CN108251405A - Complex enzyme and additive and their application and the method for removing mycotoxin - Google Patents
Complex enzyme and additive and their application and the method for removing mycotoxin Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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Abstract
The present invention relates to mycotoxins to remove field, discloses a kind of complex enzyme and additive and their application and the method for removing mycotoxin.Specifically, being related to a kind of complex enzyme, which contains amidase and esterase, which can remove mycotoxin, particularly fumonisin, ochratoxin and T2 toxin；Additive containing the complex enzyme and they in application of the removing mycotoxin particularly in fumonisin, ochratoxin and T2 toxin and the method for removing mycotoxin.Pass through above-mentioned technical proposal, as above amidase and esterase are applied in combination, 2 toxin of Ochratoxin A, fumonisin and T can be removed simultaneously, and removal efficiency greatly improves than a kind of efficiency of enzyme of single use, and also is able to a certain extent remove vomitoxin, aflatoxin, zearalenone toxin.
Description
Technical field
The present invention relates to mycotoxins to remove field, and in particular, to a kind of complex enzyme, the addition containing the complex enzyme
The application and a kind of method for removing mycotoxin of agent, the complex enzyme and/or additive in mycotoxin is removed.
Background technology
Mycotoxin is a kind of secondary metabolites that Toxigenic fungi generates in the process in harm, mainly includes aspergillus flavus poison
Element, deoxynivalenol (also known as vomitoxin, DON), zearalenone (ZEN) and fumonisin (FUM), reddish brown song
Mould toxin and T-2 toxin etc..
In recent years, frequently occurring due to China's extreme climate results in head blight to endanger wheat, based on corn
It is very popular, mycetogenetic fumonisin (FUM), ochratoxin and T-2 pollutions aggravate, and pollute exceeded wheat and corn not
It is disconnected to increase.The safe utilization of the exceeded grain of toxin is studied, can not only protect the kind grain interests of China peasant, while prevent inflow entrance
Safeguard that national food Safety of Food Quality is extremely urgent in grain market.
Removing for mycotoxin, current domestic and international more universal method mainly have physical removal and absorption, chemistry
Processing etc..Adsorbent also largely adsorbed feed and food while mycotoxin is adsorbed in trace nutrient, sticked
Toxin after soil absorption, can not be decomposed, can cause secondary pollution.
It is one of most important means, but toxin currently to pollute grain to produce ethyl alcohol as fermenting raw materials using mycotoxin
It is further concentrated in a large amount of by-product vinasse obtained in fermenting and producing, fumonisin, ochratoxin and T-2 contents are significantly
It cannot be utilized more than national limit standard.
Toxin efficiently can be converted into non-toxic products, Environmental Safety by biological eliminating.Have become current mycotoxin to cut
Subtract treatment technology approach most promising in technology, it is not only safe and environment-friendly, efficient, but also utilize modern biotechnology
It conducts a research and meets very much the development trend of current China's energy-saving and emission-reduction.But at present in terms of biological eliminating, it is difficult to simultaneously efficiently
Ground removes a variety of toxin, when using compound enzyme agent, often will appear a kind of removal efficiency of toxin and improves, and its
He but has dropped the removal efficiency of toxin.When being related to fumonisin (FUM), ochratoxin and T-2 toxin, due to these three
Content of toxins is much lower relative to vomitoxin, aflatoxin and zearalenone toxin, therefore, research relatively also compared with
It is few, it is more difficult to realize to efficient removal while them.
Therefore there is an urgent need for develop one kind can energy conservation and environmental protection and a variety of mycotoxins of efficient removal can particularly take off simultaneously
The complex enzyme formulation removed.
Invention content
The purpose of the invention is to overcome the disadvantages described above of the prior art, providing one kind can while efficiently remove more
The compounding complex enzyme formulation of kind mycotoxin particularly fumonisin, ochratoxin and T2 toxin, contains the complex enzyme formulation
Additive, the complex enzyme formulation and/or additive are in removing mycotoxin particularly fumonisin, ochratoxin and T2 poison
Application and a kind of method for removing mycotoxin particularly fumonisin, ochratoxin and T2 toxin in element.
