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A kind of zearalenone degrading enzyme mutant with improved thermostability and application thereof

A technology of zearalenone and thermal stability, applied in the field of bioengineering, can solve problems such as low removal efficiency, unfavorable environmental protection, animal and plant health, and secondary pollution

Active Publication Date: 2021-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, physical removal is to remove zearalenone by heating, irradiation or adsorption, but it often destroys nutrients and the removal efficiency is low; chemical decomposition uses acid / alkali hydrolysis, ammoniation or ozonation, etc. Detoxification of zearalenone by means of detoxification, the removal efficiency is not high and will cause secondary pollution, which is not conducive to environmental protection and animal and plant health

Method used

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  • A kind of zearalenone degrading enzyme mutant with improved thermostability and application thereof
  • A kind of zearalenone degrading enzyme mutant with improved thermostability and application thereof
  • A kind of zearalenone degrading enzyme mutant with improved thermostability and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 Preparation of Zearalenone Degrading Enzyme Mutant

[0029] Select zearalenone-degrading enzymes from different microbial sources, analyze the differences in their thermal stability, and compare their respective amino acid sequences; at the same time, based on the crystal structure that has been resolved, analyze the The residues are exactly in the "cap" region and the core catalytic region in the ligase structure. For this reason, different ways of site-directed mutagenesis were chosen for the 134-position and 136-position residues.

[0030] Construction of pET-22b(+)-H134L / S136L mutant plasmid:

[0031] According to the gene encoding zearalenone degrading enzyme derived from Gliocladium roseum MA918 (accession number: KR363960.1), the gene Glro encoding the wild enzyme of zearalenone degrading enzyme was synthesized and linked to the pET-22b (+) Recombinant plasmid pET-22b(+)-Glro was obtained between restriction site Nde I and Xho I; using pET-22b(+)-G...

Embodiment 2

[0050] Example 2 Expression and purification of zearalenone degrading enzyme mutant

[0051] Transform the mutant plasmid pET-22b(+)-H134L / S136L into Escherichia coli BL21(DE3) cells, pick the positive transformants and culture them overnight in LB medium at 37°C and 200rpm in shake flasks, and then insert them into LB medium at 37°C Cultivate for 3-4 hours until the OD value is 0.6-0.8, cool down to 28°C, and add IPTG at a final concentration of 0.6mM to induce for 6 hours.

[0052] The fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 min to obtain bacterial cells. Add 20mL buffer (50mM Tris, 200mM NaCl, HCl to adjust the pH to 8.5) to fully resuspend the bacteria, then place the centrifuge tube in an ice bath and put it into an ultrasonic cell disruptor. The conditions for ultrasonic disruption are: working time 1s , stop time 2s, a total of 18min. The obtained crushed solution was subjected to low-temperature high-speed centrifugation at 4°C and 10,000 rpm ...

Embodiment 3

[0054] Example 3 Determination of Thermal Stability of Zearalenone Degrading Enzyme Mutant H134L / S136L

[0055] The wild enzyme and the mutant H134L / S136L were incubated at 53°C and 58°C for a period of time to measure the remaining enzyme activity. The initial enzyme activity at 0 time of incubation at 53°C and 58°C was 100%, and the specific enzyme activity was 118U / mg and 47U / mg. Such as figure 1 and figure 2 As shown, compared with the wild enzyme, the optimal catalytic conditions of mutant H134L / S136L did not change, but the residual enzyme activity of mutant H134L / S136L increased by 45% after incubation at 53°C for 2 minutes; the residual enzyme activity after incubation for 5 minutes Increased by 38%; after incubation for 7 minutes, the residual enzyme activity increased by 38%. After incubation at 58°C for 2 minutes, the residual enzyme activity increased by 41%, and after incubation for 5 minutes, the residual enzyme activity increased by 32%.

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Abstract

The invention discloses a zearalenone-degrading enzyme mutant with improved thermal stability and an application thereof, belonging to the technical field of bioengineering. In the present invention, the zearalenone degrading enzyme whose amino acid sequence is shown in SEQ ID NO.2 is used as a parent enzyme, and the histidine His at position 134 and the serine Ser at position 136 are simultaneously replaced with leucine Leu to obtain a bis Mutant H134L / S136L. After H134L / S136L was incubated at 53°C for 2 minutes, the residual enzyme activity increased by 45%; after incubation for 5 minutes, the residual enzyme activity increased by 38% respectively; after incubation for 7 minutes, the residual enzyme activity increased by 38%. After incubation at 58°C for 2 minutes, the residual enzyme activity increased by 41%, and after incubation for 5 minutes, the residual enzyme activity increased by 32%. This discovery has important application value for the preparation of industrialized zearalenone degrading enzyme with high thermal stability.

Description

technical field [0001] The invention relates to a zearalenone-degrading enzyme mutant with improved thermal stability and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Zearalenone is a non-steroidal estrogenic mycotoxin with the chemical name of 6-(10-hydroxy-6oxy-undecenyl)β-rapenolide. Zearalenone was first discovered in moldy corn in 1962. It is a toxic secondary metabolite produced by Fusarium spp. It is widely found in moldy corn, wheat, barley, and other grains and grain by-products , has become a worldwide food and feed contaminant. Usually, unsuitable storage conditions (temperature of 10-30° C. and ambient relative humidity of 40-50%) are the main reasons for the production of zearalenone. In addition, zearalenone also has a variety of toxic derivatives such as: α / β-zearalenol (α / β-Zearalenol, α / β-ZOL), α / β-zearalenol (α / β-Zearalanol, α / β-ZAL), Zearalenone (Zearalanone, ZAN) and so on. As an exogenous ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21A23L5/20
CPCA23L5/25C12N9/16C12N15/70
Inventor 沐万孟张文立张振霞徐炜
Owner JIANGNAN UNIV
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