CN110292105B - Cucumber mediator for improving degradation rate of laccase to mycotoxin and application thereof - Google Patents
Cucumber mediator for improving degradation rate of laccase to mycotoxin and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of agricultural biology, and particularly relates to a cucumber mediator for improving the degradation rate of laccase to mycotoxin and application thereof. The invention provides a laccase mediator for mycotoxin degradation, which comprises a cucumber aqueous extract. The mediator can assist laccase to effectively degrade mycotoxins with different structural types, and is widely applied to the field of food and feed mycotoxin detoxification.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a cucumber mediator for improving the degradation rate of laccase to mycotoxin and application thereof.
Background
Mycotoxins are secondary metabolites produced by fungi, mainly pollute stored grain and oil food and feed, and seriously harm human and livestock health. According to the structural characteristics, the mycotoxins can be divided into two main classes of aromatic rings and non-aromatic rings, wherein the aromatic rings comprise aflatoxin, zearalenone, citrinin, ochratoxin, patulin, trichothecene toxins and the like; the non-aromatic ring only comprises fumonisins, wherein aflatoxin and zearalenone are the most common and most harmful mycotoxins.
At present, the detoxification method of the feed polluted by mycotoxin mainly comprises a physical method, a chemical method, an adsorption method, a biological method and the like. Physical and chemical detoxification methods have the defects of difficult operation, unstable effect, large loss of nutrient components, influence on the palatability of the feed and the like. Although the adsorption method is simple and easy, the method has the defects of large dosage, low economy, easy secondary pollution and the like. The microbial detoxification method has the advantages of mild action conditions, little influence on sensory properties, palatability and the like of the raw materials, increase in the nutritive value of the raw materials and the like, and is considered to be the optimal detoxification method. Biological detoxification mainly refers to the enzymatic reaction of degrading enzymes to convert toxins into low-toxicity or non-toxicity products, wherein the degrading enzymes comprise oxidases such as laccase, manganese peroxidase, hydrolases (such as esterase) and the like.
In the process of realizing large-scale application of the biological detoxification technology, bacterial strains capable of degrading the mycotoxin are searched and screened, characteristic research is carried out on extracellular degrading enzymes produced by the bacterial strains, and the degrading enzyme genes are cloned and expressed, so that the bacterial strains are important breakthrough points and development directions in the field of biological degradation research of the mycotoxin. The degradation rate of the existing laccase to the mycotoxin is generally low, so that how to effectively improve the degradation rate of the laccase to the mycotoxin is the key point of a biological detoxification technology.
It has been shown that laccases can oxidize a variety of refractory substrates with the aid of mediators. Laccase mediators are compounds with low molecular weight and low redox potential, are easy to obtain lost electrons, can form a high-activity and stable intermediate under the action of laccase, and act on a substrate to be oxidized. Currently, commonly used mediators can be classified into 3 classes: 1) synthetic mediators, such as: 2, 2-hydrazine-bis (3-ethyl-benzothiazole-6-sulfonic Acid) (ABTS), violuric acid (VIO), 1-Hydroxybenzotriazole (HBT), 2 ', 6, 6' -tetramethylpiperidine oxide (TEMPO), and the like; 2) natural mediators, such as: syringaldehyde (Syringaldehyde, SA), Acetosyringone (Acetosyringone, AS), Acetovanillone (Acetovanillone), p-coumaric acid (p-coumaric acid), and the like; 3) other types of mediators, such as Polyoxometalates (POM), and the like. However, the synthesis of mediators suffers from the following disadvantages: 1) the stability is poor; 2) the price is high; 3) has potential toxicity, and some mediators can generate some toxic byproducts after being oxidized to inactivate enzymes; 4) most synthetic mediators are not regenerated after oxidation. The above disadvantages limit the use of synthetic mediators. The natural mediator is easy to source, low in cost, environment-friendly and high in economic value, so that more and more attention is paid.