To achieve these goals, on the one hand, the present invention provides a kind of complex enzyme, wherein, which contains amide
Enzyme and esterase, the complex enzyme can remove mycotoxin, particularly fumonisin, ochratoxin and T2 toxin.
Second aspect, the present invention also provides a kind of additive, wherein, which contains complex enzyme as described above.
The third aspect, the present invention provides complex enzyme as described above and/or additive as described above in removing fungi
Application in toxin particularly fumonisin, ochratoxin and T2 toxin.
Fourth aspect, the present invention provides a kind of method for removing mycotoxin, wherein, this method includes：To as above institute
The complex enzyme and/or additive as described above stated are contacted with the sample that mycotoxin pollutes, to remove the poison of the fungi in sample
Element, the mycotoxin are preferably fumonisin, ochratoxin and T2 toxin.
Through the above technical solutions, amidase as above and esterase are applied in combination, can remove simultaneously Ochratoxin A,
Fumonisin and T-2 toxin, and removal efficiency greatly improves, and also be able to certain than a kind of efficiency of enzyme of single use
Vomitoxin, aflatoxin, zearalenone toxin are removed in degree.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Specific embodiment
The specific embodiment of the present invention is described in detail below.It is it should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood to comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It between the endpoint value of a range and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
In a first aspect, the present invention provides a kind of complex enzyme, wherein, which contains amidase and esterase, this is compound
Enzyme can remove mycotoxin.
According to the present invention, amidase and esterase are carried out with the use of can be removed to a variety of mycotoxins, but work as
When the mycotoxin is related to fumonisin, ochratoxin and T2 toxin, removal efficiency significantly improves, and compared to single
Enzyme preparation, the removal efficiency of complex enzyme formulation provided by the invention will greatly promote.
Although amidase and esterase to be used cooperatively and can achieve the object of the present invention, it was found by the inventors of the present invention that
By using the amidase with following amino acid sequence, the complex enzyme is for mycotoxin, particularly fumonisin, reddish brown song
The removal efficiency of mould toxin and T2 toxin can be promoted further.Specifically, the amidase has：(a)SEQID NO：2
Shown amino acid sequence is (by SEQ ID NO：It is nucleotide sequence coded shown in 1)；(b)SEQ ID NO：Amino shown in 2
One or several in the amino acids residue of the 11st, 89,179 and 239 still have after replacing, lacking or add in acid sequence
SEQ ID NO：The amino acid sequence of amino acid sequence activity shown in 2 preferably has by SEQID NOs：3rd, shown in 4 and 5
Nucleotide sequence coded amino acid sequence.More preferably, the amino acid sequence of the amidase is SEQ ID NO：Shown in 2
Amino acid or for by SEQ ID NOs：3rd, the nucleotide sequence coded amino acid sequence shown in 4 and 5.
Although amidase and esterase to be used cooperatively and can achieve the object of the present invention, it was found by the inventors of the present invention that
By using the esterase with following amino acid sequence, the complex enzyme is for mycotoxin, particularly fumonisin, Aspergillus ochraceus
The removal efficiency of toxin and T2 toxin can be promoted further.Specifically, the esterase have SEQ ID NOs: 6,7,8,9,
10th, the amino acid sequence encoded described in nucleotide sequence shown at least one of 11,12,13,14,15,16 and 17.Preferably,
The amino acid sequence of the esterase is at least one of SEQ ID NOs: 6,7,8,9,10,11,12,13,14,15,16 and 17 institutes
Show the amino acid sequence encoded described in nucleotide sequence.
Wherein, it after as above amidase and the preferred amino acid sequence of esterase has been understood, can be pushed away according to codon is counter
Method be readily available the nucleotide sequence for encoding the amidase and esterase.Simultaneously as there are degeneracies for codon, it can
To obtain a plurality of coding same amino acid sequence but the different nucleotide sequence of nucleotide sequence, these should be the present invention's
Within protection domain.
