Method for preparing levodopa by virtue of enzymic method

A technology for L-dopa and enzymatic preparation, applied in the field of microorganisms, can solve the problems of less than 70% tyrosine molar conversion rate, inability to achieve effective conversion, low tyrosinase activity, etc., and achieve significant economic benefits and industrial applications. Value, improved tyrosine conversion, high enantioselectivity effect

Active Publication Date: 2015-06-24
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Peng Zhenrong's method, the activity of tyrosinase is low, and the highest can only convert 8.6g / L tyrosine into 7.8g / L levodopa, and adding high content of tyrosine can not achieve effective conversion, and the product concentration Low, difficult to separate and extract
Yoshitake Tanaka of Japan (Agi. Biol. Chem. 38(3), 633-639, 1974) also improved the enzyme production performance by mutagenesis breeding, and the concentration of levodopa in the transformation solution after transformation was 21g / L , but the molar conversion rate of tyrosine in this method is less than 70%, a large amount of substrate tyrosine remains, separation and extraction are difficult, and there is a considerable distance from industrial application
[0015] From this it can be seen that the currently disclosed method of converting tyrosine into levodopa with tyrosinase has the problem of low yield or low conversion rate, making the product Low yield, difficult separation and extraction, high production cost, complicated operation process, unfavorable for industrialization

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Pick a ring of Pseudomonas maltophilia (Pseudomonas maltophilia) ATCC17806 (commercially available, the same below) and insert it into a 500ml Erlenmeyer flask containing 50ml of seed medium, culture at 30°C and 180r / min 24h, become a first-class seed;

[0045] (2) Put the seed liquid into an automatic fermenter with 5% inoculation amount into an automatic fermenter equipped with 3L fermentation medium. During the fermentation, the main parameters are controlled as follows: stirring speed 500-700r / min, dissolved oxygen 40-50%, temperature 30 ℃, pH 7.0, cultured for 36 hours to obtain fermentation broth, centrifuged at 8000r / min, 0℃ for 10 minutes to harvest 210g of wet cells (that is, bacterial cells, the same below);

[0046] (3) Prepare 1000mL 0.2mol acetic acid-sodium acetate buffer solution, pH 5.0, add 10g tyrosine, 50g wet cells and mix well, then add 1g triton, 10g ascorbic acid, 0.2g copper sulfate in sequence, at 30℃, The enzymatic reaction was carried out...

Embodiment 2

[0050] (1) Pick a ring of Pseudomonas maltophilia (Pseudomonas maltophilia) ATCC17806 strain slope into a 500ml Erlenmeyer flask with 50ml seed culture medium, cultivate at 30°C, 180r / min for 24h, and become first-class seeds;

[0051] (2) Put the seed liquid into an automatic fermenter with 10% inoculation amount of 3L fermentation medium. During the fermentation, the main parameters are controlled as follows: stirring speed 200-300r / min, dissolved oxygen 20-30%, temperature 30 ℃, pH 7.0, cultured for 36 hours to obtain fermentation broth, centrifuged at 8000r / min, 0℃ for 10min to harvest 220g of wet cells;

[0052] (3) Prepare 1000mL 0.2mol acetic acid-sodium acetate buffer solution, pH 5.4, add 20g tyrosine, 50g wet cells and mix well, then add 1g triton, 16g ascorbic acid, 0.2g copper sulfate in sequence, at 30℃, The enzymatic reaction was carried out under the condition of pH 5.5 for 14 hours, and the content of levodopa was 19.6g / L measured by HPLC after sampling and aci...

Embodiment 3

[0055] (1) Pick a ring of Pseudomonas maltophilia (Pseudomonas maltophilia) ATCC17806 strain slope into a 500ml Erlenmeyer flask with 50ml seed culture medium, cultivate at 30°C, 180r / min for 24h, and become first-class seeds;

[0056] (2) Put the seed liquid into the automatic fermenter with 2L fermentation medium at the inoculum amount of 7%. ℃, pH 7.0, cultured for 36 hours, after the end of fermentation, centrifuged at 8000r / min, 0℃ for 10min, and harvested 150g of wet cells;

[0057] (3) Prepare 1000mL 0.2mol acetic acid-sodium acetate buffer solution, pH 5.4, add 20g tyrosine (tyrosine is added in 5 times, add once every 2.5h, 4g each time), 40g wet cells and mix well, Add 1 g of triton, 15 g of ascorbic acid, and 0.2 g of copper sulfate in sequence, and carry out an enzymatic reaction at 25° C. and pH 5.4 for 14 hours. The acid molar conversion rate was 92.3%. The chromatographic conditions are the same as in Example 1.

[0058] Seed medium and fermentation medium ar...

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PUM

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Abstract

The invention discloses a method for preparing levodopa by virtue of an enzymic method. The method comprises the following steps: picking up an annular stenotrophomonas maltophilia strain slant for inoculation into a seed culture medium for culturing, thereby obtaining a primary seed liquid; inoculating a fermentation culture medium with the primary seed liquid at the inoculum size of 3-10% for fermentation culturing, centrifuging after the fermentation is ended and collecting bacterial cells; and adding tyrosine and the bacterial cells to a buffer solution to have an enzymatic reaction under the conditions of 18-30 DEG C and the pH of 5.0-6.0, thereby converting the tyrosine into the levodopa. According to the method, due to the resting cell transformation technology, and the preferred intermittent weak ventilation technology, adopted in the transformation process, the catalytic efficiency of the enzyme is improved; the concentration of the levodopa is 27g/L and the molar transformation rate of the tyrosine is above 99%; and besides, the method is mild in conditions, high in enantio-selectivity and free from racemization effect, is more suitable for industrial production, and has remarkable economic benefit and industrial application value.

Description

technical field [0001] The invention relates to a method for preparing levodopa, in particular to a method for enzymatically preparing levodopa, which belongs to the technical field of microorganisms. Background technique [0002] Levodopa (L-DOPA), whose chemical name is 3,4-dihydroxyphenylalanine, is an important intermediate product in the biochemical metabolic pathway from L-tyrosine to catechol or melanin. Dopamine, a derivative of L-DOPA, 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. And decarboxylation in the brain tissue to form dopamine, so that the content of dopamine in the brain tissue increases to achieve the purpose of treatment. Birkmayer in 1961 with levodopa treatment achieved significant results. L-DOPA and compound levodopa (such as Madopar) have become the most effective d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/22C12R1/38
Inventor 冯志彬王东阳蔡传康
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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