CN110684754A - Mycotoxin ZEN degrading enzyme mutant and application thereof - Google Patents
Mycotoxin ZEN degrading enzyme mutant and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The invention discloses a mycotoxin ZEN degrading enzyme mutant and application thereof, and belongs to the technical field of biological engineering. The invention discloses zearalenone degrading enzyme (ZENG enzyme for short) derived from Gliocladium roseum MA918 as a parent, and a double mutant H134F/S136F is obtained by simultaneously replacing histidine His at a 134 th position and serine Ser at a 136 th position with phenylalanine Phe by using a gene mutation technology. Under the most suitable catalysis condition, the residual enzyme activity of the enzyme is improved by 36 percent after the enzyme is kept at 53 ℃ for 2 min; the residual enzyme activity after 5min of heat preservation is improved by 33 percent; after the heat preservation is carried out for 7min, the residual enzyme activity is improved by 12 percent. The residual enzyme activity is improved by 34 percent after the temperature is kept for 2min at 58 ℃, and the residual enzyme activity is improved by 13 percent after the temperature is kept for 5 min.
Description
Technical Field
The invention relates to a mycotoxin ZEN degrading enzyme mutant and application thereof, belonging to the technical field of biological engineering.
Background
Zearalenone (ZEN), also known as F-2 toxin, is a fungal toxin produced by many fusarium species such as fusarium graminearum, fusarium flavum and fusarium graminearum and released into the soil environment. The chemical structure of ZEN was determined by Urry in 1966 using techniques such as nuclear magnetic resonance, classical chemistry and mass spectrometry and was named: 6- (10-hydroxy-6-oxy-undecenyl) beta-resorcinolic acid lactone. ZEN has wide pollution in grains and byproducts thereof all over the world, brings huge loss to the planting industry and the breeding industry, and also poses serious threat to food safety.
Currently, the degradation method of ZEN can be divided into three categories, namely physical, chemical and biological.
The physical method mainly comprises manual removal, water washing, shelling, high temperature, pressure boiling, adsorbent adsorption and the like. Removing ZEN from slightly ZEN-polluted grains by means of removing, washing and the like; because the ZEN has good thermal stability and does not decompose after being heated for 4 hours at 120 ℃, the modes of heat treatment, pressure cooking and the like mainly have a killing effect on fungi generating the ZEN, the damage effect on the ZEN is small, and the high temperature damages the nutritional value of food and feed and influences the taste of the food; the adsorption method mainly utilizes the hydrophobic effect to achieve the purpose of removing mycotoxin in food, and common adsorbents comprise yeast cell walls, activated carbon, montmorillonite, particularly modified montmorillonite, kaolin, cholestyramine and the like, but the adsorbents can combine some important nutrient substances such as amino acid, vitamin and the like in feed while adsorbing the toxin, influence nutrient substances in food, reduce the product quality, and can not be completely degraded, so that the environment can be seriously influenced.
The degradation principle of the chemical method is oxidative degradation, and the chemical structure of ZEN is O3，H2O2And the like, and finally becomes a nontoxic ZEN byproduct. However, the chemical method has large workload and long operation time, and the ZEN is oxidized and degraded and can damage the nutrient components in the feed, so the improvement is still needed.
The principle of the biological method is to degrade ZEN into a nontoxic product by utilizing the adsorption of microbial cells and the degradation of the ZEN by the microbes or enzymes generated by the microbes. Particularly, the latter, the key enzyme gene with the ability to degrade ZEN is the focus of the current biological method research. At present, the research on the zearalenone biodegradation technology is still incomplete, for example, the principle of enzyme degradation and conversion of ZEN is not deeply researched, and meanwhile, the problems that the function of ZEN degrading bacteria is easy to decline and is unstable are endless and need to be improved.
Therefore, an effective method for biodegrading ZEN is provided, and the method has a wide application prospect.
Disclosure of Invention
One of the purposes of the invention is to provide a fungal toxin ZEN degrading enzyme mutant with improved thermal stability, and the amino acid sequence of the mutant is as follows:
(a) as shown in SEQ ID NO. 4;
(b) or (b) the protein which is derived from the (a) and has ZEN degrading enzyme activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by the (a).
One of the objects of the present invention is to provide a gene encoding the above-mentioned mycotoxin ZEN degrading enzyme mutant.
