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Construction method and application of mutant of fusion enzyme capable of simultaneously degrading aflatoxin B1 and zearalenone

A technology for zearalenone and aflatoxin, applied in the fields of biotechnology and genetic engineering, can solve the problems of low efficiency, complicated experiments, poor integrity of detoxification effect, etc., and achieve the effect of improving the degradation rate and high degradation efficiency

Active Publication Date: 2021-10-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, a large number of studies are mainly focused on the detoxification research of a single type of microorganism and its metabolites to degrade a toxin. The detoxification method is not universal, the detoxification object is highly targeted, and the overall detoxification effect is poor.
There is also a method of combining multiple probiotics to achieve simultaneous degradation of two toxins, but this method is inefficient, complicated in experiments, and narrow in scope of application, and is not suitable for natural toxin-producing foods and grains
The biotechnology of linking multiple different functional proteins with connecting peptides to realize the multifunctional research of enzymes has been studied in depth in some research fields, but it has not been widely used in the degradation of mycotoxins.

Method used

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  • Construction method and application of mutant of fusion enzyme capable of simultaneously degrading aflatoxin B1 and zearalenone
  • Construction method and application of mutant of fusion enzyme capable of simultaneously degrading aflatoxin B1 and zearalenone
  • Construction method and application of mutant of fusion enzyme capable of simultaneously degrading aflatoxin B1 and zearalenone

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] The selection and mutation method of embodiment 1 fusion enzyme mutant mutation site

[0066] The unmutated sequence of the fusion enzyme is shown in SEQ ID NO.1. The enzyme is a fusion enzyme amino acid sequence obtained by linking the enzyme ZHD101.1 that degrades zearalenone from Clonostachys rosea and the manganese peroxidase PhcMnp enzyme from Phanerochaete chrysosporium through a unit of flexible linking peptide GGGGS As shown in SEQ ID NO.1. Mutation hotspots were determined by analysis, and the following sites were selected and correspondingly mutated: Q45V, S103A, H125R, H125E, H134D, H134A, V172R, V172T, P188D, P188H, G205Q, W210D, W210E, T211A, H248D, H248E, D250H, R301A、E328A、I334L、H339D、N374G、N374A、N374L、S379I、S379A、N381D、L407V、K443S、R447E、F448M、S461A、H466D、H466P、H466Y、V468I、R470A、K473V、K473E、V474I、T512Q、T512P、 L569M.

[0067] The specific steps are: construct the recombinant plasmid pKLAC1-zpf1: connect the nucleotide sequence encoding the fusion protei...

Embodiment 2

[0078] Example 2 Construction and Identification of Fusion Enzyme Mutant Recombinant Yeast

[0079] The recombinant plasmid constructed in Example 1 was linearized with restriction endonuclease SacII, and the digested product was purified and recovered; the linearized plasmid was transformed into Kluyveromyces lactis GG799 competent cells by electric pulse method; Immediately add 1.0 mL of pre-cooled sorbitol solution to the yeast cells after electric shock, incubate at 30°C for 1-3 hours, collect the yeast cells by centrifugation, leave 100 μL of resuspended cells, and evenly spread them on the YCB plate containing acetamide. Cultivate at ℃ for 3-5 days until a single clone grows. Pick multiple well-growing single colonies, inoculate them in 10.0 mL YEPD liquid test tubes, and culture overnight at 30°C and 200 rpm. The bacteria were collected by centrifugation, and the genome of the recombinant bacteria was extracted using a fungal genome extraction kit. The extracted genom...

Embodiment 3

[0080] The secretion expression method of embodiment 3 fusion enzyme mutant and mutant to aflatoxin B 1 and the degradation method of zearalenone

[0081] The recombinant yeast glycerol tubes carrying mutant genes that were verified to be correct by sequencing and stored in Example 2 were taken out for activation. Pick a single colony in 10.0mL YEPD medium, culture at 30°C and 200rpm for 18-22h; when OD 600When it reached 1.0, it was transferred to an inoculum containing 0.5mmol / L MnSO with 1% (1mL / 100mL) 4 and 0.2mmol / L hemin in YEPG induction medium, induce enzyme production (secretory expression) at 30°C, 200rpm for 72h. Centrifuge at 8000rpm after the end of fermentation, collect the fermentation supernatant, and concentrate it with an ultrafiltration centrifuge tube with a molecular weight cut-off of 10kDa. Protein quantification was performed on the concentrated fermentation supernatant with a protein quantification kit.

[0082] Degradation of Aflatoxin B Using Secr...

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Abstract

The invention discloses a construction method and application of a mutant of a fusion enzyme capable of simultaneously degrading aflatoxin B1 and zearalenone, and belongs to the technical field of biotechnology and genetic engineering. According to the construction method and application of the mutant of the fusion enzyme capable of simultaneously degrading the aflatoxin B1 and the zearalenone, the zearalenone hydrolase ZHD101.1 and the manganese peroxidase PhcMnp are fused, the obtained fusion enzyme can simultaneously degrade the zearalenone and the aflatoxin B1, mutation is carried out on the fusion enzyme, the fusion enzyme mutant is produced through fermentation of food-grade yeast Kluyveromyces lactis, the obtained fusion enzyme mutant has the advantages that the degradation efficiency of the zearalenone and the aflatoxin B1 is remarkably improved compared with the starting enzyme, and the expressed product has food-grade safety, is suitable for the fields of food, feed and the like, and has important significance for food safety.

Description

technical field [0001] The invention relates to a method capable of simultaneously degrading aflatoxin B 1 The invention relates to a method for constructing a mutant of a fusion enzyme with zearalenone and its application, belonging to the technical fields of biotechnology and genetic engineering. Background technique [0002] Aflatoxin B 1 (Aflatoxins B 1 , AFB 1 ) and Zearalenone (Zearalenone, ZEN) are two common mycotoxins in corn, peanuts and other grains, which can cause liver cancer, growth retardation, reproductive system damage and other diseases, and pose a huge threat to the health of humans and livestock . Aflatoxin B 1 It is a toxic secondary metabolite produced by some Aspergillus flavus and Aspergillus parasiticus; Zearalenone is mainly a non-steroidal estrogenic mycotoxin produced by a variety of Fusarium fungi. Both toxins are highly toxic, carcinogenic and teratogenic, contaminating a variety of economic crops and causing food and feed safety issues. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/81C12N1/19A23L5/20C12R1/645
CPCC12N9/0065C12N15/815A23L5/25C12Y111/01013C07K2319/00
Inventor 夏雨吴梓凤秋杨煜何瑞程倩倩王周平
Owner JIANGNAN UNIV
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