Disclosure of Invention
The invention aims to provide a cucumber mediator for improving the degradation rate of laccase to mycotoxin.
It is a further object of the present invention to provide a use of the above mediator.
It is a further object of the present invention to provide a method for increasing the degradation rate of a mycotoxin by a laccase.
It is a further object of the present invention to provide a mycotoxin degrading agent.
Cucumber mediators, according to embodiments of the present invention, that increase the rate of degradation of mycotoxins by laccases include cucumber aqueous extracts.
Wherein, the laccase BsCotA amino acid sequence is shown as SEQ ID No. 1:
MTLEKFVDALPIPDTLKPVQQSKEKTYYEVTMEECTHQLHRDLPPTRLWGYNGLFPGPTIEVKRNENVYVKWMNNLPSTHFLPIDHTIHHSDSQHEEPEVKTVVHLHGGVTPDDSDGYPEAWFSKDFEQTGPYFKREVYHYPNQQRGAILWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSDEYDVPLLITDRTINEDGSLFYPSAPENPSPSLPNPSIVPAFCGETILVNGKVWPYLEVEPRKYRFRVINASNTRTYNLSLDNGGDFIQIGSDGGLLPRSVKLNSFSLAPAERYDIIIDFTAYEGESIILANSAGCGGDVNPETDANIMQFRVTKPLAQKDESRKPKYLASYPSVQHERIQNIRTLKLAGTQDEYGRPVLLLNNKRWHDPVTETPKVGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARYQESGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVWHCHILEHEDYDMMRPMDITDPHK
according to the cucumber mediator for improving the degradation rate of laccase to mycotoxin, the cucumber aqueous extract is prepared by the method comprising the following steps:
(1) crushing the cucumber;
(2) extracting cucumber with water as extractant at a ratio of 33-35g/L, adjusting pH to 8.7-9.3, and performing ultrasonic extraction;
(3) carrying out rotary evaporation on the supernatant obtained by the extraction in the step (2);
(4) and (4) dissolving the product obtained by rotary evaporation in the step (3), and freeze-drying to obtain the cucumber aqueous extract.
According to the cucumber mediator for improving the degradation rate of the laccase to the mycotoxins, in the step (2), ultrasonic extraction is carried out for 20-25min at the temperature of 45-55 ℃ and the power of 600-800W.
According to the cucumber mediator for improving the degradation rate of the laccase to the mycotoxin, in the step (3), rotary evaporation is carried out at the temperature of 45-55 ℃ and the rotating speed of 50-60 rpm.
According to the cucumber mediator for improving the degradation rate of laccase to mycotoxin, which is a tris buffer solution of a cucumber aqueous extract, the concentration of the buffer solution is 50mM, and the pH value is 7.0.
According to the cucumber mediator for improving the degradation rate of the laccase to the mycotoxin, disclosed by the embodiment of the invention, the mycotoxin comprises aflatoxin B1 or zearalenone.
According to an embodiment of the present invention, the method for substantially increasing the degradation rate of a mycotoxin by a laccase comprises the step of degrading the mycotoxin by placing the laccase in the above-mentioned mediator.
A mycotoxin degrading agent according to an embodiment of the present invention includes the above mediator and laccase.
The mediator can efficiently degrade mycotoxin, has low cost and wide application range, and can be widely applied to the field of feed toxin degrading enzymes.
Drawings
FIG. 1 shows the degradation of aflatoxin B1 and zearalenone by laccase-cucumber water extract systems;
FIG. 2 shows the HPLC analysis results of the degradation of aflatoxin B1 by laccase-cucumber water extract system;
FIG. 3 shows the HPLC analysis results of zearalenone degradation by laccase-cucumber water extract system.
Detailed Description
Test materials and reagents
1. The strain is as follows: an engineered strain of Escherichia coli for producing laccase BsCotA derived from Bacillus subtilis.
2. Biochemical reagents: aflatoxin B1, zearalenone, methyl syringate; chromatographic purity acetonitrile, trifluoroacetic acid and Tris.
