Method of preparing chiral phenylalanine by catalysis and asymmetric reduction of marine strain

A chiral phenylalanine and asymmetric technology, applied in biochemical equipment and methods, methods based on microorganisms, bacteria, etc., can solve the problems of low yield, many side reactions, and long production cycle of chemical synthesis methods. Achieve the effect of high optical purity of the product, good industrial application prospects, and simple equipment

Active Publication Date: 2014-11-19
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The yield of the chemical synthesis method is low; it needs to use highly toxic cyanide, which pollutes the environment; there are many side reactions; the process route is long and other disadvantages
The microbial fermentation method has the advantages of being environmentally friendly, energy-saving and green, but the disadvantage is that the concentration of fermentation products is low, the production cycle is long, and the process management requirements are strict.

Method used

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  • Method of preparing chiral phenylalanine by catalysis and asymmetric reduction of marine strain
  • Method of preparing chiral phenylalanine by catalysis and asymmetric reduction of marine strain
  • Method of preparing chiral phenylalanine by catalysis and asymmetric reduction of marine strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] (1) Cultivation of Bacillus nanhaiensis DSF-15A2: Inoculate the strain into 500 mL of 2216L medium with an inoculum amount of 1%. The composition of 2216L medium is 10.0g / L peptone, 5.0g / L yeast powder, 0.5g / L beef extract, 0.5g / L sodium citrate, 0.2g / L NH 4 NO 3 , 1.0g / L NaAc, configured with sea water. The culture conditions are as follows: the initial pH is 7.0, the volume fraction of the filling volume is 10%, the culture temperature is 25° C., the rotation speed of the shaker is 200 rpm, and the culture time is 24 hours.

[0026] Preparation of cells: The fermentation broth obtained at the end of the culture was centrifuged in a refrigerated centrifuge (4°C, 8000rpm, 15min) to obtain the cells, the supernatant was discarded, the pellet was resuspended with Tris-HCl buffer (pH7.5), and washed thoroughly After centrifugation, the operation was repeated 3 times to obtain cells.

[0027] (2) Whole-cell catalytic reaction: the reaction system is 0.1mol / L phenylpyruva...

Embodiment 2

[0030] (1) The experimental procedure is as in the step (1) of Example 1.

[0031](2) Whole-cell catalytic reaction: the reaction system is 0.5mol / L phenylpyruvate, 1mol / L ammonium chloride, 1mol / L glucose, 0.01mol / L Tris-hydrochloric acid buffer, pH8.0, 60g / L Cells, the reaction volume is 50mL, and the reaction time is 48h at the reaction temperature of 30°C and shaking at 200rpm.

[0032] (3) Separation and detection: the reaction solution was centrifuged to remove the precipitate, the supernatant was added with an equal volume of methanol, mixed and oscillated evenly, the precipitate was discarded by centrifugation, and the supernatant was diluted for later use. Detected by high performance liquid chromatography, the calculated yield of the product phenylalanine is 85.2%, and the e.e. value is 99.9%.

Embodiment 3

[0034] (1) The experimental procedure is as in the step (1) of Example 1.

[0035] (2) Whole-cell catalytic reaction: the reaction system is 0.5mol / L phenylpyruvate, 1mol / L histidine, 1mol / L isopropanol, 0.01mol / L carbonate buffer, pH9.5, 50g / L L of cells, the reaction volume is 50mL, and the reaction time is 48h at the reaction temperature of 35°C and shaking at 200rpm.

[0036] (3) Separation and detection: the reaction solution was centrifuged to remove the precipitate, the supernatant was added with an equal volume of methanol, mixed and oscillated evenly, the precipitate was discarded by centrifugation, and the supernatant was diluted for later use. Detected by high performance liquid chromatography, the calculated yield of the product phenylalanine is 52.1%, and the e.e. value is 99.9%.

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Abstract

The invention discloses a method of preparing chiral phenylalanine by catalysis and asymmetric reduction of a marine strain, and relates to chiral phenylalanine. Bacillus nanhaiensis DSP-15A2 is preserved in CGMCC (China General Microbiological Culture Collection Center) with a preservation number of CGMCC No. 8969. The method comprises the steps of adding the bacillus nanhaiensis to a 2216L culture medium for culture to obtain a fermentation solution, centrifuging to obtain a cell, resuspending and washing with a buffer solution, preparing a cell solution, adding phenylpyruvic acid and an amino donor to the cell solution as substrates, adding an auxiliary substrate for cyclic regeneration of coenzyme, performing a reaction to obtain a reaction solution containing a product namely L-phenylalanine, centrifuging to remove deposit, adding equivalent methanol to a supernate, shaking and mixing uniformly, centrifuging to remove deposit (protein), diluting the supernate, then measuring, detecting the concentration of phenylpyruvic acid by high performance liquid chromatography, and measuring the concentration of the product namely L-phenylalanine and an excessive value of an antipode by the high performance liquid chromatography.

Description

technical field [0001] The invention relates to chiral phenylalanine (L-phenylalanine), in particular to a method for preparing chiral phenylalanine by catalytic asymmetric reduction of marine strains. Background technique [0002] Chiral amino acids (natural L-amino acids and unnatural D-amino acids) are key intermediates for the synthesis of fine chemicals such as chiral drugs, chiral pesticides and chiral food additives. The preparation of many new drugs (such as protease inhibitors, anti-AIDS drugs, etc.) requires the use of artificially synthesized unnatural chiral amino acids. The synthesis of many chiral amino acids is inseparable from the catalysis of oxidoreductases, so the search for highly selective oxidoreductases has become a hot spot in the research of chiral amino acids. [0003] Phenylalanine is mainly used as a reagent and parenteral nutrition infusion in medicine, and also used as an ingredient in nutritional supplements such as synthetic diets and essenti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/22C12R1/07
Inventor 方柏山任红王世珍
Owner XIAMEN UNIV
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