20 kinds of amino acid residues of constitutive protein matter, four classes are segmented into according to pendant polar：1st, nonpolar amino acid：
Alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine
(Phe), tryptophan (Trp) and proline (Pro)；2nd, the uncharged amino acid of polarity：Glycine (Gly), serine
(Ser), threonine (Thr), cysteine (Cys), aspartic acid (Asn), glutamine (Gln) and tyrosine (Tyr)；3rd, band
The amino acid of positive charge：Arginine (Arg), lysine (Lys) and histidine (His)；4th, negatively charged amino acid：Asparagus fern ammonia
Sour (Asp) and glutamic acid (Glu) (referring to " biochemistry " (second edition) first volume, Shen Tong, Wang Jingyan, the 82-83 pages, high religion
Educate publishing house, December nineteen ninety).If it happens the amino acid residue substitution of a classification is belonged in protein, such as is taken by Arg
Replace Ile for Lys or by Leu, effect of the residue played in protein domain (for example provides positive charge or formed and dredged
The effect of water bag structure) do not change, therefore the stereochemical structure of protein can't be had an impact, therefore still can be real
The function of existing albumen.The amino acid residue substitution for belonging to a classification can be happened at any one amino acid of above-mentioned enzyme
On resi-dues.
As previously mentioned, enzyme provided by the invention can also be modified or is mutated, derivative protein is obtained.Institute of the present invention
It states " derivative protein " to refer to the enzyme with above-mentioned amino acid sequence with the difference on amino acid sequence, it is possibility to have not shadow
It rings the difference on the modified forms of sequence or haves both at the same time.These albumen include natural or induction genetic variant.It is described
Induction variant can be obtained by various technologies, such as the random mutation of radiation or mutagens generation, can also be by such as fixed
The technology of point mutation method or other known molecular biology." the derivative protein " is further included with natural L-form amino acid
Residue analog (such as D types amino acid) and with it is non-naturally occurring or synthesis amino acid (such as beta-amino acids, γ-
Amino acid etc.) analog.
Modification (not changing primary structure usually, i.e., do not change amino acid sequence) form includes：In vivo or in vitro albumen
Chemical derivative form such as acetylation or carboxylated.Modification further include glycosylation, such as those in the synthesis and processing of albumen or
Albumen that is glycosylation modified and generating is carried out in further processing step.This modification can be by being exposed to progress sugar by albumen
The enzyme (glycosylase or deglycosylation enzyme of such as mammal) of base and complete.Modified forms are further included with phosphorylation amino
The sequence of sour residue (such as phosphotyrosine, phosphoserine, phosphothreonine).It further includes and is modified to improve its anti-egg
White hydrolysis property or the albumen for optimizing solubility property.And in the present invention, in asparagine (Asn), the silk of the complex enzyme
Propylhomoserin (Ser) and threonine (Thr) residue carry out that N- and/or O- is glycosylation modified can further to improve dissolubility at any one
And thermal stability.
In order to facilitate purifying, the label that this field can also be used common (such as Poly-Arg, Poly-His, FLAG,
At least one of Strep-tag II and c-myc) modification is added to (a) or (b).The label does not interfere with the present invention
The activity of the enzyme of offer in actual application, can choose whether addition label according to demand.
In order to further improve removal effect of the enzyme compound formulation to mycotoxin, under preferable case, the amidase and
The weight ratio of esterase is 1：(0.01-100), further preferably 1：(0.05-20) is still more preferably 1：(0.1-10),
More preferably 1：(0.2-5).
The present inventor has found in the course of the study, when also containing phosphatidyl silk in the enzyme compound formulation of the present invention
In the case of propylhomoserin and/or Zn-ef ficiency, the removal efficiency of mycotoxin can be promoted further.Preferably, with described compound
On the basis of the total weight of enzyme preparation, the content of the phosphatidylserine and/or Zn-ef ficiency is 1-30 weight %, more preferably 5-
10 weight %.
