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Extracellular expression and application of tyrosine phenol lyase

A tyrosine phenol and lyase technology, which is applied in the field of extracellular expression and the synthesis of tyrosine and derivatives, can solve the problem of low activity, increase the contact efficiency of enzymes and substrates, tyrosine and derivatives Low efficiency and other problems, to achieve the effect of saving use cost, good cell permeability, and improving catalytic efficiency

Active Publication Date: 2018-10-30
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the shortcomings of the low enzyme activity of the current strains and the low efficiency of catalytic synthesis of tyrosine and derivatives, to provide a method for extracellular expression of tyrosine phenol lyase, to increase the expression of the enzyme, Simplify the membrane permeation treatment process before enzyme use, increase the contact efficiency between enzyme and substrate, and improve the efficiency of enzymatic production of tyrosine and its derivatives

Method used

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  • Extracellular expression and application of tyrosine phenol lyase
  • Extracellular expression and application of tyrosine phenol lyase
  • Extracellular expression and application of tyrosine phenol lyase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of Genetically Engineered Bacteria Secreting L-Aspartic Acid α-Decarboxylase Extracellularly The construction of engineered bacteria was carried out using a one-step cloning directional seamless cloning kit.

[0035] According to the gene sequence (D13714.1) information of TPL in Erwinia herbicola and the cloning vector pET28a, design upstream and downstream primers containing adapters (indicated in italics) and restriction endonuclease sites (indicated in bold), and the upstream primer is TPL-F: 5′-GTGCCGCGCGGCAGCCATATGATGAACTATCCTGCCGAGCC-3′; downstream primer is TPL-R: 5′

[0036] -GTGGTGGTGGTGGTGCTCGAGTTAAATAAAGTCAAAACGCG-3'. Genomic DNA of Erwinia herbicola ATCC21434 was extracted by the bacterial genomic DNA extraction kit, and the genomic DNA was used as a template for PCR amplification, and then the PCR fragment was combined with the NdeI and XhoI digested vector using the one-step directional cloning seamless cloning kit pET28a was conne...

Embodiment 2

[0048] Example 2 Obtaining tyrosine phenol lyase fermented liquid and converting L-tyrosine through secreted expression

[0049] (1) Insert the constructed tyrosine phenol lyase engineering strain E.coli BL21(DE3) / pT28a-torA-TPL into the LB slant medium containing 100 mg / L kanamycin, and cultivate it at 37°C for 10~ 20h; the LB medium formula is: yeast powder 5g / L, peptone 10g / L, NaCl 5g / L, agar 20g / L, pH7.0, sterilized at 121°C for 30min;

[0050] (2) 50mL of LB liquid medium in a 500mL Erlenmeyer flask, add 100mg / L of kanamycin, connect 1 ring of strains cultured in slant medium to LB liquid medium, and cultivate at 37°C and 200r / min for 4-8h, of which The liquid LB medium formula is: yeast powder 5g / L, peptone 10g / L, NaCl 5g / L, pH7.0, sterilized at 121°C for 30min;

[0051] (3) 3.0L fermentation medium is loaded into the 5L fermenter, the seed liquid is inserted into the fermentation medium with 5% seed amount, the speed and ventilation are adjusted to control the dissolve...

Embodiment 3

[0055] Obtaining Tyrosine Phenol Lyase Fermentation Broth to Convert L-Tyrosine Through Secreted Expression

[0056](1) Insert the constructed tyrosine phenol lyase engineering strain E.coli BL21(DE3) / pT28a-torA-TPL into the LB slant medium containing 100mg / L kanamycin and culture it at 37°C for 10-20h, wherein The formula of LB slant medium is: yeast powder 5g / L, peptone 10g / L, NaCl 5g / L, agar 20g / L, pH7.0, sterilized at 121°C for 30min;

[0057] (2) 50mL of LB liquid medium in a 500mL Erlenmeyer flask, add 100mg / L of kanamycin, connect 1 ring of slant medium cultured strains in LB liquid medium at 37°C and 200r / min for 4-8h, and the liquid The formula of LB medium is: yeast powder 5g / L, peptone 10g / L, NaCl 5g / L, pH7.0, sterilized at 121°C for 30min;

[0058] (3) 3.0L fermentation medium is loaded into the 5L fermenter, the seed liquid is inserted into the fermentation medium with 5% seed amount, the speed and ventilation are adjusted to control the dissolved oxygen value be...

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Abstract

The invention discloses extracellular expression and application of tyrosine phenol lyase and belongs to the technical field of biology. A method for extracellular expression includes: cloning high-activity tyrosine phenol lyase gene, co-expressing with signal peptide signal, converting Escherichia coli competent cells, and building a tyrosine phenol lyase extracellular secretion engineering strain mediated by signal peptide to realize extracellular expression, wherein obtained tyrosine phenol lyase fermentation liquid is used for converting and synthesizing tyrosine and derivatives. An extracellular expression mode is adopted to build the tyrosine phenol lyase engineering strain, and enzyme release quantity reaches higher than 50%, so that toxicity caused to cells due to excessive accumulation of protein in the cells is reduced, and total enzyme activity is improved by 36% when compared with common expression. Part of enzyme generated by fermentation is released into the fermentationliquid, thallus cells are high in permeability, contact of a substrate with enzyme is facilitated, and catalytic efficiency of the enzyme is improved substantially. The fermentation liquid can be directly adopted to catalyze the substrate to product L-tyrosine and the derivatives, and the cells do not need to be broken to extract pure enzyme for reaction, so that using cost of the enzyme is saved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the extracellular expression of a tyrosine phenol lyase and its application in the synthesis of tyrosine and its derivatives. Background technique [0002] Tyrosine phenol-lyase (TPL), also known as β-tyrosinase, catalyzes the cleavage of L-tyrosine in vivo to generate phenol, pyruvate and ammonia, but in vitro can phenol, Pyruvate and ammonia are converted to L-tyrosine. Since tyrosine phenol lyase is not strictly substrate-specific, it can also catalyze structural analogs of phenol such as catechol, o-fluorophenol, and guaiacol to generate tyrosine derivatives levodopa, 3-fluoro-L-tyrosine, 3-methoxy-L-tyrosine. [0003] Tyrosine and its derivatives have important industrial uses. For example, tyrosine is an important protein amino acid, which can be used as a raw material for amino acid infusion and amino acid compound preparations, and as a nutritional supplement. ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P13/22C12R1/19
CPCC12N9/88C12N15/70C12P13/225C12Y401/99002
Inventor 冯志彬张娟
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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