Furthermore, the nucleotide sequence of the gene is shown as SEQ ID NO. 3.
It is an object of the present invention to provide plasmids containing the above genes, including but not limited to pET series plasmids.
Further, the plasmid includes pET-22b (+).
One of the purposes of the invention is to provide Escherichia coli expressing the fungal toxin ZEN degrading enzyme mutant.
Further, the Escherichia coli is Escherichia coli BL21(DE 3).
One of the purposes of the invention is to provide a method for improving the thermal stability of ZEN degrading enzyme, which is characterized in that histidine His at the 134 th position and serine Ser at the 136 th position of zearalenone degrading enzyme parent enzyme with amino acid sequences shown as SEQ ID NO.2 are simultaneously replaced by phenylalanine Phe. The nucleotide sequence of the gene for coding the parent enzyme of the zearalenone degrading enzyme is shown as SEQID NO.1, and the number of the gene is KR363960.1 in GeneBank
One of the purposes of the invention is to provide a method for degrading ZEN, which takes ZEN as a substrate and adds crude enzyme solution or whole cells containing the mycotoxin ZEN degrading enzyme mutant.
Further, the addition amount of the mycotoxin ZEN degrading enzyme mutant is 1-10000U/g substrate.
One of the purposes of the invention is to provide the application of the ZEN degrading enzyme mutant in the field of agricultural products.
One of the purposes of the invention is to provide the application of the ZEN degrading enzyme mutant in the field of feed.
The invention has the beneficial effects that: the invention provides a mutant enzyme H134F/S136F of ZENG, the optimum catalytic condition of which is not changed, compared with the parent enzyme ZENG, the optimum catalytic condition of the mutant enzyme H134F/S136F of ZENG is not changed, but the residual enzyme activity is improved by 36 percent after the mutant enzyme is insulated for 2min at 53 ℃; the residual enzyme activity after 5min of heat preservation is improved by 33 percent; after the heat preservation is carried out for 7min, the residual enzyme activity is improved by 12 percent. The residual enzyme activity is improved by 34 percent after the temperature is kept for 2min at 58 ℃, and the residual enzyme activity is respectively improved by 13 percent after the temperature is kept for 5 min. The finding has important value for the industrial application of the ZEN in degrading the lactonohydrolase.
Drawings
FIG. 1: comparison of the thermostability of the parent enzyme and the mutant enzyme H134F/S136F at 53 ℃.
FIG. 2: comparison of the thermostability of the parent enzyme and the mutant enzyme H134F/S136F at 58 ℃.
Detailed Description
Enzyme activity determination method of (I) ZEN degrading enzyme
The reaction system was 250. mu.L, and consisted of 5. mu.L of ZEN in methanol (4mg/mL), 5. mu.L of the enzyme solution (0.5mg/mL) and 240. mu.L of phosphate buffer (50mM, pH 7.0) and stopped by adding 50. mu.L of hydrochloric acid (1mol/L) and 300. mu.L of methanol at 38 ℃ for 10 min.
1U total enzyme activity is defined as the amount of enzyme required to consume 1. mu.g of substrate per minute for the reaction at pH 7.0, 38 ℃.
(II) protein purification method
A nickel ion affinity chromatography column was prepared by first pumping deionized water into the column (about 6-12 column volumes) using a constant flow pump at room temperature, and then equilibrating the column environment with buffer A (500mmol/L NaCl, 50mM Tris-HCl, pH 8.0). When the effluent at the lower end of the column and buffer A pumped into the column have the same pH value (about 5 column volumes of buffer), the resulting membrane-passed crude enzyme solution is added to the column. The heteroproteins are first washed with buffer B (500mmol/L NaCl, 50mmol/L imidazole, 50mM Tris-HCl, pH 8.0) to baseline equilibrium and then eluted with an eluent containing high concentrations of imidazole (500mmol/L NaCl, 500mmol/L imidazole, 50mM Tris-HCl, pH 8.0). Collecting the eluate of the absorption peak, and determining the enzyme activity to obtain the target protein.
Example 1: preparation method of ZENG enzyme mutant
Selecting ZENG degrading enzymes from different microbial sources, analyzing the difference condition of the thermal stability of the ZENG degrading enzymes, and comparing respective amino acid sequences; at the same time, based on the crystal structure that has been resolved, the "cap" region and the core catalytic region where residues at positions 134 and 136 are exactly in the ligase structure are analyzed. For this purpose, site-directed mutagenesis of residues 134 and 136 was chosen in different ways.
