CN107099520A - A kind of zearalenone digestive enzyme and its encoding gene and application - Google Patents
A kind of zearalenone digestive enzyme and its encoding gene and application Download PDFInfo
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Abstract
The present invention relates to a kind of zearalenone digestive enzyme and its encoding gene, the digestive enzyme has the amino acid sequence shown in SEQ ID NO.1, or the digestive enzyme is the conservative variant for being lacked on the basis of the amino acid sequence shown in SEQ ID NO.1, replacing, insert or/and adding a conservative mutation to several amino acid and obtain.Zearalenone digestive enzyme of the present invention has the advantages that high enzyme activity, pH better tolerances, the enzymolysis of zearalenone and its several derivatives is can be widely applied to, it is relatively good particularly with the substrate specificity of zearalenone and β zearalenols.
Description
Technical field
The invention belongs to biological technical field, in particular to a kind of zearalenone digestive enzyme and its coding base
Cause and application.
Background technology
Zearalenone is separated from corn first, is the one kind that can be produced by many Fusarium sps
Non steroidal estrogen mycotoxin, crop can all produce before and after harvest.Zearalenone always including corn, barley,
It is found in many crops such as wheat and cereal by-products, especially in the environment for being adapted to fungi growth.
The derivative of zearalenone has a lot, such as zearalenol, and they can be entered by the crop of pollution
Food chain is simultaneously accumulated in human body and animal body, and biology is caused damage.The chemical constitution of zearalenone and its derivative
Similar to natural estrogen, therefore they can competitively combine ERs, cause outwardly and inwardly genitals change
And breeding difficulty, cause high female hormone disease and infertility, this toxoid can also stimulate the growth of breast cancer cell line and small
It is carcinogenic in mouse.
Given this harm of toxoid, the content in cereal, food and feed such as zearalenone have to be lower than one
Calibration is accurate.Because zearalenone etc. is extremely stable, removing this toxoid using traditional physics and chemical method is
Poorly efficient.In order to solve these problems, a promising strategy for reducing such endotoxin contamination is enzyme degraded.Enzyme is degraded not only
Toxin efficiently can be converted into non-toxic product, safety and environmental protection, and enzymic catalytic reaction selectivity is strong, degradation efficiency is high,
The nutriment of cereal will not be destroyed.
So far, there are some researchs for zearalenone digestive enzyme, obtained some jade that can degrade
The enzyme of Zearlenone toxin, they can specifically combine zearalenone and degrade it.But, at present to screening
The enzyme related to zearalenone degraded research is few in obtained microorganism.
The content of the invention
It is an object of the invention to provide a kind of zearalenone digestive enzyme and its encoding gene, and it is beautiful in hydrolysis
Application in Zearlenone and its derivative.
In order to realize the purpose of the present invention, inventor is finally obtained as follows by lot of experiments research and unremitting effort
Technical scheme：
A kind of zearalenone digestive enzyme, the digestive enzyme has the amino acid sequence shown in SEQ ID NO.1 in sequence table
Row；Or the digestive enzyme is to lack, replace on the basis of the amino acid sequence shown in SEQ ID NO.1, inserting or/and addition one
To several amino acid conservative mutation and the conservative variant that obtains.
It should be noted that zearalenone digestive enzyme provided by the present invention is a kind of lactone hydrolase.SEQ ID
Amino acid sequence shown in NO.1 is made up of 266 amino acid residues.
In order that above-mentioned degraded zymoprotein is easy to purifying, the amino for the protein that can be constituted in above-mentioned amino acid sequence
End or the upper label as shown in table 1 of carboxyl terminal connection.
The sequence of the label of table 1
| Label | Residue | Amino acid sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned degraded zymoprotein also can first be synthesized its encoding gene, then carry out biological expression and obtained with artificial synthesized.
The encoding gene of protein in above-mentioned can also be by lacking, replacing, inserting in the amino acid sequence shown in SEQ ID NO.1
Enter or add one to several and keep original enzymatic activity, or connect the coded sequence of the label shown in table 1 to obtain.
A kind of encoding gene of zearalenone digestive enzyme, the gene code：
(a) there is the protein of the amino acid sequence shown in SEQ ID NO.1；Or
(b) have derived from shown in missing, displacement, one SEQ ID NO.1 to several amino acid of insertion or/and addition
Amino acid sequence and protein with zearalenone and its derivative degrading activity.
It should also be noted that, zearalenone and its derivative degrading activity refer to that the lactone of substrate can be cut
Key, then produces dihydroxy benzenes radical derivative and release carbon dioxide with open side chain, act on zearalenone,
This several substrate of α-zearalenol, β-zearalenol, α-zearalanol, β-ZER.
