Preparation method of D-phenylalanine

A technology of phenylalanine and phenylalanine deaminase, which is applied in the field of enzyme engineering and can solve the problems of high production cost, complicated preparation process of D-phenylalanine, and difficult availability of raw materials.

Inactive Publication Date: 2018-02-02
ANHUI UNIVERSITY OF TECHNOLOGY AND SCIENCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the above-mentioned prior art, the purpose of the present invention is to overcome the complicated preparation process of D-phenylalanine in the prior art, and the preparation cost is high, and the problems such as raw mat

Method used

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  • Preparation method of D-phenylalanine
  • Preparation method of D-phenylalanine
  • Preparation method of D-phenylalanine

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preparation example Construction

[0023] The present invention provides a method for preparing D-phenylalanine, wherein the preparation method includes: oxidizing trans-cinnamic acid, phenylalanine deaminase, and L-amino acid in the presence of a buffer Enzyme, ammonia water and ammonia borane are mixed to produce high-purity D-phenylalanine.

[0024] Through the above technical solutions, the present invention uses trans-cinnamic acid as the substrate material, which is cheap and has a wide range of sources. Trans-cinnamic acid is catalyzed by phenylalanine deaminase to add ammonia (amination) to L-phenylalanine. Acid and D-phenylalanine. In the reaction system, L-phenylalanine is oxidized by L-amino acid oxidase to generate imine, which is in the reducing agent ammonia borane (NH 3 BH 3 ) Is reduced to L-phenylalanine and D-phenylalanine under the existing conditions, so that the L-phenylalanine produced in the reaction process is continuously converted into D-phenylalanine. Through the continuous cycle convers...

preparation example

[0033] The gene sequence of phenylalanine deaminase in Anabaena variabilis (for details, see the sequence published by NCBI with the accession number LF643444.1) and the gene sequence of L-amino acid oxidase in Proteus mirabilis (for details, see the accession number published by NCBI) For EU669819.1) submitted to Shanghai Shenggong Bioengineering Technology Co., Ltd. for synthesis, the gene for phenylalanine deaminase was named pal, and the gene for L-amino acid oxidase was named laao. The two genes pal and laao were respectively Connect with the cloning vector pUC vector to obtain cloning vectors pUC-pal and pUC-laao.

[0034] The pUC-laao and empty expression vector pET28a were double digested with restriction enzymes EcoRI and NdeI, respectively. pUC-pal and empty expression vector pET28a were double digested with BamH I and Not I, respectively, and the target gene fragment and pET28a vector, then ligate the double digestion product overnight at 16°C, transfer the ligation pr...

Embodiment 1

[0038] Take 50mg of the mixture of phenylalanine deaminase and L-amino acid oxidase prepared above and add it to the substrate trans-cinnamic acid containing 10mmol / L, 5mol / L ammonia (NH 3 H 2 O), 40mmol / L NH 3 BH 3 In the Tris hydrochloric acid buffer solution (pH8.5), the reaction was carried out at 35°C for 16 hours. The content of D-phenylalanine and L-phenylalanine was determined by HPLC, and the conversion rate and D of the substrate trans-cinnamic acid were calculated. -Optical purity of phenylalanine (ee D ). Where ee D Calculate according to the following formula:

[0039]

[0040] HPLC determination results such as Figure 4 Shown.

[0041] It can be seen from the figure that after the conversion, L-phenylalanine is not detected, so the optical purity of D-phenylalanine (ee D ) Over 99%.

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Abstract

The invention discloses a preparation method of D-phenylalanine. The preparation method comprises the steps of mixing trans-cinnamic acid, phenylalanine deaminase, L-amino-acid oxidase, ammonia waterand ammonia borane and performing reaction to prepare the D-phenylalanine. Through the adoption of the technical scheme, the trans-cinnamic acid serves as a substrate raw material, ammonification of the trans-cinnamic acid is catalyzed by the phenylalanine deaminase, L-phenylalanine and D-phenylalanine are generated, the L-phenylalanine in the reaction system is under the oxidation action of the L-amino-acid oxidase to generate imidic acid, and the imidic acid is reduced into the L-phenylalanine and the D-phenylalanine under the existence of a reducing agent ammonia borane (NH3BH3), so that the L-phenylalanine generated in the reaction process is continuously converted into the D-phenylalanine. Through continuous circulating conversion, the yield of the D-phenylalanine is greatly increased, the yield reaches to 96 percent, and the optical purity of the D-phenylalanine exceeds 99 percent.

Description

Technical field [0001] The present invention relates to the field of enzyme engineering and technology, in particular, to a method for preparing D-phenylalanine. Background technique [0002] D-Phenylalanine is an important pharmaceutical intermediate used to produce nateglinide, anticoagulant (PPACK) and tumor treatment drugs for the treatment of type 2 diabetes. The production methods of D-phenylalanine include chemical synthesis, microbial fermentation and enzymatic conversion synthesis. Because D-phenylalanine is a kind of unnatural aromatic amino acid, microbial fermentation needs to construct a complex metabolic pathway through metabolic engineering, and the yield is too low, and there are no reports of industrial production. At present, chemical asymmetric synthesis is mainly used And enzymatic transformation. [0003] Chemical asymmetric hydrogenation synthesis requires expensive chiral reagents, high temperature and high pressure conditions, and strong acid and strong ba...

Claims

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Application Information

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IPC IPC(8): C12P13/22
CPCC12P13/222
Inventor 朱龙宝杨瑾陶玉贵葛飞李婉珍宋平堵国成
Owner ANHUI UNIVERSITY OF TECHNOLOGY AND SCIENCE
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