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Recombinant bacterium providing improved efficiency in convertive production of Alpha-phenylpyruvic acid

A technology of phenylpyruvate and recombinant bacteria, applied in the field of recombinant bacteria, can solve the problems of low efficiency, low selectivity, toxicity of co-substrates, etc., achieve high yield, improve transformation efficiency, and improve regeneration efficiency

Active Publication Date: 2016-06-08
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, this whole-cell catalyzed oxidative deamination reaction requires the coenzyme FAD as an electron carrier, and the intracellular regeneration system of FAD requires multi-enzyme catalysis, which is inefficient; and FAD is expensive and not suitable for external addition, so it is necessary to construct a one-step regeneration system , to increase conversion efficiency
[0005] Existing FAD regeneration systems include electrochemical regeneration, chemical regeneration, and enzymatic regeneration. The defect of these methods is the generation of H 2 o 2 , enzyme inactivation, low selectivity, toxic co-substrate, etc.

Method used

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  • Recombinant bacterium providing improved efficiency in convertive production of Alpha-phenylpyruvic acid
  • Recombinant bacterium providing improved efficiency in convertive production of Alpha-phenylpyruvic acid
  • Recombinant bacterium providing improved efficiency in convertive production of Alpha-phenylpyruvic acid

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Embodiment 1

[0025] Cloning of embodiment 1 rebH and aad gene and construction of recombinant whole cell catalyst

[0026] The aad gene was synthesized, the nucleotide sequence of which is shown in SEQ ID NO.1, and further digested with BamHI and HindIII, and then cloned into the multi-cloning 1 site of the plasmid pRSFDuet-1 to construct the recombinant plasmid pRSFDuet-AAD. Using the Lechevalieria aerocolonigenes genome as a template and using rebH-MCS2-S and rebH-MCS2-A as primers, the rebH gene was amplified by PCR, and its nucleotide sequence is shown in SEQ ID NO.2. The PCR product was double digested with NdeI and XhoI, and then cloned into the multi-cloning 2 site of the plasmid pRSFDuet-AAD to construct the plasmid pRSFDuet-AAD-RebH. The recombinant plasmids pRSFDuet-AAD and pRSFDuet-AAD-RebH were transformed into Escherichia coli BL21(DE3), and BL21(pRSFDuet-AAD) and BL21(pRSFDuet-AAD-RebH) were constructed.

Embodiment 2

[0027] Preparation of embodiment 2 whole cell catalyst and whole cell transformation process

[0028] The recombinant Escherichia coli in Example 1 after co-expressing L-amino acid deaminase and halogenase was inoculated into seed medium (containing 10 mg / L kanapenicillin), and cultivated overnight at 37° C. and 200 rpm. Fermentation was carried out in a 500mL Erlenmeyer flask, 1% inoculum was added to 50mL fermentation medium, cultured at 37v, when OD 600 When it reaches 0.6-0.8, add 0.4mMIPTG to induce the expression of L-amino acid deaminase and halogenase. After induction at 28°C for 24h, centrifuge at 8,000rpm for 10-15min at low temperature, collect the bacteria, and wash with 20mM Tris-HCl (pH8.0) buffer Get the bacteria twice.

[0029] The whole cell transformation system is: L-phenylalanine 20-30g / L, whole cell catalyst 4-6g / L, L-tryptophan 1-2g / L, in 0.9% NaCl at pH 8.0, in 36-37°C, 200-220rpm transformation for 6-10h. The PPA yields of BL21(pRSFDuet-AAD) and BL21...

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Abstract

The invention discloses a recombinant bacterium providing improved efficiency in convertive production of Alpha-phenylpyruvic acid and belongs to the field of biotechnology. Co-expression of L-amino acid deaminase and halogenase in Escherichia coli is successfully implemented herein, coenzyme regeneration is improved, and PPA yield is up to 18.3 g / L. By establishing this whole-cell conversion system, the problems of step complexity, low yield, environmental pollution and the like in chemical synthesis of PPA are solved, the problem that enzymatic convertive production of PPA is low in conversion efficiency is solved, pollution-free, high-yield and one-step PPA production is implemented, and certain theoretical basis is laid for following industrial production.

Description

technical field [0001] The invention relates to a recombinant bacterium with improved transformation and production efficiency of α-phenylpyruvate, which belongs to the field of biotechnology. Background technique [0002] α-Phenylpyruvate (PPA) has many applications. It can be used to synthesize heterocyclic compounds of antineoplastic drugs, as an antioxidant and to promote wound healing. At present, the production of PPA is mainly based on chemical synthesis methods. These methods require multi-step reactions, have high requirements on reaction conditions, and are prone to produce toxic and harmful products. Enzymes and whole-cell biocatalysts are increasingly used in industrial production. [0003] L-amino acid deaminase (EC1.4.3.2) catalyzes the oxidative deamination of L-amino acid to generate the corresponding α-keto acid and ammonia. The L-amino acid deaminase in ProteusmirabilisKCTC2566 has broad substrate specificity and can catalyze aliphatic and aromatic L-amin...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/40C12R1/19
CPCC12N9/0022C12N9/0071C12P7/40C12Y104/03002C12Y114/14007
Inventor 堵国成刘龙陈坚李江华侯颖
Owner JIANGNAN UNIV
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