Further, the encoding gene of the zearalenone digestive enzyme is (i), (ii) or (iii) DNA molecular：
(i) there is the DNA molecular of the nucleotide sequence shown in SEQ ID NO.2；
(ii) under strict conditions with the nucleotide sequence hybridization described in (i) and coding with zearalenone and its several
Plant the protein DNA molecule of derivative degrading activity；
(iii) there are DNA points of the nucleotide sequence of more than 90% homology with the nucleotide sequence described in (i) or (ii)
Son.
Nucleotide sequence shown in SEQ ID NO.2 is made up of 801 nucleotides.
Further, during the stringent condition is the solution that Na ion concentration is 50-300mM, reaction temperature is 50-68 DEG C.
For example：Can be the solution in 6 × SSC, the SDS that mass fraction is 0.5% during molecule hybridization is carried out
In, hybridize at 65 DEG C, then with 2 × SSC, SDS and 1 × SSC that mass fraction is 0.1%, mass fraction are 0.1%
SDS respectively washes film once.Wherein SDS Chinese be lauryl sodium sulfate, 1 × SSC include 0.15mol/L NaCl and
0.015mol/L citric acids；The SSC of SDS and various concentrations multiple is the common agents of this area.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing any of the above-described encoding gene are fallen within
Protection scope of the present invention.
The present invention provides a kind of recombinant vector, and the recombinant vector includes the coding base of above-mentioned zearalenone digestive enzyme
Cause.Specifically, the recombinant vector is that any of the above-described encoding gene insertion is set out into carrier (for example：PET28a) many grams
The recombinant expression carrier that grand site is obtained.The recombinant expression carrier of the gene can be contained with existing expression vector establishment.Make
During with the gene constructed recombinant expression carrier, before its transcription initiation nucleotides can plus any enhanced promoter or
Constitutive promoter, they can be used alone or are used in combination with other promoters；In addition, using the gene constructed of the present invention
During recombinant expression carrier, enhancer, including translational enhancer or transcriptional enhancer are it is also possible to use, these enhancer regions can be
ATG initiation codon or neighboring region initiation codon etc., but must be identical with the reading frame of coded sequence, to ensure whole sequence
The correct translation of row.The source of the translation control signal and initiation codon is extensive, can be it is natural or
Synthesis.Translation initiation region can come from transcription initiation region or structural gene.
The present invention also provides a kind of transformant, and the transformant includes above-mentioned recombinant vector.Transformant can be recombinant bacterium,
For example, any of the above-described encoding gene insertion is set out into carrier (for example：PET28a carriers) the obtained weight of multiple cloning sites
Group expression vector is converted to e. coli bl21 (DE3), obtains recombinant bacterium.
The present invention also provides a kind of primer pair, the encoding gene total length of the zearalenone digestive enzyme above-mentioned for expanding
And its any fragment.For example：The sequence of primer pair is as shown in SEQ ID NO.3 and SEQ ID NO.4.
It is any of the above-described protein, any of the above-described encoding gene, any of the above-described recombinant expression carrier, described
Any one in expression cassette, transgenic cell line or recombinant bacterium is in degrading zearalenone, α-zearalenol, β-jade
The red mould enol of rice, α-zearalanol, the application of β-ZER this several substrate fall within protection scope of the present invention.
During concrete application, it can adopt in the following method：With zearalenone, α-zearalenol,
β-zearalenol, α-zearalanol, β-ZER this it is several be substrate, in meta-alkalescence pH conditions, utilize corn
Zeranol digestive enzyme is to zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-jade
The red mould alcohol of rice is digested.
Described enzymatic hydrolysis condition includes：30-40 DEG C of the temperature of reaction system, preferably 40 DEG C, the pH value of reaction system is
6.0-9.0, preferably 8.0.
Present invention also offers a kind of method for producing zearalenone digestive enzyme, this method includes cultivating above-mentioned turn
Change body and zearalenone digestive enzyme is collected in cultured products.The zearalenone digestive enzyme of collection can further enter
Row purifying.
Compared with prior art, there is the albumen that the present invention is provided zearalenone and its degraded of several derivatives to live
Property, belong to zearalenone digestive enzyme.And the amino acid of the albumen and other zearalenone digestive enzymes characterized
Sequence is compared, and similitude is not more than 65%, belongs to a kind of brand-new zearalenone digestive enzyme, is that people's degrading maize is red mould
Ketenes adds a kind of new selection.The most suitable natural substrate of zearalenone digestive enzyme of the present invention is Gibberella zeae alkene
Ketone, and with the higher characteristic of the activity under the conditions of meta-alkalescence pH, while its stability under different pH is preferable.