In the present invention, the amidase and esterase can be prepared by existing method, for example, by that will encode correlation
The nucleotide sequence of amino acid sequence is cloned into carrier, then is transferred in host, then by conventional method from the place after proliferation
The isolated related nucleotide sequence of chief cell, can also be commercially available, the phosphatidylserine and/or Zn-ef ficiency are equal
It can be commercially available, can also be obtained by other regular approach.
According to the present invention, in order to further enhance the detoxification efficiency of the complex enzyme, the complex enzyme also containing a kind of or
A variety of other enzymes；One or more other enzymes can be selected from but be not limited to：Aflatoxin detoxifying enzymes, Aspergillus ochraceus
Toxin lactonase, fumonisin Carboxylesterase, fumonisin aminopherase, amino polyol amine oxidase, deoxidation melon wither sickle
Mykol epoxide hydrolase, carboxypeptidase, aspergillus niger aspartic protease PEPAa, PEPAb, PEPAc and PEPAd, elastic egg
White enzyme, aminopeptidase, pepsin or pepsin sample protease, trypsase or trypsin like proteases, bacterioprotein
Enzyme, be related to starch metabolism, fiber removal, lipid-metabolism enzyme, be related to glycogen metabolism protein or enzyme, amylase, Arab
Carbohydrase, arabinofuranosidase, catalase, cellulase, chitinase, renin, cutinase, DNA
Enzyme, epimerase, esterase, galactosidase, dextranase, glucan lyase, endoglucanase, glucoamylase, Portugal
Grape carbohydrate oxidase ,-glucosidase, including β-glucosyl enzym, glycuronidase, hemicellulase, hexoxidase, hydrolase,
Invertase, isomerase, lipolytic enzyme, laccase, lyases, mannosidase, oxidizing ferment, oxidoreducing enzyme, pectate lyase, pectin
Acetylesterase, pectin remove polymerase, pectinesterase, pectin decomposing enzyme, peroxidase, phenol oxidase, phytase, poly half
Lactobionic acid enzyme, protease, sandlwood-galacturonic acid enzyme, African hesperidium element, transferase, transport protein, turn ribalgilase
Glutaminase, zytase, hexoxidase, acid phosphatase and combination thereof.
It in the present invention, uses for convenience, under preferable case, each component of the enzyme compound formulation may be mixed together
Packaging, can also respective independent packaging, then mixed again when in use.
The present invention also provides the preparation method of above-mentioned enzyme compound formulation, this method includes：It will according to aforementioned weight ratio
Each ingredient is uniformly mixed.
According to the present invention, the fumonisin can include its each Type of toxin, for example, FA1, FA2, FB1, FB2, FB3,
FB4, FC1, FC2, FC3, FC4 and FP1, wherein, most preferably fumonisin FB1.The ochratoxin can also include it
Each Type of toxin, for example, ochratoxin A and ochratoxin B.
Second aspect, the invention also discloses a kind of additive, wherein, which contains complex enzyme as described above.
In situations where it is preferred, the additive is using complex enzyme provided by the invention as active constituent.In the addition
In agent, the content of the complex enzyme is 0.001-10g/kg, more preferably preferably 0.01-8g/kg, 0.1-5g/kg.In addition,
It can also be containing well known to a person skilled in the art solvent (such as glycerine, carbohydrate and protease inhibitors egg in the additive
White protective agent), agonist etc..
According to the present invention, also containing physiologically acceptable carrier in the additive, wherein, it is described physiologically to connect
The carrier received is selected from least one of following substance：Maltodextrin, lime stone (calcium carbonate), cyclodextrin, wheat, wheat bran or
Wheat component, rice or rice bran, sucrose, starch, Na2SO4, talcum powder and PVA and their mixture.
It will be apparent for a person skilled in the art that can these additives be added to pollution has the feed of mycotoxin
And/or grain and oil material is to reduce existing endotoxin level.