3. Culture medium:
(1) escherichia coli Medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH7.0)
Example 1 preparation of recombinant laccase BsCotA
BL21(DE3)/BsCotA Escherichia coli engineering strain containing recombinant plasmid is taken and inoculated into 50mL LB culture solution, after shaking culture at 37 ℃ and 220rpm for 12h, the strain is transferred into 300mL LB culture medium according to the proportion of 2 percent, shaking culture at 37 ℃ and 220rpm is carried out for about 3h (OD600 is approximately equal to 0.6), inducer IPTG with the final concentration of 1mM is added, after 15h of induction at 16 ℃, the strain is collected by centrifugation. The cells were resuspended in disodium hydrogen phosphate-citric acid buffer (20mM, pH 7.5). The cells were lysed by ultrasonication. The disrupted cell debris was centrifuged and removed, purified by Ni affinity column chromatography, and the electrophoretically pure eluate was collected and dialyzed into Tris-HCl protein stock (50mM Tris-HCl, pH7.4, 150mM NaCl).
Example 2 preparation of cucumber aqueous extract Medium
2.1 slicing cucumber and drying in an oven at 40 ℃ for 4 h. Adding the dry slices into a pulverizer to pulverize for 1min twice. Pure water is used as an extracting agent, the material-liquid ratio is 33g/L, the pH value is adjusted to about 8.7, the temperature is 45 ℃, the power is 600W, ultrasonic extraction is carried out for 20min, and the precipitate is discarded after centrifugation. The supernatant was transferred to a round bottom flask at 45 ℃ and 50rpm and rotary evaporated. About 20mL of distilled water was added to the flask, mixed well and poured into two 50mL centrifuge tubes. Freezing at-80 deg.C for 2 hr. Freeze drying to obtain cucumber water extract.
A50 mg/mL aqueous cucumber extract solution was prepared, mixed by inversion at room temperature for 1h, centrifuged at 12000rpm for 5 minutes, and the precipitate was discarded.
2.2 slicing cucumber and drying in an oven at 40 ℃ for 4 h. Adding the dry slices into a pulverizer to pulverize for 1min twice. Pure water is used as an extracting agent, the material-liquid ratio is 33g/L, the pH value is adjusted to about 8.7, the temperature is 45 ℃, the power is 600W, ultrasonic extraction is carried out for 20min, and the precipitate is discarded after centrifugation. The supernatant was transferred to a round bottom flask at 45 ℃ and 50rpm and rotary evaporated. About 20mL of distilled water was added to the flask, mixed well and poured into two 50mL centrifuge tubes. Freezing at-80 deg.C for 2 hr. Freeze drying to obtain cucumber water extract.
A50 mg/mL aqueous cucumber extract solution was prepared, mixed by inversion at room temperature for 1h, centrifuged at 12000rpm for 5 minutes, and the precipitate was discarded.
2.3 slicing cucumber and drying in an oven at 40 ℃ for 4 h. Adding the dry slices into a pulverizer to pulverize for 1min twice. Pure water is used as an extracting agent, the material-liquid ratio is 33g/L, the pH value is adjusted to about 8.7, the temperature is 45 ℃, the power is 600W, ultrasonic extraction is carried out for 20min, and the precipitate is discarded after centrifugation. The supernatant was transferred to a round bottom flask at 45 ℃ and 50rpm and rotary evaporated. About 20mL of distilled water was added to the flask, mixed well and poured into two 50mL centrifuge tubes. Freezing at-80 deg.C for 2 hr. Freeze drying to obtain cucumber water extract.
A50 mg/mL aqueous cucumber extract solution was prepared, mixed by inversion at room temperature for 1h, centrifuged at 12000rpm for 5 minutes, and the precipitate was discarded.