In addition, the zearalenone digestive enzyme Zhd518 that the present invention is provided has significant progressive, major embodiment is gone back
In the following areas：
(1) document report, the zearalenone digestive enzyme characterized is Zhd101, and two kinds are ZEN- in addition
The amino acid sequence of JJM and Zlhy-6 zearalenone digestive enzyme, itself and Zhd101 amino acid identity for 99% and
98%, property is basically identical.The amino acid identity of Zhd518 and Zhd101 in the present invention only have 65%, it is determined that being a kind of new
The zearalenone digestive enzyme of type.In addition, the zearalenone digestive enzyme Zhd101 characterized optimal reactive temperature
37 DEG C, optimal pH be 9.5.The optimum temperature of Zhd518 in the present invention is 40 DEG C, optimal pH is 8.0 so that it is not required
The high environment of alkalescence.Zhd518 still has 60% enzyme activity in the range of temperature is 30-40 DEG C, is 6.0-9.0's in pH
In the range of have more than 70% enzyme activity.This illustrates that the Zhd518 that the present invention is obtained has significant advantage.
(2) the zearalenone digestive enzyme Zhd518 that the present invention is provided has degraded to ZEN and its four kinds of derivatives
Activity, but degradation capability difference.As a result it is as follows：Enzyme liquid will be diluted, and in same concentrations, (substrate is dense eventually in reaction system respectively
Spend for 20.0 μ g/ml) different substrates under the conditions of carry out enzyme activity determination.It is that substrate measures enzyme activity as reference using zearalenone
(100%) phase, is surveyed by substrate using α-zearalenol, β-zearalenol, α-zearalanol, β-ZER
It is respectively 6.6%, 94.9%, 32.6%, 21.5% to enzyme activity.Therefore, the enzyme is to zearalenone and β-Gibberella zeae alkene
The expression activitiy of alcohol is high, and other take second place.
Brief description of the drawings
Fig. 1 is the SDS-PAGE before and after zearalenone digestive enzyme Zhd518 protein purifications.
Fig. 2 is the result of the change of zearalenone digestive enzyme Zhd518 activity with temperature.
The result for the active change with pH that Fig. 3 is zearalenone digestive enzyme Zhd518.
The stability of the activity that Fig. 4 is zearalenone digestive enzyme Zhd518 at different temperatures.
Stability of the activity that Fig. 5 is zearalenone digestive enzyme Zhd518 under different pH.
Embodiment
The principle and feature of the present invention are described below in conjunction with accompanying drawing, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The preparation and purification of embodiment 1, albumen and gene
1st, gene order is artificial synthesized
Nucleotide sequence shown in SEQ ID NO.2 entrusts Wuhan Jin Kairui bioengineering Co., Ltd according to this area
Routine techniques progress gene is artificial synthesized, in gene insertion plasmid vector pUC57, preserves, standby.
2nd, the amplification of gene order
Nucleotide sequence design primer pair according to SEQ ID NO.2 is as follows：
Forward primer：5′-CGCGGATCCATGGCCGCTACACGTACACGAGG-3 ', as shown in SEQ ID NO.3；
Reverse primer：5′-CCGCTCGAGCTATTTCAAATACTTCCGACTCG-3 ', as shown in SEQ ID NO.4；
The underscore part of forward primer is BamHI restriction enzyme site, and the underscore part of reverse primer is XhoI digestions
Site.
PCR reaction systems：
| 10 × buffer solution | 5μL |
| dNTP | 4μL |
| ExTaq archaeal dna polymerases | 0.5μL |
| Forward primer | 1μL |
| Reverse primer | 1μL |
| Template | 0.5μL |
| Water | 38μL |
PCR reaction conditions：94 DEG C of pre-degeneration 5min, then 94 DEG C are denatured 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min,
30 circulations, last 72 DEG C of extensions 10min.
PCR primer mass fraction detects yield and specificity for 0.7% agarose gel electrophoresis, and is purified with DNA
Kit (ultrathin centrifugal column type, the production of Tiangeng company) purifying.The PCR primer of purifying is sequenced, as a result shows PCR primer
Sequence include shown in SEQ ID NO.2 1-801, and be named as zhd518 DNA fragmentations.
3rd, the structure of recombinant expression carrier
1) by above-mentioned sequencing correct PCR primer BamHI and XhoI double digestions, agarose electrophoresis reclaims digestion products.
2) by plasmid pET28a (Cat.N0 69864-3, Novogen) BamHI and XhoI double digestions, agarose electrophoresis
Reclaim digestion products.