Construction of pET-22b (+) -H134F/S136F mutant plasmid:
according to the zearalenone degrading enzyme-encoding gene derived from Gliocladium roseum MA918 (accession number: KR363960.1), a ZEN lactone-degrading hydrolase gene was synthesized and ligated between Nde I and Xho I enzyme-cleavage sites of pET-22b (+), to obtain a recombinant plasmid pET22b (+) -ZENG. The pET-22b (+) -ZENG plasmid is used as a template, H134F/S136F site-directed mutation is introduced through PCRl, PCR2 and PCR3, and sequencing verification results show that random mutation does not occur except the required mutation site, so that the construction of the mutant plasmid pET-22b (+) -H134F/S136F is successful.
The H134F/S136F mutant primers were as follows: (the mutated base is underlined)
The primers on the outer side: 5'-TTATCCATATGGAGGATGCAAACATGCAT-3' the flow of the air in the air conditioner,
downstream outer primer: 5'-TATATCTCGAGCCGATAACACACTTCATT-3' the flow of the air in the air conditioner,
forward mutation primer: 5' -TGGACTTTCTTTTTAA CACCGC-3', the mutated bases underlined,
reverse mutation primer: 5' -CAGCGGTGTTAAAAAGAAAGTCCAGTAG-3', the mutated base is underlined.
PCR 1: composition of the reaction system:
a cloning vector pET22b (+) -ZENG with a zearalenone degrading enzyme target gene is used as a template.
And (3) PCR 2: composition of the reaction system:
| 10×PCR Buffer | 5μL |
| dNTP(2mmol/L) | 4μL |
| downstream outer primer (10. mu.M) | 1μL |
| Forward mutation primer (10. mu.M) | 1μL |
| pET22b(+)-ZENG | 0.5μL |
| Taq Plus DNA polymerase(5U/μL) | 0.5μL |
| ddH2O | Make up the system to 50. mu.L |
The amplification conditions of PCRl and PCR2 are as follows:
pre-denaturation at 94 ℃ for 4 min; then denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; finally, keeping the temperature at 72 ℃ for 10 min.
Detecting the amplification products of PCRl and PCR2 by agarose electrophoresis, and recovering and purifying the tapping rubber;
and (3) PCR: composition of the reaction system:
| 10×PCR Buffer | 10μL |
| dNTP(2mmol/L) | 8μL |
| upstream outer primer (10. mu.M) | 1μL |
| Downstream outer primer (10. mu.M) | 1μL |
| PCR1 purified product | 10μL |
| PCR2 purified product | 10μL |
| Taq Plus DNA polymerase(5U/μL) | 1μL |
| ddH2O | Make up the system to 100. mu.L |
The PCR3 amplification conditions were:
pre-denaturation at 94 ℃ for 4 min; then denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; finally, keeping the temperature at 72 ℃ for 10 min.
The PCR3 amplification product after recovery and purification by agarose gel electrophoresis is cut by restriction endonucleases Nde I and Xho I, then is connected to a vector pET-22b (+), is transformed into an escherichia coli DH5 alpha competent cell, is cultured overnight in an LB solid culture medium containing 50 mu g/mL of ampicillin, is picked to be singly cloned in an LB liquid culture medium containing 50 mu g/mL of ampicillin for culture, and then a mutant plasmid pET-22b (+) -H134F/S136F is extracted, and is identified as a correct mutation by sequencing.
Example 2: expression and purification method of mutant enzyme H134F/S136F
And (3) transforming the mutant plasmid pET-22b (+) -H134F/S136F subjected to sequencing verification into an escherichia coli BL21(DE3) cell, selecting a positive transformant, carrying out shake-flask culture in an LB culture medium at 37 ℃ and 200rpm overnight, inoculating the positive transformant into the LB culture medium at 37 ℃ for 3-4H until the OD value is 0.6-0.8, cooling to 28 ℃, and adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.6mM for induction for 6H.
