A kind of process of enzymatic conversion of phenylalanine to produce phenylpyruvate

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: 2020-09-04
JIANGNAN UNIV +1
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  • 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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  • A kind of process of enzymatic conversion of phenylalanine to produce phenylpyruvate
  • A kind of process of enzymatic conversion of phenylalanine to produce phenylpyruvate
  • A kind of process of enzymatic conversion of phenylalanine to produce phenylpyruvate

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, further optimized by codons suitable for the expression system of Escherichia coli, the nucleotide sequence of the mutant is shown in SEQ ID NO: 2, the gene before optimization 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 increased to 1.0. The nucleotide sequence of SEQ ID NO: 2 was synthesized through the whole gene, connected to the pET24a vector, and expressed recombinantly in Escherichia coli E.coli BL21(DE3), and the strain was named E.coli-PM-LAAD. After codon optimization, the enzyme activity of L-AAD increased from 2.37U / mL to 3.21U / mL, and the enzyme activity increased by 35.4%.

Embodiment 2

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

[0030] (1) The composition of fermentation medium a is: 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 ionic liquid 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, soybean 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 feeding medium are: glycerol 400-600g / L, yeast powder 5-8g / L, MgSO 4 ·7H 2 O 6~10g / L.

[0034] (2) Inoculate E.coli-PM-LAAD in Example 1 into LB seed medium (kanamycin sulfate 100mg / L), culture at 37°C, shake at 200rpm for 10-12h, and inoculate inoculations according to 5% inoculum size For fermentation...

Embodiment 3

[0039] Example 3: Effects of Additives on Whole Cell Transformation Production of Phenylpyruvate

[0040] The E. coli-PM-LAAD wet thallus obtained in the fermentation medium a of Example 2 was used as a cell catalyst for converting phenylalanine to produce phenylpyruvate. In a 1L transformation system, use pH 7.5 Tris-HCl buffer to dissolve 75g / L of L-phenylalanine, 30g / L of wet bacteria, 4mol / L NaOH solution to control pH 7.5-8.0, temperature 25°C, stirring speed 400rpm , the ventilation volume was controlled at 0.5vvm, and 0.1% CTAB, 0.1% Triton-100, 0.1% Tween-80, 2mmol / L MnCl were added 2 , 2mmol / L MgCl 2 , 2mmol / L CaCl 2 , to detect the effect of additives on the yield of PPA. Experimental results such 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 surfactant...

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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 producing phenylpyruvate by enzymatically converting phenylalanine, which 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-keto acid tablets; PPA is the raw material for the synthesis of D-phenylalanine, which is hand Synthetic intermediates of sexual 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 currently mainly produced by chemical synthesis and biological methods. The chemical synthesis usually includes the hydrolysis of α-acetamide aminocinnamic acid, the synthesis of hydantoin and benzaldehyde, and the dihydroxylation of benzyl chloride. React three routes. However, the chemical synthesis met...

Claims

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Application Information

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Patent Type & Authority Patents(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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