3) by step 1) digestion products and step 2) digestion products be attached, the electroporated large intestine bar of connection product
It is coated on the LB flat boards containing 50 μ g/mL kanamycins after bacterium DH5 α, 37 DEG C of incubated overnights, by obtained transformant with above-mentioned
Forward primer and reverse primer carry out bacterium colony PCR, screen the recombinant bacterium containing zhd518 genes, extract the plasmid of recombinant bacterium,
Sequence verification is carried out, as a result shows, zhd518 DNA fragmentations is inserted between pET28a BamHI and XhoI restriction enzyme sites,
The fragment includes the SEQ ID NO.2 nucleotides of the 1st to 801 from holding 5 ', and direction of insertion is correct, and the recombinant plasmid is ordered
Entitled pET28a-zhd518.
4th, the preparation of engineering bacteria
By the electroporated e. coli bl21s of plasmid pET28a-zhd518 (DE3) (Cat.N0CD601, Quan Shi King Company)
The LB flat boards containing 50 μ g/mL kanamycins are coated on afterwards, and 37 DEG C of incubated overnights are obtained containing plasmid pET28a-zhd518's
Engineering bacteria, is denoted as BL21/pET28a-zhd518.
PET28a-zhd518 is replaced with pET28a, conversion e. coli bl21 (DE3), step ibid, is contained
PET28a recombinant bacterium, is used as control bacterium.The positive restructuring bacterium for being transferred to BL21 (DE3) is denoted as BL21/pET28a.
5th, the expression and purifying of target protein
His60Ni Superflow resin purification columns are purchased from TaKaRa companies, and catalog number is 635660.
GE HiTrap Desalting purification columns are purchased from GE Healthcare companies, and catalog number is respectively 17-
1408-01、17-5053-01。
Positive restructuring bacterium BL21/pET28a-zhd518 prepared by above-mentioned steps 4 is incubated at containing 50 μ g/mL cards that is mould
In the LB culture mediums of element, 37 DEG C of culture 3h；OD600When=0.7, IPTG is added to its final concentration 0.8mM in LB culture mediums,
18 DEG C are gone to continue to cultivate 16h.
Thalline is collected by centrifugation under the conditions of 3800rpm, 15min, be suspended in PBS solution (50mM Tris-HCl, pH7.4,
0.5M NaCl) in, ultrasonication (60w, the 10min in ice bath；Ultrasonic 1s, stops 2s), 12000rpm centrifuges 10min afterwards
Cell fragment is removed, supernatant is taken；Supernatant is crossed into His60Ni Superflow resin purification columns, rushed with 5mL ultra-pure waters
Wash, then rinsed with 10mL solution As (50mM Tris-HCl, pH8.0,25mM imidazoles), finally with 5mL solution Bs (50mM Tris-
HCl, pH8.0,500mM imidazoles) elution, collect eluent.Then eluent desalting column GE HiTrap Desalting are entered
Row desalting processing, is eluted with solution C (50mM Tris-HCl, pH 8.0), obtains the pure enzyme liquids of Zhd518.
Control bacterium prepared by step 4 is cultivated and purified using identical step, and obtained solution is used as control enzyme
Liquid.
The molecular weight of the Zhd518 albumen of SDS-PAGE electrophoresis showeds purifying is about 29kDa, meets theoretical implications
29.4kDa.As a result as shown in figure 1, in Fig. 1, swimming lane M represent Protein Marker (250,150,100,75,50,37,
25kDa)；Swimming lane 1 represents that e. coli bl21/pET28a-zhd518 breaks the supernatant after bacterium；Swimming lane 2 represents that Ni-NTA posts are pure
Zhd518 albumen after change；Swimming lane 3 represents the Zhd518 albumen of GE Desalting desalting columns after purification.It can be seen that obtaining
Obtained Zhd518 albumen.The experiment of control group has been carried out simultaneously, but control bacterium does not obtain destination protein.
Embodiment 2, the checking protein function by substrate of zearalenone
Enzyme-activity unit is defined as enzyme amount in 1min required for 1 μ g substrate zearalenones of degraded as an enzyme activity list
Position U.
(1) optimum temperature
The pure enzyme liquids of Zhd518 in the step 5 of embodiment 1 are diluted with pH8.0 50mM Tris-HCl buffer solutions, with dilution
Enzyme liquid afterwards carries out enzyme activity determination.Enzyme liquid after dilution is denoted as to dilute enzyme liquid.
Solution A is constituted：By 50mM, pH8.0Tris-HCl buffer solutions and zearalenone solution composition；Substrate corn is red
Final concentration of 20.0 μ g/ml of the mould ketenes in reaction system 0.5mL.
Experimental group：Determination of activity reaction system is 0.5mL, and enzyme liquid is diluted by 0.45mL solution As and 0.05mL；Reaction system
PH value be 8.0；Reaction system is incubated after 10min in specific range of temperatures (20-55 DEG C), and 0.5mL hplc grade methanols are terminated
Reaction, degradation of substrates amount is determined after cooling using high performance liquid chromatograph (HPLC).