Example 3 BsCotA-cucumber aqueous extract mediator System degradation of Aflatoxin B1
Dissolving aflatoxin B1 into dimethyl sulfoxide to prepare a mother solution with the concentration of 50mg/L, and reacting according to the following reaction system: 50mM Tris-HCl (pH 7.0), 20. mu.L aflatoxin B1 solution (50mg/L), 20. mu.L cucumber aqueous extract aqueous solution (50mg/mL), 20. mu.L BsCotA (300U/L). The reaction system was set to 3 replicates with the system without laccase BsCotA added as a control. The reaction was carried out at 30 ℃ and after 10h three volumes of acetonitrile was added to stop the reaction and the degradation rate of aflatoxin B1 was analyzed by High Performance Liquid Chromatography (HPLC). The liquid chromatography is Shimadzu Nexera UHPLC high performance liquid chromatography analysis system, and the chromatographic separation column is Zorbax SB-C18 (4.6X 250mm,5 μm), mobile phase A (water of 0.06% TFA), mobile phase B (acetonitrile of 0.05% TFA); gradient elution conditions 0% B for 4 min, 0% -100% B for 15 min, 100% B for 6 min; the detection wavelength is 365 nm.
As a result, as shown in FIGS. 1 and 2, a part of aflatoxin B1 was degraded, and the degradation rate was 30.5%.
In a water and Tris-HCl buffer solution degradation system without adding a mediator, the degradation rate of laccase to aflatoxin B1 is 2.5% and 8.3%, and in a system with a cucumber aqueous extract as a mediator, the degradation rate of aflatoxin B1 is improved by about 4-15 times. Therefore, the degradation rate of laccase to aflatoxin B1 is remarkably improved by the system with the cucumber aqueous extract mediator.
Example 4 BsCotA-cucumber aqueous extract mediator System for degradation of zearalenone
Dissolving zearalenone in dimethyl sulfoxide to prepare a mother solution with the concentration of 50mg/L, and reacting the mother solution according to the following reaction system: 50mM Tris-HCl (pH 7.0), 20. mu.L aflatoxin B1 solution (50mg/L), 20. mu.L cucumber aqueous extract aqueous solution (50mg/mL), 20. mu.L BsCotA (300U/L). The reaction system was set to 3 replicates with the system without laccase BsCotA added as a control. The reaction is carried out at 30 ℃, acetonitrile with three times of volume is added after 10 hours to stop the reaction, and the degradation rate of the zearalenone is analyzed by High Performance Liquid Chromatography (HPLC). The liquid chromatography is Shimadzu Nexera UHPLC high performance liquid chromatography analysis system, and the chromatographic separation column is Zorbax SB-C18 (4.6X 250mm,5 μm), mobile phase A (water of 0.06% TFA), mobile phase B (acetonitrile of 0.05% TFA); gradient elution conditions 0% B for 4 min, 0% -100% B for 15 min, 100% B for 6 min; the detection wavelength was 316 nm.
As shown in FIGS. 1 and 3, part of zearalenone was degraded at a rate of 26.4%.
The degradation rate of laccase to zearalenone in a water and Tris-HCl buffer solution degradation system without adding a mediator is 1.7 percent and 2.6 percent respectively, and the degradation rate of zearalenone in a system with a cucumber aqueous extract as a mediator is improved by about 10-15 times. Therefore, the system added with the cucumber aqueous extract mediator remarkably improves the degradation rate of laccase on zearalenone.