The Feed Material of the present invention can include：A) cereal, for example, granule cereal (such as wheat, barley, naked barley, oat with
And combination thereof) and/or big grain cereal such as maize or sorghum；B) by-product from cereal, for example, it is corn protein powder, dry
Vinasse and soluble matter (DDGS), wheat bran, sizing, wheat wheat-middlings, rice bran, rice husk, oat shell, palm kernel and citrus pulp；c)
Ensilage；D) protein derived from following source：Such as soybean, sunflower, peanut, lupin, pea, broad bean, cotton, card
Nola, fish meal, dry plasma albumen, meat and bone meal, potato protein, whey, copra, sesame；E) from plant and animal source
The oil ＆ fat of acquisition；F) minerals and vitamins.
The grain and oil of the present invention refer to the grains such as cereal, beans and oil plant and its general designation of processing finished product and semi-finished product, special
Do not refer to the edible product of the mankind.For example, the grain and oil can be the edible grain and oil production of the common mankind in this field
Product, specifically, the grain and oil can include in cereal and its agricultural and sideline product, oil ＆ fat product, drinks, milk and its product etc.
It is at least one.
It will be apparent to one skilled in the art that feed according to the present invention or additive can also include other groups
Divide such as stabilizer and/or incremental agent and/or enzyme.
Preferably, the method for preparing additive according to the present invention includes mixing step, which includes mixing such as
The upper complex enzyme, carrier optionally physiologically acceptable at least one, solvent, agonist, stabilizer, incremental agent or enzyme
Class is mixed together.
It is apparent to the skilled person that, as precautionary step, any feed and/or grain and oil can be added an additive to
In material.
For example, when the additive is used for the feed of cultivated animals, in the additive also containing bacillus licheniformis,
Bacillus subtilis, bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, lactoenterococcus, lactobacillus acidophilus, Lactobacillus casei,
German-style Lactobacillus lactate subspecies, lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, candida utili, baby's bifid bar
Bacterium, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, animal bifidobacteria, rice
Aspergillus, bacillus lentus, bacillus pumilus, lactobacillus cellobiosas, lactobacillus fermenti and Lactobacillus delbrueckii
At least one of subspecies.
For example, when the additive is used for ensilage or ox feed, also containing production propionic acid propionic acid in the additive
At least one of bacillus, lactobacillus buchneri and Lactobacillus paracasei.
For example, when the additive be used for poultry, growing-finishing pig and aquiculture animal feed when, the feed or
Also contain bacillus coagulans and/or Brevibacillus laterosporus in additive.
Based on description as above, the present invention also provides a kind of grain and oil or feed, wherein, the grain and oil or feed contain above-mentioned
Additive.
The third aspect is true in removing the present invention also provides complex enzyme as described above and/or additive as described above
Application in verticillium toxin particularly fumonisin, ochratoxin and T2 toxin.
Fourth aspect, the present invention also provides a kind of method for removing mycotoxin, wherein, this method includes：It will as above
The complex enzyme and/or additive is contacted with the sample that mycotoxin pollutes, described true to remove the mycotoxin in sample
Verticillium toxin is preferably fumonisin, ochratoxin and T2 toxin.
According to the present invention, contact each enzyme component preferably in the complex enzyme can play under conditions of enzymatic activity into
Row.Wherein, term " condition for playing enzymatic activity " refers to meet the conditions such as temperature and the pH of enzymatic activity performance.
According to the present invention, the sample of the mycotoxin pollution can include any sample containing mycotoxin, for example,
Can be grain and oil and/or feed etc..It is described compound on the basis of the total weight of the sample of mycotoxin pollution under preferable case
The dosage of enzyme preparation is is at least 0.1ppm, more preferably preferably 0.1-15ppm, further preferably 1-10ppm, 4-
8ppm.In the present invention, when the sample of mycotoxin pollution is liquid, that " ppm " is represented is " μ g/mL "；Work as vomitoxin
When the sample of pollution is solid, that " ppm " is represented is " μ g/g ".
In following embodiment and comparative example, ochratoxin A, fumonisin FB1 and T2 toxin standard items are purchased from Sigma
Company；Phosphatidylserine is purchased from upper Hisense's sail bio tech ltd.