As a result it is as shown in Figure 2.Fig. 2 shows that zearalenone digestive enzyme has the activity of degrading zearalenone.
Under the conditions of 40 DEG C, zearalenone digestive enzyme has highest enzymatic activity；By the substrate of the enzyme activity reaction system at a temperature of this
Zearalenone degradation amount is as relative activity 100%, the substrate zearalenone of enzyme activity reaction system at a temperature of other
The ratio of degradation amount and the substrate zearalenone degradation amount of this highest enzyme activity system is used as relative activity.In 30-40 DEG C of bar
More than 60% activity is respectively provided with part.
Control group：Above-mentioned experiment is carried out with the albumen (being denoted as compareing enzyme liquid) for compareing bacterium BL21/pET28a acquisitions, as a result not
Pipe is under which temperature conditions, control enzyme liquid all activity without degrading zearalenone.
Experiment sets 3 repetitions, as a result unanimously.
(2) optimal pH
Dilution enzyme liquid in following each group is the Zhd518 in the step 5 for use the buffer solution dilution embodiment 1 in each group
What pure enzyme liquid was obtained.
Experimental group：Determination of activity reaction system be 0.5mL, respectively by 0.45mL solution Bs (B1, B2, B3, B4, B5, B6,
B7, B8, B9, B10, B11, B12, B13, B14 and B15) and 0.05mL dilution enzyme liquid compositions, substrate zearalenone is in reaction
Final concentration of 20.0 μ g/ml in system 0.5mL.
The composition of solution B 1：50mM glycine-HCl buffer solutions and substrate zearalenone composition；The pH of solution B 1
It is worth for 2.0.
The composition of solution B 2：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 0.2M Na2HPO4- citrate buffer solution.The pH value of solution B 2 is 3.0.
The composition of solution B 3：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 0.2M Na2HPO4- citrate buffer solution.The pH value of solution B 3 is 4.0.
The composition of solution B 4：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 0.2M Na2HPO4- citrate buffer solution.The pH value of solution B 4 is 5.0.
The composition of solution B 5：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 0.2M Na2HPO4- citrate buffer solution.The pH value of solution B 5 is 6.0.
The composition of solution B 6：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 0.2M Na2HPO4- citrate buffer solution.The pH value of solution B 6 is 7.0.
The composition of solution B 7：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 0.2M Na2HPO4- citrate buffer solution.The pH value of solution B 7 is 7.5.
The composition of solution B 8：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 50mM Tris-HCl buffer solutions.The pH value of solution B 8 is 7.5.
The composition of solution B 9：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 50mM Tris-HCl buffer solutions.The pH value of solution B 9 is 8.0.
The composition of solution B 10：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 50mM Tris-HCl buffer solutions.The pH value of solution B 10 is 8.5.
The composition of solution B 11：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 50mM Tris-HCl buffer solutions.The pH value of solution B 11 is 9.0.
The composition of solution B 12：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 50mM glycine-NaOH buffer solutions.The pH value of solution B 12 is 9.0.
The composition of solution B 13：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 50mM glycine-NaOH buffer solutions.The pH value of solution B 13 is 9.5.
The composition of solution B 14：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 50mM glycine-NaOH buffer solutions.The pH value of solution B 14 is 10.0.
The composition of solution B 15：It is identical with the composition of solution B 1, the difference is that 50mM Glycine-HCl buffer solutions are replaced
For 50mM glycine-NaOH buffer solutions.The pH value of solution B 15 is 11.0.
By reaction system after 40 DEG C incubate 10min, 0.5mL hplc grade methanol terminating reactions are added, height is used after cooling
Effect liquid phase chromatogram instrument (HPLC) determines degradation of substrates amount.
Experiment sets three repetitions.
As a result it is as shown in Figure 3.
Zearalenone digestive enzyme is respectively provided with hydrolysed corn zeranol under conditions of being between 3.0 to 10.0 in pH
Activity, you can with degrading zearalenone.
Fig. 3 shows that zearalenone digestive enzyme has highest enzymatic activity under the conditions of pH8.0.With this highest enzymatic activity body
The substrate zearalenone degradation amount of system drops as relative activity 100%, the substrate zearalenone of other reaction systems
Solution amount and the ratio of the substrate zearalenone degradation amount of this highest enzymatic activity system are used as respective relative activity.In pH
More than 70% activity is respectively provided with the conditions of 6.0-pH 9.0.
Control group：Above-mentioned experiment is carried out with the albumen (being denoted as compareing enzyme liquid) for compareing bacterium BL21/pET28a acquisitions, as a result not
Pipe is under the conditions of which pH, control enzyme liquid all activity without degrading zearalenone.
Experiment sets 3 repetitions, as a result unanimously.
