M phosphate buffer (pH7.2) with 20% of the volume of the fermentation broth, and the wall was broken by a high-pressure homogenizer at a pressure of 600bar for 30min; The wall liquid passes through the ceramic membrane with a pore size of 100nm. Add 30% of the volume of the feed liquid to clean the ceramic membrane and collect the filtrate; pass the filtrate through the ultrafiltration membrane with a molecular weight cut-off of 150KD, add 30% of the feed liquid volume to clean the ultrafiltration membrane membrane to collect the retentate; the retentate was treated with ion-exchange decolorization resin (A-722), the flow rate was controlled to 1BV, and the effluent was collected to obtain tyrosine phenol lyase with an enzyme activity of 53.1U / mL.

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PUM

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Abstract

The invention provides a conversion and extraction method for producing levodopa by an enzyme method. The method comprises the following steps of 1, preparing tyrosine phenol lyase, that is, centrifuging and collecting escherichia coli by a fermentation liquid, resuspending thalli by using a phosphate buffer solution, and carrying out wall breaking treatment through a high-pressure homogenizer; passing a wall-breaking liquid through a ceramic membrane, removing thallus fragments, and collecting filtrate; passing the filtrate through an ultrafiltration membrane, removing small-molecule impurities in the cells, and collecting entrapment liquid; and treating the retentate through decolorization resin, and collecting the effluent to obtain the tyrosine phenol lyase; 2, producing levodopa through enzymatic conversion, adding tyrosine phenol lyase into a reaction system based on 3000-10000U / L, adding a reaction bed charge, and supplementing catechol, sodium pyruvate and ammonium chloride inthe conversion process in different patches, wherein the yield of levodopa in the conversion liquid is 120-150g / L; and 3, carrying out extraction of levodopa, that is, adjusting the pH of the conversion liquid to be 3-4, and performing standing for 8-16h at a temperature of 4-8 DEG C; carrying out centrifugation collection of precipitation, carrying out pulping by 5-10 times of pure water and thencarrying out suction filtration, and repeating the steps for 2-5 times; eluting crystals by ethyl alcohol for 1-5 times and performing suction filtration to a dry state; and placing the crystals at atemperature of 40-80 DEG C and performing drying 1-4h to obtain a levodopa. The content of the levodopa reaches 98% or above, and the total yield reaches 80% or above.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a conversion and extraction method for producing levodopa by conversion with tyrosine phenol lyase. Background technique [0002] Levodopa (L-DOPA), also known as 3,4-dihydroxyphenylalanine, can pass through the blood-brain barrier and be converted into dopamine under the action of decarboxylase in the central nervous system, thereby increasing the content of dopamine in brain tissue to achieve therapeutic effect. Purpose of Parkinsonism. In 1970, it was approved by the US Food and Drug Administration (FDA) as a drug for the treatment of Parkinson's syndrome. So far, levodopa has been the first choice and the most effective drug for the treatment of Parkinson's syndrome at home and abroad. [0003] In the 1960s, many foreign scholars began to devote themselves to the research of microbial enzymatic synthesis of L-DOPA. Compared with plant extraction and chemical synthesi...

Claims

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Application Information

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IPC IPC(8): C12P13/22C12N9/88C07C227/40C07C229/36C12R1/19
CPCC12P13/225C12N9/88C07C227/40C12Y401/99002C07C229/36
Inventor 任明刘建民孙荣张雷
Owner 山东惠仕莱生物科技有限公司
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