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Engineered escherichia coli for expressing lignin peroxidase, preparation method and application of engineered escherichia coli

A technology of peroxidase and Escherichia coli is applied in the field of preparation of Escherichia coli engineering bacteria expressing lignin peroxidase, and can solve the problems of difficult lignin waste biodegradable pulp bleaching, limited promotion and use, low reproduction speed and the like , to achieve high protein yield, facilitate gene cloning, and increase solubility

Inactive Publication Date: 2014-01-29
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although white rot fungi are widely used in the research of lignin degradation relying on their strong lignin degradation ability, compared with bacteria, their reproduction speed is generally lower than that of bacteria, which makes them subject to certain restrictions in the actual promotion and use. limit
In addition, the amount of lignin peroxidase secreted by white rot fungi is limited, and it is difficult to apply large-scale biodegradation of lignin waste and pulp bleaching in industrial production, relying only on a small amount of lignin secreted by existing microorganisms Degradation by peroxidase is unrealistic

Method used

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  • Engineered escherichia coli for expressing lignin peroxidase, preparation method and application of engineered escherichia coli
  • Engineered escherichia coli for expressing lignin peroxidase, preparation method and application of engineered escherichia coli
  • Engineered escherichia coli for expressing lignin peroxidase, preparation method and application of engineered escherichia coli

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Cloning the cDNA of lignin peroxidase gene

[0034] The protein sequence (P21764) of lignin peroxidase obtained from the database UniProtKB screening is SEQ ID: NO: 01, which is Met Ala Phe Lys Gln Leu Phe Ala Ala Ile Ser Leu Ala Leu Ser Leu Ser Ala Ala Asn Ala Ala Ala Val Ile Glu Lys Arg Ala Thr Cys Ser Asn Gly Lys Thr Val Gly Asp Ala Ser Ser Cys Ala Trp Phe Asp Val Leu Asp Asp Ile Gln Gln Asn Leu Phe His Gly Gly Gly Gln Cys Gly Ala Glu Ala His Glu Ser Ile Arg Leu Val Phe His Asp Ser Ile Ala Ile Ser Pro Ala Met Glu Ala Gln Gly Lys Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Met Ile Phe Asp Asp Ile Glu Thr Ala Phe His Pro Asn Ile Gly Leu Asp Glu Ile Val Lys Leu Gln Lys Pro Phe Val Gln Lys His Gly Cys Thr Pro Gly Asp Phe Ile Ala Phe Ala Gly Ala Val Ala Leu Ser Asn Cys Pro Gly Ala Pro Gln Met Asn Phe Phe Thr Gly Arg Ala Pro Ala Thr Gln Ala Ala Pro Asp Gly Leu Val Pro Glu Pro Phe His Thr Val Asp Gln Ile Ile Asn Arg Val Asn Asp Ala Gly Glu Phe Asp Glu Leu Glu Leu Val Trp Me...

Embodiment 2

[0050] Application of the above-mentioned Escherichia coli engineering bacteria in expressing lignin peroxidase

[0051] The Escherichia coli engineering bacteria prepared in Example 1 were inoculated in LB liquid medium, containing 100 mg / L of ampicillin, 37 ° C, 200 rpm, and cultivated to OD 600 About 0.6, add protein inducer IPTG, 16°C, 200 rpm, induce recombinant protein expression for 12-16 h. The formula of the LB liquid medium is: 0.5 g of peptone, 0.25 g of yeast extract, 0.5 g of sodium chloride, and 50 ml of water.

Embodiment 3

[0053] Separation and Purification of Lignin Peroxidase

[0054] Put the engineered Escherichia coli bacteria induced and cultured in Example 2 into a centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, add PBS buffer to resuspend, ultrasonicate, add 1 mL of nickel column, and purify.

[0055] The Escherichia coli engineering bacteria induced and cultured in Example 2 were centrifuged at 10000rpm for 5min, collected by centrifugation and analyzed for protein solubility, ultrasonically crushed, and the total protein, supernatant and precipitate were respectively taken for SDS-PAGE protein electrophoresis analysis. The specific results are as follows image 3 As shown, it shows that the lignin peroxidase protein can be highly expressed in Escherichia coli BL21.

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Abstract

The invention relates to engineered escherichia coli, and also provides a preparation method of engineered escherichia coli. The preparation method comprises the following steps: cloning cDNA (complementary Deoxyribose Nucleic Acid) of a gene of phanerochaete chrysosporium lignin peroxidase; performing double digestion on the cDNA; connecting the digestion fragments with a pCold-TF vector; authenticating whether the obtained plasmid pCold-TF-LiP is subjected to positive expression; and introducing the positive plasmid pCold-TF-LiP into escherichia coli BL21 competent cells to obtain the engineered escherichia coli for expressing lignin peroxidase. The invention also provides an application of the engineered escherichia coli, namely, inoculating the engineered escherichia coli into a liquid culture medium, adding ampicillin to the liquid culture medium, culturing until OD600 (Optical Density) reaches 0.58 to 0.62, and adding a protein inductive agent IPTG (Isopropyl Thiogalactoside) to induce the expression of recombinant protein. The engineered escherichia coli is high in expression quantity and extremely high in activity, and the enzyme activity reaches 3,528U / L.

Description

technical field [0001] The invention relates to an Escherichia coli engineering bacterium, in particular to an Escherichia coli engineering bacterium expressing lignin peroxidase, a preparation method and application thereof. Background technique [0002] Lignin is the second most abundant biopolymer known on the earth. It has a very complex structure and is not easy to degrade (Martinez, A. T., et al. 2005."Biodegradation of lignocellulars:microbial,chemical,and enzymatic aspects of the fungal attack of lignin." Int Microbiol 8:195-204). Due to lack of proper disposal, much lignin becomes waste, causing various environmental problems. With the increasing shortage of energy sources, researchers at home and abroad have tried to produce ethanol by converting lignin as an alternative energy source and have made some progress. Lignocellulose is also used as raw material for making paper, cultivating edible fungi and animal feed (Sanchez, C. 2009."Lignocellular residues: biodeg...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12N9/08C12R1/19
Inventor 陈明晏铭曾光明杜长青张嘉超鲁伦慧朱艺赖萃许飘武海鹏
Owner HUNAN UNIV
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