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A method for producing extracellular ferulic acid esterase from Escherichia coli

A technology of ferulic acid esterase and Escherichia coli, applied in the direction of biochemical equipment and methods, using vectors to introduce foreign genetic material, enzymes, etc., to achieve the effect of simplifying purification and utilization, and promoting industrial production

Inactive Publication Date: 2019-01-11
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The ferulic acid esterases expressed in the above documents are all expressed intracellularly, and the subsequent complicated purification process is still unavoidable

Method used

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  • A method for producing extracellular ferulic acid esterase from Escherichia coli
  • A method for producing extracellular ferulic acid esterase from Escherichia coli
  • A method for producing extracellular ferulic acid esterase from Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Cloning of Lactobacillus amylophagous ferulic acid esterase gene

[0058] Take 1-3ml of Lactobacillus amylophagicus cultured overnight and centrifuge at 6000rpm for 3min to collect the bacteria. Resuspend in 567μl TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0), add 25μL 50mg / ml lysozyme, mix well, and treat in 37℃ water bath for 1h. Add 30 μL of 10% (mass percentage) SDS, 3 μL of 20 mg / ml proteinase K, and treat in a water bath at 37° C. for 1 h. Add 100 μL of 5M NaCl and 80 μL of CTAB / NaCl solution (10% (mass percentage) CTAB, 0.7 mol / L NaCl) and treat in a water bath at 65° C. for 10 minutes. Add an equal volume of chloroform isoamyl alcohol (volume ratio 24:1) for extraction, centrifuge at 12,000 rpm for 5 min, and retain the supernatant. Add RNase A solution with a final concentration of 100 μg / mL, and treat in a water bath at 37°C for 30 minutes. Then use an equal volume of phenol-chloroform-isoamyl alcohol (volume ratio 25:24:1) and chloroform-isoamy...

Embodiment 2

[0060] Embodiment 2: Construction of ferulic acid esterase gene recombinant Escherichia coli expression strain

[0061] Plasmid pET-22b was extracted and digested with NdeI and XhoI, and the digested products were recovered by gel. Mix the digested target gene with the vector pET-22b at a molar ratio of 1:5, and use T 4 DNA ligase was ligated overnight at 16°C, and then the ligated products were added to E. coli DH5α competent cells for heat shock transformation. The recovered cultured bacterial solution was concentrated and spread on LB (Amp resistant) plates, and cultivated overnight at 37. Pick the transformant and check it by enzyme digestion. After extracting the plasmids from the successfully verified DH5α transformants, they were transformed into E.coli BL21(DE3), and the BL21(DE3) transformants were screened out on LB (Amp resistant) plates.

Embodiment 3

[0062] Embodiment 3: Utilize IPTG to induce Escherichia coli genetic engineering strain to secrete and express ferulic acid esterase

[0063] Pick BL21(DE3) transformants into 5 mL LB containing Amp antibiotics, transfer to 100 mL LB medium at 2% after overnight culture, and culture at 37°C until the OD value is about 0.6, add IPTG with a final concentration of 0.5 mM, Continue to culture for 24h to induce protein expression.

[0064] Preliminary test of ferulic acid esterase activity secreted by Escherichia coli genetically engineered strains: the induced expression of Escherichia coli culture fluid was centrifuged at 13000rpm for 10min, and the collected supernatant was filtered with a 0.22μm filter membrane to be its extracellular secretion fraction. Take 200uL of the secretory fraction and add it to the Oxford cup placed on the ferulic acid esterase activity detection medium. In 10% ethyl ferulic acid solution of N,N-dimethylformamide, after standing at 37°C for 6 hours, ...

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Abstract

The invention relates to a method for producing extracellular ferulic acid esterase from Escherichia coli. The method comprises the following steps: (1) preparing a recombinant Escherichia coli containing a ferulic acid esterase gene, wherein the nucleotide sequence of the ferulic acid esterase gene is shown in SEQ ID NO. 1; (2) subjecting the recombinant Escherichia coli obtained in the step (1)to IPTG or lactose induced fermentation, removing the bacterial cell, wherein the ferulic acid esterase is purified to obtain the ferulic acid esterase. The invention firstly adopts the restriction enzyme Nde I to digest the ferulic acid esterase gene, and the first enzyme digestion site after the ribosome binding site is changed into the Nde I enzyme digestion site, so that the obtained ferulic acid esterase has a natural N-terminal sequence; this special structure enables ferulic acid esterase to be secreted to extracellular after Escherichia coli synthesis, which greatly simplifies the purification and utilization of ferulic acid esterase, and has a significant role in promoting the industrial production of ferulic acid.

Description

technical field [0001] The invention relates to a method for producing extracellular ferulic acid esterase by using Escherichia coli, and belongs to the technical field of biotechnology. Background technique [0002] Ferulic acid esterase was first used in the degradation of lignocellulose. With the development of society and energy crisis, more and more researches focus on the use of lignocellulose to produce bioethanol. The key technology is to convert lignocellulose into a fermentable carbon source. Due to the cross-linked network structure of the plant cell wall, its degradation requires the synergistic action of a series of cellulase and hemicellulase. Ferulic acid esterase belongs to the hemicellulase family, which can open the ester bonds between hemicelluloses and between hemicelluloses and lignin, thereby facilitating the binding of other enzymes to lignocellulose. According to substrate specificity and sequence similarity, ferulic acid esterase can be divided int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/70C12N15/66C12P7/42
CPCC12N9/18C12N15/66C12N15/70C12P7/42C12Y301/01073
Inventor 徐振上王婷刘新利
Owner QILU UNIV OF TECH
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