The fermentation broth was centrifuged at 4 ℃ and l0000rpm for 20min to obtain the cells. 20mL of buffer (50mM Tris, 200mM NaCl, HCl to adjust pH to 8.5) was added to fully resuspend the cells, and then the centrifuge tube was placed in an ice bath and placed in an ultrasonic cell disrupter under the conditions: working time 1s and stopping time 2s, and the total time is 18 min. And centrifuging the obtained crushed solution at low temperature and high speed for 30min at 4 ℃ and 10000rpm to obtain a crude enzyme solution. Filtering with 0.45 μm microporous membrane. The purified ZENG mutant enzyme H134F/S136F is electrophoretically pure.
Example 3: thermostability assay of mutant enzyme H134F/S136F.
The parent enzyme and the mutant enzyme H134F/S136F are respectively subjected to heat preservation at 53 ℃ and 58 ℃ for a period of time, the residual enzyme activity is determined, the initial enzyme activity at 0 time of heat preservation at 53 ℃ and 58 ℃ is 100%, and the specific enzyme activity is 69U/mg and 29U/mg respectively. Compared with a parent enzyme ZENG, the optimum catalytic condition of the mutant enzyme H134F/S136F of the ZENG is not changed, but the residual enzyme activity of the enzyme is improved by 36 percent after the enzyme is kept at 53 ℃ for 2 min; the residual enzyme activity after 5min of heat preservation is improved by 33 percent; after the heat preservation is carried out for 7min, the residual enzyme activity is improved by 12 percent. The residual enzyme activity is improved by 34% after the temperature is kept for 2min at 58 ℃, and the residual enzyme activity is improved by 13% after the temperature is kept for 5min (see a figure 1 and a figure 2).
Comparative example 1
Taking zearalenone degrading enzyme with an amino acid sequence shown as SEQ ID NO.2 as parent enzyme, and simultaneously replacing histidine His at the 134 th position and serine Ser at the 136 th position with alanine Ala to obtain a mutant H134A/S136A, wherein the amino acid sequence is consistent with that in the rest examples. The results show that mutant H134A/S136A was only 5min inactive when incubated at 53 and 58 ℃.
Comparative example 2
The zearalenone degrading enzyme with the amino acid sequence shown as SEQ ID No.2 is used as a parent enzyme, the histidine His at the 134 th position and the serine Ser at the 136 th position are replaced by other non-hydrophobic amino acids, and other conditions are kept consistent with those in the embodiment. The results show that incubation at 53 and 58 ℃ for more than 2min lost almost all viability.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> mycotoxin ZEN degrading enzyme mutant and application thereof
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Claims (10)
1. A fungal toxin ZEN degrading enzyme mutant with improved thermostability, characterized in that its amino acid sequence is:
(a) as shown in SEQ ID NO. 4;
(b) or (b) the protein which is derived from the (a) and has ZEN degrading enzyme activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by the (a).
2. A gene encoding the mycotoxin ZEN degradative enzyme mutant of claim 1.
3. A plasmid containing the gene of claim 2.
4. The plasmid of claim 3, wherein the plasmid comprises, but is not limited to, pET series plasmids.
5. A cell expressing the mycotoxin ZEN degrading enzyme mutant of claim 1.
6. The cell of claim 5, wherein the cell is E.coli BL21(DE 3).
7. A method for improving the heat stability of ZEN degrading enzyme is characterized in that histidine His at the 134 th position and serine Ser at the 136 th position of zearalenone degrading enzyme with an amino acid sequence shown as SEQ ID NO.2 are simultaneously replaced by phenylalanine Phe.
8. A method for degrading ZEN is characterized in that ZEN is used as a substrate, and crude enzyme solution or whole cells containing the mycotoxin ZEN degrading enzyme mutant are added.
9. Use of the ZEN degrading enzyme mutant of claim 1 in the field of agricultural products.
10. The use of the ZEN degrading enzyme mutant of claim 1 in the field of feed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708604A (en) * | 2021-01-29 | 2021-04-27 | 潍坊康地恩生物科技有限公司 | Zearalenone toxin degrading enzyme mutant and high-yield strain thereof |
| CN113308449A (en) * | 2021-06-24 | 2021-08-27 | 江南大学 | Mutant S162P of zearalenone lactone hydrolase with improved thermal stability and application thereof |
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| CN113308449B (en) * | 2021-06-24 | 2022-09-27 | 江南大学 | Mutant S162P of zearalenone lactone hydrolase with improved thermal stability and application thereof |
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