Sequence listing
<110> institute of feed of Chinese academy of agricultural sciences
<120> cucumber mediator for improving degradation rate of laccase to mycotoxin and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 513
<212> PRT
<213> Bacillus subtilis
<400> 1
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu
1 5 10 15
Lys Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met
20 25 30
Glu Glu Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu
35 40 45
Trp Gly Tyr Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg
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Asn Glu Asn Val Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His
65 70 75 80
Phe Leu Pro Ile Asp His Thr Ile His His Ser Asp Ser Gln His Glu
85 90 95
Glu Pro Glu Val Lys Thr Val Val His Leu His Gly Gly Val Thr Pro
100 105 110
Asp Asp Ser Asp Gly Tyr Pro Glu Ala Trp Phe Ser Lys Asp Phe Glu
115 120 125
Gln Thr Gly Pro Tyr Phe Lys Arg Glu Val Tyr His Tyr Pro Asn Gln
130 135 140
Gln Arg Gly Ala Ile Leu Trp Tyr His Asp His Ala Met Ala Leu Thr
145 150 155 160
Arg Leu Asn Val Tyr Ala Gly Leu Val Gly Ala Tyr Ile Ile His Asp
165 170 175
Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp Glu Tyr Asp Val Pro
180 185 190
Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly Ser Leu Phe Tyr
195 200 205
Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn Pro Ser Ile
210 215 220
Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys Val Trp
225 230 235 240
Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile Asn
245 250 255
Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly Asp
260 265 270
Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys
275 280 285
Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile
290 295 300
Asp Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Ala
305 310 315 320
Gly Cys Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln
325 330 335
Phe Arg Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro
340 345 350
Lys Tyr Leu Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn
355 360 365
Ile Arg Thr Leu Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro
370 375 380
Val Leu Leu Leu Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr
385 390 395 400
Pro Lys Val Gly Thr Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg
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Gly Thr His Pro Ile His Leu His Leu Val Ser Phe Arg Val Leu Asp
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Arg Arg Pro Phe Asp Ile Ala Arg Tyr Gln Glu Ser Gly Glu Leu Ser
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Tyr Thr Gly Pro Ala Val Pro Pro Pro Pro Ser Glu Lys Gly Trp Lys
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Asp Thr Ile Gln Ala His Ala Gly Glu Val Leu Arg Ile Ala Ala Thr
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Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His Cys His Ile Leu Glu
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His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile Thr Asp Pro His
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Lys
Claims (5)
1. The method for improving the degradation rate of laccase to mycotoxin is characterized by comprising the step of placing the laccase in a mediator to degrade the mycotoxin, wherein the mycotoxin is aflatoxin B1 or zearalenone, the amino acid sequence of the laccase is shown in SEQ ID No.1, the mediator is a cucumber aqueous extract, and the cucumber aqueous extract is prepared by the method comprising the following steps:
(1) crushing the cucumber;
(2) extracting cucumber with water as extractant at a ratio of 33-35g/L, adjusting pH to 8.7-9.3, and performing ultrasonic extraction;
(3) carrying out rotary evaporation on the supernatant obtained by the extraction in the step (2);
(4) and (4) dissolving the product obtained by rotary evaporation in the step (3), and freeze-drying to obtain the cucumber aqueous extract.
2. The method for improving degradation rate of laccase to mycotoxin according to claim 1, wherein in step (2), ultrasonic extraction is performed at 45-55 ℃ and 600-800W for 20-25 min.
3. The method for improving the degradation rate of laccase to mycotoxins according to claim 1, wherein in step (3), the rotary evaporation is performed at 45-55 ℃ and 50-60 rpm.
4. The method for increasing the degradation rate of a mycotoxin by a laccase according to claim 1, characterized in that the mediator is a tris buffer solution of cucumber aqueous extract, the buffer solution having a concentration of 50mM and a pH of 7.0.
5. The mycotoxin degrading microbial inoculum is characterized by comprising a mediator and laccase, wherein the mycotoxin is aflatoxin B1 or zearalenone, an amino acid sequence of the laccase is shown as SEQ ID No.1, the mediator is a cucumber aqueous extract, and the cucumber aqueous extract is prepared by the method comprising the following steps:
(1) crushing the cucumber;
(2) extracting cucumber with water as extractant at a ratio of 33-35g/L, adjusting pH to 8.7-9.3, and performing ultrasonic extraction;
(3) carrying out rotary evaporation on the supernatant obtained by the extraction in the step (2);
(4) and (4) dissolving the product obtained by rotary evaporation in the step (3), and freeze-drying to obtain the cucumber aqueous extract.
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