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A kind of co-production process and application of pyruvic acid and levodopa

A technology for pyruvic acid and pyruvic acid is applied in the field of fermentation and enzymatic catalysis, which can solve the problems of high conversion rate, high substrate cost, and no industrialized production, and achieve the effects of reducing three wastes and raw material costs.

Active Publication Date: 2020-06-19
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dariush Hek-mat et al. used an enzyme membrane reactor to convert lactic acid into pyruvate using the crude cell extract of Proteus vulgaris. This method has a high conversion rate, but the substrate cost is high, and industrial production has not been realized.

Method used

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  • A kind of co-production process and application of pyruvic acid and levodopa

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0031] Example 1: Obtaining pyruvate by fermentation

[0032] Activation medium (YPD liquid medium): 1% yeast extract, 2% peptone, 2% glucose.

[0033] Seed medium: 10% glucose, 3% fish peptone, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 4 mg / L nicotinic acid, 400 μg / L pyridoxal, 20 μg / L biotin and 4% CaCO3, pH 5.5.

[0034]Fermentation medium: 10% glucose, 0.1% soybean peptone, 0.6% (NH4) 2SO4, 0.1% KH2PO4, 0.05%, MgSO4 7H2O, 8 mg / L niacin, 1 mg / L pyridoxal, 20 μg / L biotin, 20 μg / L Thiamine and 4% CaCO3, pH 5.5.

[0035] Activation culture: Inoculate the strains stored in the -80°C cryopreservation tube into a 30ml screw bottle containing 5ml YPD liquid medium with a pipette tip, and activate the culture at 30°C or 40°C, 220r / min, approx. After 12 hours, the cells were grown to the logarithmic phase for transfer culture.

[0036] Seed culture: inoculate the activated bacterial liquid into a 500ml shake flask with a certain volume (10, 15, 25ml) of seed medium, and cultivate for 24 hou...

example 2

[0040] Example 2: Fermentative production of tyrosine phenol lyase

[0041] LB medium: tryptone 10g / L, yeast extract 0.5g / L, sodium chloride 10g / L, pure water.

[0042] 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.

[0043] 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;

[0044] 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;

[0045] 3) Centrifuge the bacterial liquid in step (2) to collect the bacterial cells, and place in a -20°C refrigerator.

example 3

[0046] Example 3: Extraction of Tyrosine Phenol Lyase

[0047] 1) Add 3 times the volume of water to the bacteria, and break the cells with a high-pressure homogenizer;

[0048] 2) High-speed centrifugation to obtain supernatant enzyme liquid;

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Abstract

The invention relates to a pyruvic acid and L-dopa co-production process and application. The process comprises the following steps: (1) obtaining a pyruvic acid feed liquid by fermentation with torulopsis glabrata, sterilizing the feed liquid by a ceramic membrane, and removing proteins, nucleic acids, pigments and other macromolecules by an ultrafiltration membrane; (2) adding a certain amount of catechol, ammonium acetate, EDTA, sodium sulfite and other compounds to a pyruvic acid concentrate according to the molar amount of pyruvic acid, adjusting the pH to 7.0-9.0, and preparing an enzyme-catalyzed substrate solution; (3) fermenting with tyrosine-phenol lyase genetic engineering bacteria to obtain a tyrosine-phenol lyase bacterial solution, centrifuging to collect thalli, breaking cells by a high-pressure homogenizer, centrifuging, and collecting an enzyme solution; and (4) adding a certain amount of substrate solution to the enzyme solution, stirring evenly, and at the temperature of 25 DEG C, carrying out seal shock reaction. The substrate solution is prepared from the pyruvic acid concentrate, and the concentration of catechol is controlled at 0-10 g / L by fed-batching the substrate solution, the product reaches 120 g / L or more, and fed-batch of the substrate solution is stopped; when the content of catechol in the reaction solution is less than 0.2 g / L, and the reactionis stopped.

Description

technical field [0001] The invention relates to a pyruvate and levodopa co-production process and its application, belonging to the field of fermentation and enzyme catalysis processes. 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), also known as β-tyrosinase, has a molecul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/22C12P7/40C12R1/88
CPCC12P7/40C12P13/225
Inventor 储消和林挺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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