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Method for manufacturing D-phenylalanine by bio-enzyme asymmetric transformation

A technology of phenylalanine and a production method, applied in the field of bioengineering, can solve the problems of high production cost, low substrate concentration and high cost, and achieve the effects of low production cost, simple production process and mild conditions

Inactive Publication Date: 2010-03-17
淮北新旗氨基酸有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the "aminoacylase method" is currently the conventional method for producing D-amino acids, and it is also widely used in the production of other D-amino acids. However, because aminoacylase is expensive, it can only be used for small-scale production.
The "asymmetric chemical transformation" reaction mechanism is too complicated, the process is long, and expensive pure chiral reagents are required as resolving agents, resulting in high production costs
At present, the industrial production of D-phenylalanine at home and abroad generally adopts the "hydantoin enzymatic method", which requires the participation of two enzyme catalysts, low substrate concentration, long conversion time, low conversion rate, low product purity, and low cost. very high
At present, domestic and foreign enterprises have industrial production methods for D-phenylalanine, which have the disadvantages of small scale, low yield and high cost.
Unable to cope with growing worldwide demand for D-phenylalanine

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Prepare 1000ml of medium with the mass fraction of each component as follows.

[0024] Glucose 0.5%, peptone 1%, yeast extract 1%, NaCl 0.5%, K 2 HPO 4 0.1%, L-tyrosine 0.1%, patoin 0.01%, DL-phenylalanine 0.1%, glutamic acid 0.1%.

[0025] Adjust the pH to 5.5 with sulfuric acid.

[0026] This 1000ml medium is divided into 1000ml Erlenmeyer flasks, and the volume of each bottle is 250ml. The above medium was autoclaved at 121°C for 15 minutes. Cool to 30°C, pick a loop of Rhodotorula viscosus stored on the slope with a ф1mm inoculation loop and insert it into the above medium. Incubate at 30° C., 110 rpm for 24 hours using a rotary shaker.

[0027] Combine 1 liter of the above cell culture solution, adjust the pH to 8.5, add 30 g of DL-phenylalanine, control the temperature at 45°C, and start adding DL-phenylalanine after 5 to 10 hours of reaction. The reaction continues for 40 hours, and the cumulative Add 250 g of DL-phenylalanine, stop feeding, continue to rea...

Embodiment 2

[0029] Prepare 1000ml of medium with the mass fraction of each component as follows.

[0030] Glucose 0.5%, peptone 1%, yeast extract 1%, NaCl 0.5%, K 2 HPO 4 0.1%, L-tyrosine 0.1%, patoin 0.01%, DL-phenylalanine 0.1%, glutamic acid 0.1%.

[0031] Adjust the pH to 5.5 with sulfuric acid.

[0032] This 1000ml medium is divided into 1000ml Erlenmeyer flasks, and the volume of each bottle is 250ml. The above medium was autoclaved at 121°C for 15 minutes. Cool to 30°C, pick a loop of Rhodotorula viscosus stored on the slope with a ф1mm inoculation loop and insert it into the above medium. Incubate at 30° C., 110 rpm for 24 hours using a rotary shaker.

[0033] Centrifuge the above cell culture solution, take 6 g of centrifuged bacterial cells and add 200 ml of pre-prepared DL-phenylalanine solution (concentration 3%), 45 ° C, 110 rpm, and continue to react for 10 hours. Sulfuric acid was added to the reaction solution to adjust the pH to 2, and cinnamic acid was precipitated...

Embodiment 3

[0035] Prepare primary culture containing 20g / L peptone, 20g / L yeast extract, 10g / L glucose, 5g / L sodium chloride, 18g / L corn steep liquor powder, 0.2g / L magnesium sulfate, and 1g / L potassium dihydrogen phosphate Base 150 ml, adjust the pH value to 5.5 with ammonia water or sulfuric acid, divide into 500 ml Erlenmeyer flasks, and the liquid volume of each bottle is 150 ml. The above medium was autoclaved at 121°C for 15 minutes. Cool down to 30°C, and use a ф1mm inoculation loop to pick out a loop of Rhodotorula viscosus preserved on the slant and insert it into the above-mentioned medium. Using a constant temperature shaker, culture at 30° C. and 110 rpm for 18 hours to obtain 400 ml of primary seed cell suspension.

[0036] Prepare bean cake hydrolyzate containing 20g / L (dried), malt powder 20g / L, glucose 5g / L, sodium chloride 5g / L, corn steep liquor powder 5g / L, magnesium sulfate 02g / L, potassium dihydrogen phosphate 1g / L 10 liters of fermented liquid of L, L-phenylalanin...

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PUM

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Abstract

The invention provides a biological manufacturing method of D-phenylalanine, and belongs to the enzyme engineering technology in the bioengineering field, aiming at solving the technical problem of providing a manufacturing method of D-phenylalanine by bio-enzyme asymmetric transformation which has a simple manufacturing process and can lower the manufacturing cost. The technical key points are asfollows: taking DL-phenylalanine as a raw material to take part in enzyme catalysis reaction by being used as a substrate; taking L-phenylalanine ammonialyase as a catalyst to perform catalytic conversion on L-phenylalanine in the zymolyte DL-phenylalanine to generate trans-cinnamic acid and ammonia; extracting D-phenylalanine as a target which does not take part in reaction; adding zymolyte continuously to improve concentration of the target product D-phenylalanine, forming crystal in enzymatic reaction solution and separating directly; and dissolving D-phenylalanine in the enzymatic reaction solution, regulating pH value to be 1-5, separating and removing cinnamic acid by using the water-insoluble characteristic of cinnamic acid in acid environment, concentrating and crystallizing to obtain the product. The refining purpose can be realized by membrane filtration decoloration and recrystallization of the D-phenylalanine coarse product. The product in the invention has high chemical purity and optical purity and low manufacturing cost.

Description

Technical field: [0001] The invention belongs to the enzyme engineering technology in the field of bioengineering. The invention relates to a kind of asymmetric conversion of L-phenylalanine using DL-phenylalanine as a raw material and using L-phenylalanine ammonia-lyase as a catalyst. Acid deamination produces trans-cinnamic acid and ammonia, while D-phenylalanine does not participate in the reaction at all and is extracted as a product. technical background: [0002] Amino acid biochemical products are the main substances related to human life and health, and have a wide range of uses. In recent years, the use of D-phenylalanine in food and medicine has been fully developed, especially in the field of medicine. Many companies in the world have used D-phenylalanine as raw materials to develop a series of antineoplastic drugs (Baishixin, Octreotide, etc.) etc.), type 2 diabetes treatment drugs (nateglinide), anti-AIDS drugs (indinavir) and so on. The worldwide demand for D...

Claims

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Application Information

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IPC IPC(8): C12P41/00C12P13/22C12R1/465C12R1/66C12R1/645
Inventor 黄建坡尹若春张萍萍孙旭
Owner 淮北新旗氨基酸有限公司
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