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Laccase TvLac, and coding gene and application thereof

A technology of laccase and gene, applied in the field of laccase TvLac and its coding gene and application, can solve the problems of limitation and high cost

Inactive Publication Date: 2019-09-24
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pantoacidolactone hydrolase used in the existing enzymatic mycotoxin degradation technology and some unclassified enzymes can degrade mycotoxins through oxidation or hydrolysis mechanisms, but they can only specifically degrade a single toxin. The application is greatly limited due to the disadvantages of high cost

Method used

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  • Laccase TvLac, and coding gene and application thereof
  • Laccase TvLac, and coding gene and application thereof
  • Laccase TvLac, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of embodiment 1 laccase TvLac coding gene

[0041] The laccase TvLac gene was derived from Trametes versicolor. Specific primers were designed and amplified by PCR. The primer sequences were as follows:

[0042] TvLac-F: 5'-GCTGAAGCTTACGTAGAATTCGCCATTGGGCCCGTCACCGA-3';

[0043] TvLac-R: 5'-AAGGCGAATTAATTCGCGGCCGCGAGGTCGGACGAGTCCA-3'.

[0044] The PCR product was separated by 1% agarose gel electrophoresis, recovered and purified, and its nucleotide sequence was shown as SEQ ID No.4 after analysis.

[0045] Using restriction endonucleases EcoRI and NotI to carry out double enzyme digestion on the product obtained from the PCR amplification gene and the pPIC9 vector, and the digestion products were separated by 1% agarose gel electrophoresis and recovered and purified. T4 DNA ligase was used to ligate the digested vector and the gene fragment and transform the Pichia cloning host to obtain the recombinant Pichia expression plasmid pPIC9-TvLac.

Embodiment 2

[0046] Embodiment 2 Preparation of recombinant laccase TvLac

[0047] The obtained recombinant Pichia expression plasmid pPIC9-TvLac containing laccase TvLac was transformed into Pichia GS115 to obtain recombinant Pichia GS115 / TvLac.

[0048] Take the Pichia pastoris GS115 engineering strain containing the target gene TvLac, inoculate it in 50mL YPD culture medium, and culture it with shaking at 220rpm at 30°C for 48h, then transfer it to 300mL BMGY medium at a ratio of 2%, and culture it with shaking at 220rpm at 30°C for 48h. Replace the BMMY medium (containing 0.2mM CuSO4), and use methanol to induce the expression of exogenous laccase protein. Supplement 0.5% methanol every 24h, a total of 72h induction. The cells were removed by centrifugation at 12000 rpm, the supernatant was concentrated to 5 ml by using a membrane bag, and dialyzed in 10 mM disodium hydrogen phosphate-citric acid buffer (pH 6.5) overnight. Anion column chromatography was used for purification, and th...

Embodiment 3

[0049] Example 3 Degradation of aflatoxin B1 by TvLac-methyl syringate mediator system

[0050] Dissolve aflatoxin B1 in dimethyl sulfoxide to prepare a 50mg / L mother solution, as follows: 50mM Tris-HCl (pH 7.0), 20μL aflatoxin B1 solution (50mg / L), 20μL syringic acid Methyl ester (10mM), 20μL TvLac (300U / L). The system without adding laccase TvLac was used as a control, and the reaction system was set in 3 repetitions. The reaction was carried out at 30° C., and three times the volume of acetonitrile was added to terminate the reaction after 10 hours. The degradation rate of aflatoxin B1 was analyzed by high performance liquid chromatography (HPLC). The liquid chromatography is a Shimadzu Nexera UHPLC high-performance liquid chromatography analysis system, and the chromatographic separation column is Zorbax SB-C18 (4.6×250mm, 5μm), mobile phase A (water with 0.06% TFA), mobile phase B (water with 0.05% TFA Acetonitrile); Gradient elution condition 0% B elution 4 minutes, 0%...

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Abstract

The present invention belongs to the field of agricultural biology and particularly relates to fungal-derived laccase TvLac and a coding gene and an application thereof. The present invention provides the laccase TvLac derived from trametes versicolor. The laccase can effectively degrade mycotoxins of different structure types with an aid of a mediator and can be widely used in fields of mycotoxin detoxification of food and feeds.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to laccase TvLac and its encoding gene and application. Background technique [0002] Mycotoxins are secondary metabolites of fungi that are harmful to the health of livestock, poultry and humans. Common mycotoxins include aflatoxin, zearalenone, deoxynivalenol, citrinin, ochratoxin, fumonisins, patulin, and trichothecenes, which can Divided into two types of toxins with ring structure and without ring structure. Most mycotoxins, such as aflatoxins and zearalenone, belong to the subgroup with cyclic structures and are usually synthesized via the fungal polyketide pathway. For example, aflatoxin (aflatoxin) B1 produced by Aspergillus flavus has a core coumarin ring flanked by five-membered carbon rings and two juxtaposed dihydrofuran rings, which is a strong liver carcinogen. In addition, the structure of zearalenone is m-dihydroxybenzoate phenolic lactone. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19A23L5/20C12R1/84
CPCA23L5/25C12N9/0061C12N15/815C12Y110/03002
Inventor 姚斌苏小运王晓璐罗会颖柏映国黄火清王亚茹王苑涂涛
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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