(3) enzyme heat stability
The pure enzyme liquids of Zhd518 in the step 5 of embodiment 1 are diluted with pH8.0 50mM Tris-HCl buffer solutions, with dilution
Enzyme liquid afterwards carries out enzyme activity determination by substrate of zearalenone.Enzyme liquid after dilution is denoted as to dilute enzyme liquid.
Dilution enzyme liquid is placed 10 minutes in 20 DEG C, 30 DEG C, 37 DEG C, 40 DEG C, 45 DEG C, 50 DEG C of water-baths respectively, enzyme is determined
Residual activity.As a result show, enzyme loses 80% activity for 10 minutes in 20 DEG C to 40 DEG C stabilizations in 45 DEG C of processing.
(4) pH tolerances
Enzyme liquid will be diluted respectively under the conditions of pH 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0, temperature
Placed under the conditions of 4.0 DEG C of degree after 16h, remnant enzyme activity is determined by substrate of zearalenone.As a result pH 6.0-10.0 bars are shown
Still remained under part more than 60% relative enzyme activity.Illustrate that the enzyme has good pH tolerances.
(5) substrate specificity
Enzyme liquid will be diluted respectively in same concentrations (the final concentration of 20.0 μ g/ml of substrate in reaction system) different substrate bars
Enzyme activity determination is carried out under part, substrate is respectively zearalenone, α-zearalenol, β-zearalenol, α-corn
Red mould alcohol, β-ZER.
It is that substrate measures enzyme activity as reference (100%) using zearalenone, with α-zearalenol, β-Gibberella zeae
Enol, α-zearalanol, β-ZER by substrate survey relative enzyme activity be respectively 6.6%, 94.9%, 32.6%,
21.5%.Therefore, the enzyme is high to the expression activitiy of zearalenone and β-zearalenol.
Embodiment 3, the checking protein function by substrate of β-zearalenol
Enzyme-activity unit is defined as enzyme amount in 1min required for 1 μ g substrate zearalenones of degraded as an enzyme activity list
Position U.
Dilution enzyme liquid as described below is the Zhd518 in the step 5 for diluted embodiment 1 with 50mM Tris-HCl buffer solutions
What pure enzyme liquid was obtained.
Experimental group：It is living using β-zearalenol as substrate (final concentration of 20.0 μ g/ml of the substrate in reaction system)
Property to determine reaction system be 0.5mL, enzyme liquid is diluted by 0.45mL substrate solutions and 0.05mL；The pH value of reaction system is 8.0；Instead
Answer system at 40 DEG C of optimum temperature after reaction 10min, 0.5mL hplc grade methanol terminating reactions use efficient liquid phase after cooling
Chromatograph (HPLC) determines degradation of substrates amount.
Experiment sets three repetitions, as a result unanimously.
As a result 40 DEG C are shown in, under the conditions of pH8.0, enzyme activity is surveyed as 823U/mg by substrate using β-zearalenol.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
SEQUENCE LISTING
<110>Hubei University
<120>A kind of zearalenone digestive enzyme and its encoding gene and application
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 266
<212> PRT
<213>Artificial sequence
<400> 1
Met Ala Ala Thr Arg Thr Arg Gly Tyr Val Thr Thr Lys Asp Gly Ile
1 5 10 15
Lys Trp Tyr Tyr Glu Gln Glu Gly Ser Gly Pro Asp Val Val Leu Ile
20 25 30
Pro Asp Gly Leu Gly Glu Cys Gln Met Phe Asp Lys Pro Met Ser Leu
35 40 45
Ile Ala Ser Asn Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met
50 55 60
Ser Arg Ser Ser Asp Ala Pro Pro Glu Thr Tyr Gln Asp Ile Thr Gly
65 70 75 80
Arg Lys Leu Ala Gly Tyr Ile Ile Thr Leu Leu Asp Thr Leu Asp Ile
85 90 95
Lys Ile Ala Ser Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Leu
100 105 110
Ala Leu Cys Ser Asp Tyr Pro Glu Arg Val Arg Asn Gly Met Pro His
115 120 125
Glu Val Pro Thr Glu Asn Pro Asp Ile Leu Leu His Ile His Glu Val
130 135 140
Asp Pro Ala Thr Ile Ser Gln Glu Met Ala Ala Asn Ser Arg Ala Tyr
145 150 155 160
Ser Gly Asn Val Glu Ala Trp Asp Ala Leu Gly Pro Glu Val His Ala
165 170 175
Arg Leu His Asp Asn Tyr Pro Arg Trp Ala Tyr Gly Tyr Pro Arg Thr
180 185 190
Ile Pro Pro Ser Ala Pro Val Lys Thr Glu Asp Leu His Lys Val Pro