Fumonisin FB1 is examined by high performance liquid chromatography according to the method in GB 5009.240-2016 standards
It surveys, vomitoxin is detected according to the method in GB/T 30956-2014 standards, according to GB/T 23501-2009 standards
In method T2 toxin is detected；
Toxin degradation rate %=(before reaction in sample after quality-reaction of toxin in sample toxin quality)/reaction before
Quality × 100% of toxin in sample.
Embodiment
The present invention will be described in detail by way of examples below, but is not intended to limit the present invention.Following preparation example,
In embodiment and comparative example, unless otherwise instructed, each material is commercially available, and each method is the conventional method of this field.
Preparation example 1
This preparation example is used for the preparation for illustrating amidase provided by the invention
(1) acquisition of gene
(precious bioengineering (Dalian) Co., Ltd of commission, similarly hereinafter) following nucleotide is synthesized by artificial chemistry synthetic method
Segment；In SEQ ID NO：Addition Nde I restriction enzyme sites, 3 ' ends after 5 ' end initiation codon ATG of the nucleotide sequence shown in 1
Add Xho I restriction enzyme sites and terminator codon TAG.
(2) structure of recombinant plasmid
(there is His labels, purchase to PET30a plasmids using restriction enzyme Nde I and Xho I (being purchased from NEB companies)
In Invitrogen companies of the U.S.) double digestion is carried out, in 37 DEG C of water-bath digestion 4h, digestion system (50 μ L) is as follows：
By digestion products into after row agarose gel electrophoresis, purifying recycling.Then, (it is purchased from Takara using T4 ligases
Company) reaction is attached, 4h is connected at room temperature, obtains recombinant plasmid.The linked system (10 μ L) used is as follows：
(3) acquisition of recombinant bacterial strain
Turn DH5 α competence using the recombinant plasmid electricity obtained by step (2) on Bio-Rad Gene Pulse electroporations
Cell (is purchased from Takara companies), and electric conversion condition is：Voltage 1500V, 25 μ F of capacitance, 200 Ω of resistance, transformation time is with DNA
The difference of sample and change, and provided automatically by instrument, usually in the range of 3.5-4s.Then, the cell after conversion is coated with
It is screened on the LB solid plates containing kanamycins, is inverted culture 2 days in 37 DEG C, picking positive bacterium colony is inoculated in LB liquid
In body culture medium, to obtain recombinant bacterial strain.
Then make with method known in this field to extract plasmid, then entrust precious bioengineering (Dalian) limited public affairs
Department is sequenced, the results show that gene as above is successfully transformed into Escherichia coli, shows the recombinant bacterial strain of the present invention
It builds successfully.
(4) preparation of amidase
Obtained recombinant bacterial strain is inoculated in LB fluid nutrient mediums, and (beef extract 5g, peptone 10g, sodium chloride 5g, moisturizing is extremely
1000mL, 115 DEG C of sterilizing 20min are spare, pH 7) in, 3 days are cultivated in 37 DEG C to OD600Between 2 and 6, bacterium is collected by centrifugation in value
Body, with phosphate buffer (PBS, 135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, 8mM K2HPO4, pH 7.2) and solution
After suspension, ultrasonic disruption, centrifuging and taking supernatant obtains crude enzyme liquid.
Crude enzyme liquid is placed in the ammonium sulfate powder for, being slowly added to grind on ice, it is stirring while adding, it is added to ammonium sulfate saturation
Until.Left and right for 24 hours is stood at 4 DEG C, 12000r/min centrifugation 50min abandon supernatant, dissolved and precipitated with a small amount of PBS (pH 7.2).It will
The precipitation dialysis of PBS dissolvings, removes ammonium sulfate, is resuspended in buffer solution (pH 7.4,50mM NaCl, imidazoles containing 10mM).According to
The recombinase given expression to contains His labels, affinitive layer purification is carried out using Ni columns, after 1mL/min balances Ni columns, with flow
0.5mL/min is directly by the crude enzyme liquid loading of resuspension；It is continuing with buffer solution (pH7.4,50mM NaCl, imidazoles containing 10mM)
1mL/min elutes unadsorbed or absorption non-specific foreign protein；With buffer solution (pH 7.4,50mM NaCl, 500mM imidazoles)
Destination protein is collected in elution, to obtain the amide enzyme solution of purifying, and is freeze-dried and is obtained amidase dry powder X1.
