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Method for improving efficiency of catalytic production of L-dopa by tyrosine phenol lyase

A technology of levodopa and tyrosine phenol, which is applied in the field of enzyme catalysis, can solve the problems of low yield of L-DOPA, difficult control of reaction conditions, poor stability, etc., and achieve improved conversion rate and product concentration, low cost, The effect of improving product quality

Pending Publication Date: 2019-07-26
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, the reaction conditions are difficult to control, the stability is poor, there are many by-products, and the yield of L-DOPA is low

Method used

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  • Method for improving efficiency of catalytic production of L-dopa by tyrosine phenol lyase

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0022] tyrosine phenol lyase

[0023] LB medium: tryptone 10g / L, yeast extract 0.5g / L, sodium chloride 10g / L, pure water.

[0024] Fermentation medium: tryptone 12g / L, yeast extract 24g / L, glycerin 5g / L, potassium dihydrogen phosphate 2.31g / L, dipotassium hydrogen phosphate trihydrate 16.43g / L, pure water.

[0025] 1) Pick a single colony and inoculate it into a 4ml LB medium test tube, add kanamycin (50mg / L), 37°C, 220rpm, and cultivate for 12h to obtain first-grade seeds;

[0026] 2) The primary seeds were inoculated into 100ml of fermentation medium shake flask, 37°C, 220rpm, cultured for 4h, added IPTG to a final concentration of 1mM, 25°C, 220rpm, cultured for 12h;

[0027] 3) Centrifuge the bacterial liquid in step (2) to collect the bacterial cells, and place in a -20°C refrigerator.

example 2

[0028] Example 2: Extraction of Tyrosine Phenol Lyase

[0029] 1) Add 3 times the volume of water to the bacteria, and ultrasonically disrupt the cells for 20 minutes;

[0030] 2) High-speed centrifugation to obtain supernatant enzyme liquid;

example 3

[0031] Example 3: Conversion of tyrosine phenol lyase to produce L-dopa

[0032] 1) 1L substrate solution: 14g / L sodium pyruvate, 10g / L catechol, 40g / L ammonium chloride, 2g / L sodium sulfite, 1g / L EDTA, adjust pH to 8.0;

[0033] 2) Add 10-100g of enzyme solution, add 1L of substrate solution, stir well, seal and shake at 25°C;

[0034] 3) Add a substrate (an equivalent amount of sodium pyruvate and quinone) every half hour, and control the substrate concentration of the two substrates to not be higher than 10g / L;

[0035] 4) When the product concentration reaches more than 20g / L, add 20g / L of crystals to induce a large amount of crystallization of the product in advance;

[0036] 5) When the residual concentration of catechol falls below 0.2g / L, stop the reaction, and the accumulated L-dopa concentration reaches above 130g / L.

[0037] 6) The reaction feed liquid is acidified with dilute sulfuric acid or dilute hydrochloric acid to dissolve the crystals, and then centrifuged...

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Abstract

The invention relates to a method for improving the efficiency of catalytic production of L-dopa by tyrosine phenol lyase. The method is characterized by comprising the steps: (1) selecting a single colony, inoculating a test tube containing an LB culture medium with the single colony, adding kanamycin (50 mg / L) at the temperature of 30-37 DEG C, at the speed of 220 rpm, culturing for 12-16 h, andthus obtaining primary seeds; (2) inoculating a shaking flask containing a fermentation culture medium with the primary seeds, and culturing for 10-12 h; (3) centrifuging, collecting bacteria to obtain the bacteria, breaking cells by ultrasound, and centrifuging at high speed to obtain a supernatant enzyme liquid; and (4) adding an enzyme solution into a substrate solution, stirring evenly, and carrying out sealed oscillation reaction at the temperature of 25 DEG C, wherein the substrate solution comprises 14-16 g / L of sodium pyruvate, 10-12 g / L of catechol, 50-60 g / L of ammonium acetate, 2-5g / L of sodium sulfite, and 1-3 g / L of EDTA, and the pH is adjusted to 7.5-8.5. The concentration of the reaction product reaches 20 g / L or more, and 10-30 g / L of L-dopa crystals are added to induce the product to crystallize in advance. The method greatly improves the conversion rate and the product concentration, improves the product quality, has simple process, low cost and high yield, and hasthe application value of industrial production.

Description

technical field [0001] The invention relates to a method and application for improving the efficiency of tyrosine phenol lyase to catalyze the production of levodopa, and belongs to the field of enzyme catalysis technology. Background technique [0002] The chemical name of levodopa (3,4-dihydroxyphenyl-L-ananine, referred to as L-DOPA) is 3,4-dihydroxyphenylalanine, and its structural formula is: [0003] [0004] As an important biologically active substance, L-DOPA is an important intermediate product in the biochemical metabolic pathway from L-tyrosine to catechol or melanin. [0005] In the 1960s, many foreign scholars began to devote themselves to the research of microbial enzymatic synthesis of L-DOPA. In order to increase the yield of L-DOPA and the conversion rate of the substrate, researchers have conducted a lot of research on the process of microbial enzymatic synthesis of L-DOPA. [0006] Tyrosine phenol lyase (Tyrosine phenol lyase, TPL, E.C.4.1.99.2), als...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/22C12N9/88
CPCC12N9/88C12P13/22C12Y401/99002
Inventor 储消和吴黎诚生英涛陈万河程跃徐顺清沈建方明山余炜柳鹏福孙俊杰
Owner ZHEJIANG UNIV OF TECH
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