195 200 205
Ile Asp Trp Thr Val Gly Ala Ser Thr Pro Thr Lys Leu Phe Phe Glu
210 215 220
Asn Ile Val Ile Ala Ala Arg Glu Gly Ile Asn Ile Gly Thr Leu Pro
225 230 235 240
Gly Asn His Phe Pro Tyr Val Ser His Pro Glu Glu Phe Ala Lys Tyr
245 250 255
Val Val Glu Thr Ser Arg Lys Tyr Leu Lys
260 265
<210> 2
<211> 801
<212> DNA
<213>Artificial sequence
<400> 2
atggccgcta cacgtacacg aggatatgtt accactaaag acggaataaa atggtactat 60
gagcaagagg gttccgggcc tgatgttgtc ttgattccag acggcctagg cgaatgtcag 120
atgtttgata agccaatgtc tctaattgcc agcaacggct tcagagtaac gacattcgac 180
atgcctggaa tgtcaagatc atccgatgca ccaccagaaa cgtatcagga cattacaggt 240
cgaaaattag ctggctacat tatcacactg ttggacacac tggacatcaa aatcgcttcg 300
gtctggggct gtagctcagg agcctcgacc gtccttgcac tctgctcaga ctacccagag 360
cgagtccgca atggaatgcc ccacgaggtc ccgacagaaa acccagacat tcttctgcat 420
atccacgagg tcgaccctgc cacaatctcg caagaaatgg cagccaactc gagggcctac 480
tcaggaaatg tagaggcatg ggacgctctc gggccagaag tccacgcaag gcttcatgac 540
aactacccgc gatgggcata tggctatcca cgtaccatac ccccgtccgc gccggtgaaa 600
actgaagatc tacacaaggt tccaatagat tggaccgttg gcgcttcaac tcccaccaag 660
ttgttcttcg aaaacatcgt catcgcggcc agggagggca tcaatattgg aactctgccg 720
ggcaatcatt tcccatatgt ctcgcacccg gaagagtttg cgaagtatgt cgttgagacg 780
agtcggaagt atttgaaata g 801
<210> 3
<211> 32
<212> DNA
<213>Artificial sequence
<400> 3
cgcggatcca tggccgctac acgtacacga gg 32
<210> 4
<211> 32
<212> DNA
<213>Artificial sequence
<400> 4
ccgctcgagc tatttcaaat acttccgact cg 32
Claims (10)
1. a kind of zearalenone digestive enzyme, it is characterised in that the digestive enzyme has the amino acid sequence shown in SEQ ID NO.1
Row；Or the digestive enzyme is to lack, replace on the basis of the amino acid sequence shown in SEQ ID NO.1, inserting or/and addition one
To several amino acid conservative mutation and the conservative variant that obtains.
2. a kind of encoding gene of zearalenone digestive enzyme, it is characterised in that the gene code：(a) there is SEQ ID
The protein of amino acid sequence shown in NO.1；Or (b) have derived from missing, replace, insertion or/and addition one be to several
Amino acid sequence shown in the SEQ ID NO.1 of amino acid and the active egg with degrading zearalenone and its derivative
White matter.
3. a kind of encoding gene of zearalenone digestive enzyme according to claim 2, it is characterised in that the gene is
(i), (ii) or (iii) DNA molecular：
(i) there is the DNA molecular of the nucleotide sequence shown in SEQ ID NO.2；
(ii) under strict conditions with the nucleotide sequence hybridization described in (i) and coding with degrading zearalenone and its spreading out
Biological active protein DNA molecule；
(iii) there is the nucleotide sequence of more than 90% homology with the nucleotide sequence of (i) or (ii) described DNA molecular
DNA molecular.
4. a kind of encoding gene of zearalenone digestive enzyme according to claim 3, it is characterised in that the strict bar
Part is：Na ion concentration is in 50-300mM solution, reaction temperature is 50-68 DEG C.
5. a kind of recombinant vector, it is characterised in that include the zearalenone digestive enzyme described in any one of claim 2 to 4
Encoding gene.
6. a kind of transformant, it is characterised in that include the recombinant vector described in claim 5.
7. a kind of primer pair, it is characterised in that for expanding the zearalenone digestive enzyme described in claim any one of 2-4
Encoding gene total length and its any fragment；It is preferred that the sequence of primer pair is as shown in SEQ ID NO.3 and SEQ ID NO.4.
8. a kind of zearalenone digestive enzyme as claimed in claim 1 is in hydrolysed corn zeranol and its derivative
Using.
9. application according to claim 8, it is characterised in that described derivative includes α-zearalenol, β-jade
The red mould enol of rice, α-zearalanol, β-ZER.
10. a kind of method for producing zearalenone digestive enzyme, this method is including cultivating the transformant described in claim 6 simultaneously
Zearalenone digestive enzyme is collected in cultured products.
