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Zearalenone lactone hydrolase mutant S162P with improved thermal stability and application thereof

A technology of giaralenone lactone and hydrolase, applied in the field of enzyme engineering, can solve the problems of secondary pollution, low removal efficiency, unfavorable environmental protection, animal and plant health, etc., and achieve the effect of improving thermal stability

Active Publication Date: 2021-08-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, physical removal is to remove ZEN 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 to remove ZEN. Toxin, the removal efficiency is not high and it will cause secondary pollution, which is not conducive to environmental protection and animal and plant health

Method used

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  • Zearalenone lactone hydrolase mutant S162P with improved thermal stability and application thereof
  • Zearalenone lactone hydrolase mutant S162P with improved thermal stability and application thereof
  • Zearalenone lactone hydrolase mutant S162P with improved thermal stability and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Preparation of mutant enzyme S162P

[0042] Construction of pET-22b(+)-S162P plasmid:

[0043] Construction of recombinant plasmid pET22b-Glro containing wild-type Glro: According to the gene encoding the ZEN lactonohydrolase of GliocladiumroseumMA918 (GenBank: KR363960.1), the gene fragment Glro of ZEN lactonohydrolase was synthesized and connected to the vector pET-22b (+) was cut between Nde I and Xho I to obtain recombinant plasmid pET22b(+)-Glro.

[0044] Using the pET-22b(+)-Glro plasmid as a template, through PCR 1 and PCR 2, the S162P site-directed mutation was introduced, and the sequencing verification results showed that there was no random mutation except the required mutation site, indicating that the mutant plasmid pET-22b( +)-S162P built successfully.

[0045] Mutation primers are as follows: (underlined are mutants)

[0046] Forward mutation primer: 5'-GTCTGGAGGC CCG GAGGCGTGGCAAGCCATG-3',

[0047] Reverse mutation primer: 5'-CCACGCCTC ...

Embodiment 2

[0055] Embodiment 2: the expression purification method of mutant enzyme

[0056] The mutant plasmid pET-22b(+)-S162P verified by sequencing was transformed into large E.coli BL21(DE3) cells, and the positive transformants were picked and cultured overnight in LB medium at 37°C and 200rpm, and then cultured in LB Base cultured at 37°C for 3h-4h to OD value of 0.6-0.8, cooled to 28°C, added IPTG so that its final concentration was 0.6mM, and induced for 6h.

[0057] The fermentation broth was centrifuged at 4°C and 10000 rpm for 20 min, 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 1 s, The stop time is 2s, a total of 18min. The obtained crushed solution was subjected to low-temperature high-speed centrifugation at 4°C and 10,000 rp...

Embodiment 3

[0059] Example 3: Thermostability determination of mutant enzyme S162P

[0060] The thermal stability changes of the enzymes before and after the mutation were compared, wherein the wild enzyme refers to the ZEN lactonohydrolase (GenBank: ALI16790.1) derived from Gliocladium roseum MA918, and the mutant enzyme refers to the mutant enzyme S162P prepared in Examples 1 and 2.

[0061] (1) Determination of ZEN lactone hydrolase enzyme activity:

[0062] Standard reaction system: 5 μL of ZEN in methanol solution (4 mg / mL ZEN), 5 μL of enzyme (0.5 mg / mL) and 240 μL of phosphate buffer (50 mM, pH 7.0), reacted at 38°C for 10 min, added 50 μL of 1N hydrochloric acid and 300 μL of methanol was used to stop the reaction. 1U of total enzyme activity is defined as the amount of enzyme required to consume 1 μg of substrate per minute at pH 7.0 and 38°C.

[0063] The synthetic amount of ZEN was detected by HPLC, and the specific enzyme activity of mutant S162P was calculated to be 320U / mg...

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Abstract

The invention discloses a zearalenone lactone hydrolase mutant S162P with improved thermal stability and application thereof, and belongs to the technical field of enzyme engineering. According to the invention, zearalenone lactone hydrolase derived from a microorganism GliocladiumroseumMA918 is adopted as a parent, and a gene mutation technology is adopted to mutate serine (S) at the 162 site into proline (P) so as to obtain the single-site mutant S162P. Compared with a wild enzyme, on the basis that the original catalytic activity is not obviously changed, the thermal stability of the mutant enzyme is obviously improved: after the mutant enzyme is kept at 50 DEG C for 2 minutes, the residual enzyme activity is improved to be 1.52 times of that of the wild enzyme; and after heat preservation is conducted for 10 min and 20 min at the temperature of 48 DEG C respectively, about 80% and 58% residual enzyme activities are still remained, and the half-life period at the temperature of 48 DEG C is also increased by 3.6 times that of the wild enzyme. The invention lays a certain foundation for industrial application of the ZEN lactone hydrolase.

Description

technical field [0001] The invention relates to mutant S162P of zearalenone hydrolase with improved thermal stability and application thereof, belonging to the technical field of enzyme engineering. Background technique [0002] Zearalenone (Zearalenone, ZEN) is a non-steroidal estrogen mycotoxin, the chemical name is 6-(10-hydroxy-6oxy-undecenyl) β-resorcinolactone . In 1962, ZEN was first discovered in moldy corn. It is a toxic secondary metabolite produced by Fusarium. It widely exists in moldy corn, wheat, barley and other grains and grain by-products. a worldwide food and feed contaminant. Usually, unsuitable storage conditions (10-30°C temperature and 40-50% ambient relative humidity) are the main cause of ZEN. In addition, ZEN also has a variety of toxic derivatives such as: α / β-Zearalenol (α / β-Zearalenol, α / β-ZOL), α / β-Zearalanol (α / β-Zearalanol , α / β-ZAL), zearalenone (Zearalanone, ZAN), etc. As an exogenous estrogen analogue, ZEN and its derivatives can compet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21A23L5/20C12R1/19
CPCC12N9/16C12N15/70A23L5/25
Inventor 徐炜张文立张振霞光翠娥沐万孟
Owner JIANGNAN UNIV
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