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Technology for producing phenylpyruvic acid by phenylalanine through enzymatic conversion

A technology of phenylpyruvate and amino acids, applied in the field of bioengineering, can solve the problems of low PAA yield, low enzyme activity of metabolic pathway, complex separation and purification process, etc., to meet the needs of industrialization and reduce production costs

Active Publication Date: 2018-12-18
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the long path of PPA in the bacteria and the low enzyme activity of the metabolic pathway, the yield of PAA in the direct fermentation method is relatively low, and the fermentation broth contains a large amount of impurities such as bacteria, protein, and inorganic salts, and the downstream separation and purification process of PPA is complicated.

Method used

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  • Technology for producing phenylpyruvic acid by phenylalanine through enzymatic conversion
  • Technology for producing phenylpyruvic acid by phenylalanine through enzymatic conversion
  • Technology for producing phenylpyruvic acid by phenylalanine through enzymatic conversion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of recombinant strains producing amino acid deaminase mutants

[0028] The nucleotide sequence of the L-AAD mutant is shown in SEQ ID NO: 1, which is further optimized by codons suitable for the E. coli expression system, and the nucleotide sequence of the mutant is shown in SEQ ID NO: 2. The GC content was 44.0%, and the codon adaptability index (CAI) in E. coli was 0.269. After optimization, the GC content was 51.4, and the CAI was improved to 1.0. The nucleotide sequence of SEQ ID NO: 2 was synthesized by whole gene, and ligated into pET24a vector, and recombinantly expressed in E. coli BL21 (DE3), and the strain was named as E. coli-PM-LAAD. After codon optimization, the L-AAD enzyme activity increased from 2.37U / mL to 3.21U / mL, and the enzyme activity increased by 35.4%.

Embodiment 2

[0029]Example 2: Influence of nutrient conditions on the production of amino acid deaminase by fermentation

[0030] (1) The components of fermentation medium a are: glycerol 6g / L, yeast powder 20g / L, soybean peptone 5g / L, K 2 HPO 4 ·12H 2 O 2.5g / L, KH 2 PO 4 5.0g / L, metal ion solution 10mL / L.

[0031] The composition of the metal ionic liquid is: FeSO 4 ·7H 2 O 5g / L, CaCl 2 2g / L, ZnSO 4 ·7H 2 O 1.2g / L, MnSO 4 ·4H 2 O 0.4g / L, (NH 4 ) 6 MoO 24 ·4H 2 O 0.1g / L, H 3 BO 3 0.5g / L.

[0032] Fermentation medium b is TB medium: glycerol 4g / L, yeast powder 24g / L, soy peptone 12g / L, KH 2 PO 4 2.31g / L, K 2 HPO 4 .3H 2 O 12.54g / L.

[0033] The ingredients of the feed medium are: glycerol 400-600g / L, yeast powder 5-8g / L, MgSO 4 ·7H 2 O 6~10g / L.

[0034] (2) E.coli-PM-LAAD in Example 1 was inoculated into LB seed medium (Kanamycin sulfate 100mg / L), 37°C, 200rpm shaking culture for 10-12h, respectively inoculated according to 5% inoculation amount on Fermentation me...

Embodiment 3

[0039] Example 3: Effects of additives on whole cell transformation to produce phenylpyruvate

[0040] The E.coli-PM-LAAD wet cells obtained in the fermentation medium a of Example 2 were used as a cell catalyst for converting phenylalanine to produce phenylpyruvate. In 1L transformation system, use pH 7.5 Tris-HCl buffer to dissolve L-phenylalanine 75g / L, wet cell 30g / L, 4mol / L NaOH solution to control pH 7.5~8.0, temperature 25℃, stirring speed 400rpm , ventilation volume was controlled at 0.5vvm, 0.1% CTAB, 0.1% Triton-100, 0.1% Tween-80, 2mmol / L MnCl were added respectively 2 , 2mmol / L MgCl 2 , 2mmol / L CaCl 2 , to examine the effect of additives on the yield of PPA. The experimental results are as figure 2 : MgCl 2 It has a certain promotion effect on the yield of PPA, the yield increased from 70.4g / L to 73.9g / L, and the conversion rate increased from 94.4% to 99.1%; other metal ions had no obvious effect on the conversion results; the addition of surfactants had no ...

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Abstract

The invention discloses a technology for producing phenylpyruvic acid by phenylalanine through enzymatic conversion and belongs to the technical field of biological engineering. According to the technology disclosed by the invention, a proteus mirabilis derived amino acid deaminase mutant is subjected to codon optimization and fermentation condition optimization and the enzyme activity of amino acid deaminase is remarkably improved. Furthermore, a conversion condition is optimized and the yield of the phenylpyruvic acid is remarkably improved; when the adding amount of wet strains is 25 to 30g / L, the yield of the phenylpyruvic acid can reach 81.1 to 83.0g / L and the mol conversion rate of the phenylpyruvic acid reaches 98.0 percent or mor.

Description

technical field [0001] The invention relates to a process for enzymatically converting phenylalanine to produce phenylpyruvate, and belongs to the technical field of bioengineering. Background technique [0002] Phenylpyruvate PPA is a dihydroxy compound commonly used in medicine, light industry and other fields, and can be used to make compound α-ketoacid tablets; PPA is the raw material for synthesizing D-phenylalanine, and D-phenylalanine is a manual Synthetic intermediates of sex drugs and food additives; PPA can also be used to prepare phenyllactic acid, which can be used for antibacterial, antiseptic and flavor additives. [0003] As a multifunctional organic acid, PPA is mainly produced by chemical synthesis and biological methods. React three routes. However, the chemical synthesis method has problems such as high reaction requirements, low product yield, large equipment investment, long reaction time and the production of toxic and harmful substances. Biological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/06C12P7/40C12R1/19
CPCC12N9/0022C12P7/40C12Y104/03002
Inventor 刘佳吴静杨彬罗秋玲陈修来张权
Owner JIANGNAN UNIV
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