Fungaltoxin ZEN degrading enzyme mutant and application thereof

A technology of mycotoxins and degrading enzymes, applied in the field of bioengineering, can solve the problems of insufficient research on the principle of enzyme degradation and transformation of ZEN, easy decline and instability of degrading bacteria functions, etc.

Active Publication Date: 2020-01-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are still many imperfections in the research on the biodegradation technology of zearalenone. For example, the research on the principle of enzymatic degradation and tr...

Method used

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  • Fungaltoxin ZEN degrading enzyme mutant and application thereof
  • Fungaltoxin ZEN degrading enzyme mutant and application thereof
  • Fungaltoxin ZEN degrading enzyme mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: the preparation method of ZENG enzyme mutant

[0031] Select ZENG 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, it is analyzed that the residues at positions 134 and 136 are exactly The "cap" region and the core catalytic domain in the ligase structure. For this reason, different ways of site-directed mutagenesis were chosen for the 134-position and 136-position residues.

[0032] Construction of pET-22b(+)-H134F / S136F mutant plasmid:

[0033] According to the gene encoding zearalenone degrading enzyme derived from Gliocladium roseum MA 918 (accession number: KR363960.1), the gene for ZEN degrading lactone hydrolase was synthesized and linked to the restriction site of pET-22b(+) Between Nde I and Xho I, the recombinant plasmid pET22b(+)-ZENG was obtained. Using the pET-22b(...

Embodiment 2

[0053] Example 2: Expression and purification method of mutant enzyme H134F / S136F

[0054] The mutant plasmid pET-22b(+)-H134F / S136F verified by sequencing was transformed into Escherichia coli BL21(DE3) cells, and the positive transformants were picked and cultured overnight in LB medium at 37°C and 200rpm in shake flasks, and then inserted into LB Culture the medium at 37°C for 3-4h until the OD value is 0.6-0.8, cool down to 28°C, add IPTG at a final concentration of 0.6mM to induce for 6h.

[0055] The fermentation broth was centrifuged at 4°C and 10000rpm for 20min, and the cells were collected. Add 20mL of buffer solution (50mM Tris, 200mMNaCl, 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, The stop time is 2s, a total of 18min. The obtained crushed solution was subjected to low-temperature high-speed ...

Embodiment 3

[0056] Example 3: Thermostability determination of mutant enzyme H134F / S136F.

[0057] The parent enzyme and the mutant enzyme H134F / S136F 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 69U respectively. / mg and 29U / mg. Compared with the parent enzyme ZENG, the optimal catalytic conditions of the ZENG mutant enzyme H134F / S136F did not change, but the residual enzyme activity of the enzyme after incubation at 53°C for 2 minutes increased by 36%; after incubation for 5 minutes, the residual enzyme activity increased 33%; after incubation for 7 minutes, the residual enzyme activity increased by 12%. After incubation at 58°C for 2 minutes, the residual enzyme activity increased by 34%, and after incubation for 5 minutes, the residual enzyme activity increased by 13% (see figure 1 and figure 2 ).

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Abstract

The invention discloses a fungaltoxin ZEN degrading enzyme mutant and an application thereof, which belong to the technical field of bioengineering. The invention discloses a zearalenone degrading enzyme (ZENG enzyme for short) derived from Gliocladium roseum MA918, which is used as a parent, and histidine His at the 134 site and serine Ser at the 136 site are simultaneously replaced with phenylalanine Phe by using a gene mutation technology to obtain the double mutant H134F/S136F. Under the optimal catalytic condition, the residual enzyme activity of the enzyme is improved by 36% after the enzyme is subjected to heat preservation at 53 DEG C for 2 minutes; the residual enzyme activity after heat preservation for 5 min is improved by 33%; and the residual enzyme activity is improved by 12% after heat preservation for 7min, the residual enzyme activity is improved by 34% after the temperature of 58 DEG C is kept for 2 minutes, and after the temperature is kept for 5 minutes, the residual enzyme activity is improved by 13%.

Description

technical field [0001] The invention relates to a mycotoxin ZEN degrading enzyme mutant and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Zearalenone (Zearalenone, ZEN), also known as F-2 toxin, is a mycotoxin produced and released into the soil environment by Fusarium graminearum, Fusarium yellow and Fusarium graminis and other Fusarium fungi . The chemical structure of ZEN was determined by Urry in 1966 by nuclear magnetic resonance, classical chemistry and mass spectrometry, and was named: 6-(10-hydroxyl-6oxyl-undecenyl) β-rapenolide . ZEN is widely polluted in grains and their by-products around the world, causing huge losses to the planting and breeding industries, and also poses a serious threat to food safety. [0003] At present, the degradation methods of ZEN can be divided into three categories, namely physical method, chemical method and biological method. [0004] Physical methods mainly include manual ...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21A23K20/189C12R1/19
CPCC12N9/16C12N15/70A23K20/189
Inventor 沐万孟张文立徐炜张振霞
Owner JIANGNAN UNIV
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