Preparation method of L-tyrosine or L-tyrosine derivative employing enzyme conversion

A technology for enzymatic transformation and tyrosine, which is applied in the field of enzymatic transformation and preparation of L-tyrosine or L-tyrosine derivatives, and can solve the problem of high cost of L-tyrosine or L-dopa raw and auxiliary materials , pyruvic acid or L-serine have high cost, difficult to industrialize production and other problems, and achieve the effect of outstanding implementation effect, omitting separation and purification process, and reducing production cost.

Active Publication Date: 2015-02-04
NANJING UNIV
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0026] Among the above routes of enzymatic conversion and synthesis of L-tyrosine or L-dopa, tyrosine phenol lyase has the highest activity and is the closest to industrial application, but one of the reaction substrates, pyruvate or L-serine, has a relatively high cost. The high-purity pyruvate or L-serine enzyme method to prepare L-tyrosine or L-dopa raw materials is expensive and difficult to carry out industrial production. Therefore, it is necessary to find abundant and cheap pyruvate or L-serine industrial raw materials The key to the industrial production of L-tyrosine or L-dopa by enzymatic method of acid phenol lyase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Cultivate Citrobacter freundii ATCC8090 in the following 1000ml medium (g / ml): corn steep liquor 0.5%, NaCl 0.5%, KH 2 PO 4 0.05%, MgSO 4 ·7H 2 O 0.02%, beef extract 2%, glucose 1%, maltose 0.5%, pH 7.2. Shake culture at 37°C for 18h, centrifuge at 4000r / min for 15min to obtain 12g of wet cells.

[0043] 2. Add the wet bacteria to 1000ml transformation liquid, the transformation liquid is pyruvic acid fermentation liquid containing 10g / L pyruvic acid, add 10g ammonium chloride, 21ml 500g / L phenol aqueous solution, adjust pH to 8.5 with ammonia water, then add 0.2g / L Pyridoxal phosphate and 0.01g / L Tween 80 were enzymatically reacted at 45°C for 3h, and the total volume was 1050ml after the reaction. The concentration of L-tyrosine in the conversion liquid was detected by sampling alkali solution HPLC to be 18.3 g / L, and the molar conversion rate of p-phenol was 95%.

[0044] 3. Centrifuge the transformation solution at 4000r / min for 15min, collect 38.4g of the...

Embodiment 2

[0046] 1. Cultivate Citrobacter freundii ATCC8090 in the following 1000ml medium (g / ml): 0.5% corn steep liquor, 0.5% protein hydrolyzate, 2% beef extract, 1.0% maltose, 0.5% lactose, 0.5% NaCl, KH 2 PO 4 0.5%, MgSO 4 ·7H 2 O0.03%, pH7.2. Shake culture at 37°C for 16h, centrifuge at 4000r / min for 15min to obtain 14g of wet cells.

[0047] 2. Add the wet bacteria to 1000ml transformation liquid, the transformation liquid is pyruvic acid fermentation liquid containing 30g / L pyruvic acid, add 10g ammonium chloride, 20ml 500g / L phenol aqueous solution, adjust pH to 9.0 with ammonia water, then add 0.5g / L Pyridoxal phosphate and 0.1g / L Tween 80 were enzymatically reacted at 45°C, and 44ml of 500g / L phenol aqueous solution was added. After 12 hours of reaction, the total volume of the conversion solution was 1100ml. The concentration of L-tyrosine in the conversion liquid detected by HPLC after sampling and alkali dissolution was 53.8g / L, and the molar conversion rate of p-phen...

Embodiment 3

[0050] 1. Cultivate Citrobacter koseri ATCC BAA-895 in the following 1000ml medium (g / ml): peptone 1.0%, beef extract 0.5%, sucrose 0.5%, fructose 1.0%, NaCl 0.5%, KH 2 PO 4 0.3%, K 2 HPO 4 0.1%, MgSO 4 ·7H 2 O0.05%, pH7.2. Shake culture at 37°C for 16h, centrifuge at 4000r / min for 15min to obtain 15g of wet cells.

[0051] 2. Add the wet bacteria to 1000ml transformation liquid, the transformation liquid is pyruvic acid fermentation liquid containing 150g / L pyruvic acid, add 10g ammonium chloride, 20ml 500g / L phenol aqueous solution, adjust pH to 11.0 with ammonia water, then add 0.05g / L Pyridoxal phosphate and 1.0g / L TritonX-100 were enzymatically reacted at 45°C, and 300ml of 500g / L phenol aqueous solution was added, and the reaction was completed for 22 hours, and the total volume of the conversion solution was 1350ml. The concentration of L-tyrosine in the conversion liquid was detected by sampling alkali solution HPLC to be 214.5 g / L, and the molar conversion rate...

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Abstract

The invention belongs to the technical field of organisms, and particularly relates to a preparation method of L-tyrosine or L-tyrosine derivative (L-3-hydroxy tyrosine) employing enzyme conversion. The preparation method comprises the steps of: adopting feed liquid containing pyruvic acid as a substrate; adding bacterial cells with tyrosine phenol lyase activity, or crude enzyme, ammonia water, phenol or pyrocatechol; carrying out enzymatic reaction under the conditions that the temperature is 25-55 DEG C and the pH is 7-11; and separately preparing a converted product L-tyrosine or L-dioxyphenylalanine by using an isoelectric point crystallization method. By adopting the method, the L-tyrosine or L-dioxyphenylalanine with high additional value is synthetized by using cheap pyruvic acid liquid as the substrate by an enzymic method; the preparation method has the advantages of being wide in material source, high in enzymatic conversion efficiency, simple and convenient to operate, low in production cost and the like.

Description

1. Technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an enzymatic conversion preparation method of L-tyrosine or L-tyrosine derivatives (L-3-hydroxytyrosine). 2. Background technology [0002] L-tyrosine is an aromatic amino acid and one of the important amino acids needed for protein synthesis in animals. In the field of medicine, L-tyrosine is the precursor raw material for the synthesis of thyroxine, epinephrine, L-dopa, tyrosine sulfite, etc.; clinically, tyrosine sulfite is used to treat polio and In the acute stage of tuberculous meningitis, schizophrenia and other central nervous system diseases, L-tyrosine methyl ester hydrochloride can also be used in the field of cosmetics. [0003] L-tyrosine derivatives (L-3-hydroxytyrosine), also known as L-dopa, is an important active substance in the body, which is produced in the process of metabolism from L-tyrosine to catechol or melanin. important intermediate produc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/22C12P13/04C12R1/18C12R1/01
Inventor 焦庆才刘均忠常俊俊冯莹莹刘茜
Owner NANJING UNIV
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