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Phenylpyruvic acid reductase sourced from lactobacillus plantarum and application thereof

A technology of phenylpyruvate and reductase, which is applied in the field of bioengineering, can solve the problems of long reaction time, low yield of phenyllactic acid, and inability to achieve industrial production, and achieve the effect of improving conversion efficiency

Inactive Publication Date: 2017-09-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the yield of phenyllactic acid prepared by fermentation method is low, and the product extraction is difficult, while the substrate loading and yield of phenyllactic acid prepared by biotransformation method are all improved, but the reaction time is too long, still can not To meet the requirements of industrial production, it is necessary to further develop enzymes that reduce ketoacids efficiently

Method used

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  • Phenylpyruvic acid reductase sourced from lactobacillus plantarum and application thereof
  • Phenylpyruvic acid reductase sourced from lactobacillus plantarum and application thereof
  • Phenylpyruvic acid reductase sourced from lactobacillus plantarum and application thereof

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Embodiment 1

[0029] Embodiment 1 The acquisition of Lactobacillus plantarum phenylpyruvate reductase gene and the construction of expression plasmid

[0030] Design specific primers:

[0031] LpPPR-F: 5'- CATATG ATGAAAATTTTAATGTAT-3', containing Nde I restriction site.

[0032] LpPPR-R: 5'- GCGGCCGC TTAAAAGGCGTGGGCCGT-3', containing Not I restriction site.

[0033] Extract the genomic DNA of Lactobacillus plantarum, use the genomic DNA of Lactobacillus plantarum as a template, and use LpPPR-F and LpPPR-R as primers to perform PCR: denaturation at 94°C for 5min, 30 cycles (94°C for 30s, 50°C for 30s and 72°C for 60s ), and keep warm at 72°C for 10 minutes. The PCR product was analyzed by agarose gel electrophoresis, the target gene was recovered by tapping the gel, ligated with pUCm-T, transformed into E.coliJM109, screened by blue and white spots, PCR identification of bacteria solution and DNA sequencing. The correctly sequenced recombinant plasmid was named pUCm-T-lpppr. The sequ...

Embodiment 2

[0034] Induced expression and condition optimization of embodiment 2 recombinant bacteria

[0035] Inoculate a single colony of E.coli BL21-lpppr into 2 mL of LB medium containing 100 μg / mL kanamycin, and culture overnight at 37°C and 220 r / min; Cultured at 37℃ to OD 600 When it is 0.6-0.8, the expression of the target protein is induced. The induction conditions are: add IPTG to a final concentration of 0.4-0.6mmol / L, and induce at 18-22°C for 7-9h. Bacteria were collected and washed with sodium phosphate buffer (Na 2 HPO 4 -NaH 2 PO 4 , 100mmol / L, pH 7.0), washed 2-3 times, added the same buffer to suspend to obtain a bacterial suspension with a bacterial concentration of 100mg / mL. The concentration of benzene lactic acid after the reaction was measured, and the result showed that the concentration of D-phenyl lactic acid reached 7.05-7.79 mM.

Embodiment 3

[0036] The preparation of embodiment 3D-phenyllactic acid

[0037] Add 400 μL of phenylpyruvate (final concentration: 25 mM) to a 1.5 mL EP tube, then add 100 μL of glucose (final concentration: 20 mM), add 500 μL of bacterial suspension (enzyme activity: 19.7 U / mg wet bacteria), and react at 40 ° C for 5 h . Samples were regularly sampled into methanol for dilution, centrifuged and passed through a 0.22 μm organic filter membrane, and then detected by HPLC. The reaction progress curve is as figure 2 As shown, after 210 minutes of reaction, the content of phenylpyruvate was lower than 5mM. After 5 hours of reaction, the conversion rate of phenylpyruvate was 98.38%, e.e.>99.9%, and the output of D-phenyllactic acid reached 4.15g / L.

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Abstract

The invention discloses a phenylpyruvic acid reductase sourced from lactobacillus plantarum and an application thereof and belongs to the technical field of bioengineering. In the invention, for the first time, the phenylpyruvic acid reductase, which has the activity of catalyzing phenylpyruvic acid to generate phenyllactic acid, is screened and produced. The invention belongs to the fields of gene engineering and protein expression. The enzyme is successfully cloned and expressed in Escherichia coli BL21 and is applied to production of the phenyllactic acid through whole-cell catalysis of phenylpyruvic acid. After the catalytic reaction is carried out for 5 h, conversion of the substrate reaches 98.38% and ee value reaches 99.9%.

Description

technical field [0001] The invention relates to a phenylpyruvate reductase derived from Lactobacillus plantarum and its application, belonging to the technical field of bioengineering. Background technique [0002] Phenyllactic acid (PLA), also known as 2-hydroxy-3-phenylpropionic acid, is a very important class of compounds that are widely used in medicine and biological preservatives. Phenyllactic acid has the same pharmacological function as its derivative Danshensu (β-3,4-dihydroxyphenylsodium lactate), which can regulate the level of steroids in the human body and the activity of anti-platelet aggregation. Phenyllactic acid is also an important intermediate in the synthesis of many drugs, such as: hypoglycemic drug Englitazone, non-protein amino acid Statine, anti-HIV preparations, new anthelmintic drug PF1022A, etc. In addition, in recent years, it has been found that phenyllactic acid can be used as a new type of biological preservative, and it has a very broad antib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N1/21C12N15/70C12N15/66C12P7/42C12R1/19
CPCC12N9/0006C12N15/66C12N15/70C12P7/42C12Y101/01
Inventor 李剑芳袁风娇刘艳李雪晴邬敏辰李闯
Owner JIANGNAN UNIV
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