Preparation example 2-4
Preparation example 2-4 is used for the preparation for illustrating amidase provided by the invention
The preparation of amidase X2-X4 is carried out respectively according to the method for preparation example 1, unlike, preparation example 2-4 is used respectively
SEQ ID NO：3、SEQ ID NO：4 and SEQ ID NO：5 replace SEQ ID NO：1.
Preparation example 5-16
Preparation example 5-16 is used for the preparation method for illustrating esterase provided by the invention
The preparation of esterase Z1-Z12 is carried out respectively according to the method for preparation example 1, unlike, preparation example 5-16 is used respectively
SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ
ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16 and SEQ ID NO：17 replace
Change SEQ ID NO：1.
Embodiment 1-14, comparative example 1-3
For illustrating that complex enzyme provided by the invention makees the removing of fumonisin FB1, ochratoxin A and T2 toxin
With.
The preparation of complex enzyme formulation is carried out according to the proportioning of each embodiment and comparative example in table 1 using physiological saline so that
In obtained mixed liquor, a concentration of 100ng/ml of complex enzyme, the concentration point of fumonisin FB1, ochratoxin A and T2 toxin
It Wei not 1000ppb.For reaction temperature to be respectively 37 DEG C, the pH value of reaction is respectively 5, and 20 μ L reaction products are examined after reacting 30min
The residual of fumonisin FB1, ochratoxin A and T2 toxin is surveyed, and calculates degradation rate.The results are shown in Table 2.
Table 1
| Amidase | Esterase | Phosphatidylserine | Zinc | |
| Embodiment 1 | X1 50ng/ml | Z1 50ng/ml | 5ng/ml | --- |
| Embodiment 2 | X2 80ng/ml | Z2 20ng/ml | --- | 8ng/ml |
| Embodiment 3 | X3 20ng/ml | Z3 80ng/ml | 5ng/ml | 5ng/ml |
| Embodiment 4 | X1 50ng/ml | Z1 50ng/ml | --- | --- |
| Embodiment 5 | X1 50ng/ml | Z4 50ng/ml | 5ng/ml | --- |
| Embodiment 6 | X1 50ng/ml | Z5 50ng/ml | 5ng/ml | --- |
| Embodiment 7 | X1 50ng/ml | Z6 50ng/ml | 5ng/ml | --- |
| Embodiment 8 | X1 50ng/ml | Z7 50ng/ml | 5ng/ml | --- |
| Embodiment 9 | X1 50ng/ml | Z8 50ng/ml | 5ng/ml | --- |
| Embodiment 10 | X1 50ng/ml | Z9 50ng/ml | 5ng/ml | --- |
| Embodiment 11 | X1 50ng/ml | Z10 50ng/ml | 5ng/ml | --- |
| Embodiment 12 | X1 50ng/ml | Z11 50ng/ml | 5ng/ml | --- |
| Embodiment 13 | X1 50ng/ml | Z12 50ng/ml | 5ng/ml | --- |
| Embodiment 14 | X4 50ng/ml | Z1 50ng/ml | 5ng/ml | --- |
| Comparative example 1 | --- | --- | 5ng/ml | --- |
| Comparative example 2 | X1 100ng/ml | --- | 5ng/ml | --- |
| Comparative example 3 | --- | Z1 100ng/ml | 5ng/ml | --- |
Table 2
Amidase and esterase are combined use it can be seen from the data of upper table 2, can efficiently remove volt horse
Toxin and ochratoxin, and most T2 toxin can also be removed.And work as in the complex enzyme and add phosphide
When acyl serine and/or zinc, the removal efficiency of toxin can be promoted further.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, it can be combined by any suitable means.In order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
SEQUENCE LISTING
<110>Cofco Nutrition And Health Research Institute Co., Ltd.