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| CN201710516347.5A CN107099520B (en) | 2017-06-29 | 2017-06-29 | Zearalenone degrading enzyme and coding gene and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108277210A (en) * | 2017-12-18 | 2018-07-13 | 中国农业科学院饲料研究所 | Mould ketenes hydrolase ZEN214 and encoding gene and application |
| CN110029095A (en) * | 2019-04-15 | 2019-07-19 | 南京工业大学 | A kind of zearalenone degrading enzyme and its application |
| CN110511914A (en) * | 2019-09-02 | 2019-11-29 | 中国农业科学院饲料研究所 | Zearalenone hydrolase ZH9572 and its encoding gene and application |
| CN110592046A (en) * | 2019-09-30 | 2019-12-20 | 湖北大学 | Application of zearalenone degrading enzyme in hydrolysis of zearalenone and derivatives thereof |
| CN110684754A (en) * | 2019-10-25 | 2020-01-14 | 江南大学 | Mycotoxin ZEN degrading enzyme mutant and application thereof |
| CN110819608A (en) * | 2019-10-29 | 2020-02-21 | 湖北大学 | Hydrolysis method of zearalenone and derivatives thereof |
| CN114045276A (en) * | 2021-11-09 | 2022-02-15 | 北京化工大学 | Neutral zearalenone degrading enzyme mutant with improved specific enzyme activity |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199581A (en) * | 2011-03-31 | 2011-09-28 | 国家粮食局科学研究院 | Zearalenone toxin degradation enzyme and coding gene and application thereof |
| CN105755023A (en) * | 2016-03-18 | 2016-07-13 | 湖北大学 | ZEN (zearalenone) degrading enzyme gene and high-yield strain |
-
2017
- 2017-06-29 CN CN201710516347.5A patent/CN107099520B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199581A (en) * | 2011-03-31 | 2011-09-28 | 国家粮食局科学研究院 | Zearalenone toxin degradation enzyme and coding gene and application thereof |
| CN105755023A (en) * | 2016-03-18 | 2016-07-13 | 湖北大学 | ZEN (zearalenone) degrading enzyme gene and high-yield strain |
Non-Patent Citations (1)
| Title |
|---|
| EMBL-EBI: "XM_013418296", 《EMBL-EBI》 * |
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| CN108277210B (en) * | 2017-12-18 | 2021-02-23 | 中国农业科学院北京畜牧兽医研究所 | Mycetone hydrolase ZEN214, and coding gene and application thereof |
| CN108277210A (en) * | 2017-12-18 | 2018-07-13 | 中国农业科学院饲料研究所 | Mould ketenes hydrolase ZEN214 and encoding gene and application |
| CN110029095A (en) * | 2019-04-15 | 2019-07-19 | 南京工业大学 | A kind of zearalenone degrading enzyme and its application |
| CN110029095B (en) * | 2019-04-15 | 2022-06-07 | 南京工业大学 | Zearalenone degrading enzyme and application thereof |
| CN110511914A (en) * | 2019-09-02 | 2019-11-29 | 中国农业科学院饲料研究所 | Zearalenone hydrolase ZH9572 and its encoding gene and application |
| CN110511914B (en) * | 2019-09-02 | 2021-07-20 | 中国农业科学院北京畜牧兽医研究所 | Zearalenone hydrolase ZH9572 as well as encoding gene and application thereof |
| CN110592046A (en) * | 2019-09-30 | 2019-12-20 | 湖北大学 | Application of zearalenone degrading enzyme in hydrolysis of zearalenone and derivatives thereof |
| CN110592046B (en) * | 2019-09-30 | 2022-03-15 | 湖北大学 | Application of zearalenone degrading enzyme in hydrolysis of zearalenone and derivatives thereof |
| CN110684754B (en) * | 2019-10-25 | 2021-11-23 | 江南大学 | Mycotoxin ZEN degrading enzyme mutant and application thereof |
| CN110684754A (en) * | 2019-10-25 | 2020-01-14 | 江南大学 | Mycotoxin ZEN degrading enzyme mutant and application thereof |
| CN110819608A (en) * | 2019-10-29 | 2020-02-21 | 湖北大学 | Hydrolysis method of zearalenone and derivatives thereof |
| CN110819608B (en) * | 2019-10-29 | 2022-03-15 | 湖北大学 | Hydrolysis method of zearalenone and derivatives thereof |
| CN114045276A (en) * | 2021-11-09 | 2022-02-15 | 北京化工大学 | Neutral zearalenone degrading enzyme mutant with improved specific enzyme activity |
| CN114045276B (en) * | 2021-11-09 | 2023-08-29 | 北京化工大学 | Neutral zearalenone degrading enzyme mutant with specific activity improved |
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| CN107099520B (en) | 2020-10-16 |
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