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Zearalenone degrading enzyme mutants with improved enzyme activity as well as coding gene and application of zearalenone degrading enzyme mutants

A technology of zearalenone and encoding gene, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of narrow substrate application range, low catalytic activity, low enzyme production level, etc., achieves good application potential, improved degradation efficiency, The effect of good market application prospects

Active Publication Date: 2021-06-15
QINGDAO GENYUAN BIOLOGICAL TECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current engineering strains of zearalenone degrading enzymes have low enzyme production levels, low catalytic activity, and narrow substrate application range, and there are still considerable resistance to large-scale popularization and application.

Method used

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  • Zearalenone degrading enzyme mutants with improved enzyme activity as well as coding gene and application of zearalenone degrading enzyme mutants
  • Zearalenone degrading enzyme mutants with improved enzyme activity as well as coding gene and application of zearalenone degrading enzyme mutants
  • Zearalenone degrading enzyme mutants with improved enzyme activity as well as coding gene and application of zearalenone degrading enzyme mutants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Error-prone PCR construction and screening of zearalenone-degrading enzyme mutant library

[0050] refer to Clonostachys rosea The amino acid sequence (SEQ ID NO: 1) and its optimized DNA sequence (SEQ ID NO: 2) of the source zearalenone degrading enzyme were artificially synthesized, and the EcoR I restriction enzyme was designed at the 5' end Cutting site, Not I restriction enzyme cutting site was designed at the 3' end. The synthesized gene fragment was double digested with EcoR I and Not I, recovered by gel cutting, connected to the pET-21a(+) vector, transformed into Escherichia coli DH5α, positive clones were screened on the ampicillin-resistant LB plate, and the recombinant was verified by sequencing Vector pET-YMDS0, recombinant bacteria BL21-YMDS0.

[0051] Use the GeneMorph II random mutation PCR kit to use the gene shown in SEQ ID NO: 2 as a template for random mutation. The primer sequences used are as follows:

[0052] YMDS-F: A GAATTC ATGCG...

Embodiment 2

[0059] Example 2: Expression of Zearalenone Degrading Enzyme Mutant in Pichia pastoris GS115

[0060] The artificially synthesized gene YMDS0 and the zearalenone degrading enzyme mutants YMDS01, YMDS02, and YMDS03 screened in Example 1 were constructed on the pPIC9K plasmid to obtain the expression vectors pPIC-YMDS0, pPIC-YMDS01, pPIC-YMDS02, pPIC-YMDS03; the expression vectors of each mutant were digested with Sal I and linearized, then electrotransformed into Pichia pastoris GS115, and the transformants of the expression strains of each mutant were screened on the MD plate and transformed into Received into YPD plate for activation.

[0061] The activated transformants were picked and inoculated in BMGY medium, cultured with shaking at 30°C for 18 hours, and then centrifuged to obtain bacterial cells. Transfer an appropriate amount of bacteria into BMMY medium to make the concentration of bacteria reach OD600=1, continue shaking culture, and add 1% methanol of the culture ...

Embodiment 3

[0062] Embodiment 3: Fermentation and preparation of zearalenone degrading enzyme mutant in 30L fermenter

[0063] Streak the Pichia pastoris preserved in the glycerol tube on the YPD plate, and culture it upside down at 30°C for 3 days to grow a single colony. Pick a single colony that grows well and continue to streak on the YPD plate to activate the Pichia obtained from the third generation. A single colony of red yeast was inoculated in 50 mL of BMGY medium, and cultured at 30°C and 200 rpm for 24 hours. Inoculate into 1000mL BMGY medium with a 2% inoculum amount, cultivate at 30°C and 200rpm until the OD600 is 5, and use it as seed solution to inoculate the fermenter.

[0064] Fermentation production process: BSM medium, pH 4.8, temperature 30°C, stirring rate 500 rpm, ventilation rate 1.5 (v / v), dissolved oxygen controlled above 20%.

[0065] The fermentation process is divided into three stages: (1) Bacteria culture stage: 8% of the seed liquid is added, and cultured a...

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Abstract

The invention provides zearalenone degrading enzyme mutants with improved enzyme activity as well as a coding gene and application of the zearalenone degrading enzyme mutants. Zearalenone degrading enzyme genes from Clonostachys rosea are modified by an error-prone PCR method, and the zearalenone degrading enzyme mutants YMDS01, YMDS02 and YMDS03 are obtained after screening. The zearalenone degradation capability of the zearalenone degrading enzyme mutants is obviously improved, and the zearalenone degrading enzyme mutants have good market application prospects and industrial value.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and in particular relates to a zearalenone-degrading enzyme mutant with improved enzyme activity, its coding gene and its application. Background technique [0002] Zearalenone (Zearalenone), also known as F-2 toxin, was originally isolated from moldy corn. It is mainly a non-steroidal estrogenic mycotoxin produced by Fusarium. It is widely found in corn, barley, wheat and sorghum. and other grains and their by-products. Zearalenone enters the food chain through contaminated grain agricultural by-products and feed, and after being absorbed by animals, it causes livestock estrogen syndrome, resulting in excessive estrogen in livestock, causing infertility, miscarriage, stillbirth and teratogenic phenomena. It is highly carcinogenic and seriously endangers livestock and human health. At present, the main methods for detoxification of zearalenone include physical, chemica...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/81C12N1/19C12R1/84
CPCC12N9/16C12N15/815
Inventor 肖志壮张稳毕可方安然王文博张大伟付五兵
Owner QINGDAO GENYUAN BIOLOGICAL TECH GRP
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