Cofco Group Co., Ltd.
<120>Complex enzyme and additive and their application and the method for removing mycotoxin
<130> I40389COF
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 1443
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 1
atggtccgcc gaattgcttc agctacacct cgcgtgcaat cgcccatgtc gccattgggc 60
acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120
atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180
gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240
ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300
cccgtgctca tgcctggttt gtgggattgc gacatgcatt ttggcgggga tgacgattat 360
tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420
cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480
gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcattcg 540
tctggcgctg cactcagtca gacagctgga cacggcgata tcttcgctct tccagcaggc 600
gaagtactgg ggagttatgg agtaatgaac ccacgccctg ggtactgggg ggcagggccg 660
ctatgtatcg ccgatggcgt agaggaggtc cgacgagcag tgaggttgca gatcatgcgc 720
ggtgcaaagg ttatcaaagt gatggcctct gggggtgtca tgtcgcgaga cgataatccc 780
aactttgcac agttctctcc agaagaactg aaggtgatag tggaagaggc ggctcgacag 840
aaccggatcg tttctgcaca tgtgcatggc aaggcgggga ttatggctgc tatcaaagca 900
ggctgcaaga gtctggagca tgtgtcttat gctgacgagg aggtctggga gctcatgaaa 960
gagaagggaa ttttgtatgt ggccacacgc tcggttattg aaatctttct ggctagtaat 1020
ggagaggggt tggtgaaaga gtcgtgggcc aagttgcagg cccttgccga ttcgcatttg 1080
aaagcttatc agggagctat taaggcgggt gttaccattg cgttgggaac ggataccgcc 1140
cccggtggtc ctaccgcact tgagttgcag tttgccgtcg agagaggagg tatgacgccg 1200
ttggaggcca tcaaagccgc aactgcgaac gctcccctgt cagttggtcc acaagcaccg 1260
ttgacgggtc agcttcgcga ggggtatgag gcagatgtga ttgcgttgga ggagaatcca 1320
ttggaggaca tcaaagtctt tcaggagccg aaggcagtta cccacgtctg gaagggaggg 1380
aaactgttca aaggtccagg tattggtccg tggggagaag atgcacgtaa tccttttctg 1440
tag 1443
<210> 2
<211> 480
<212> PRT
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 2
Met Val Arg Arg Ile Ala Ser Ala Thr Pro Arg Val Gln Ser Pro Met
1 5 10 15
Ser Pro Leu Gly Thr Thr Tyr Cys Val Arg Pro Asn Pro Val Ser Leu
20 25 30
Asn Leu Gln Arg Arg Pro Leu Val Ile Ala Ser Thr Asp Glu Ala Lys
35 40 45
Val Thr Ile Ile Tyr Ala Gly Leu Leu Ile Pro Gly Asp Gly Glu Pro
50 55 60
Leu Arg Asn Ala Ala Leu Val Ile Ser Asp Lys Ile Ile Ala Phe Val
65 70 75 80
Gly Ser Glu Ala Asp Ile Pro Lys Asp Tyr Leu Arg Ser Thr Gln Ser
85 90 95
Thr His Arg Val Pro Val Leu Met Pro Gly Leu Trp Asp Cys Asp Met
100 105 110
His Phe Gly Gly Asp Asp Asp Tyr Tyr Asn Asp Tyr Thr Ser Gly Leu
115 120 125
Ala Thr His Pro Ala Ser Ser Gly Ala Arg Leu Ala Arg Gly Cys Trp
130 135 140
Glu Ala Leu Gln Asn Gly Tyr Thr Ser Tyr Arg Asp Leu Ala Gly Tyr
145 150 155 160
Gly Cys Glu Val Ala Lys Ala Ile Asn Asp Gly Thr Ile Val Gly Pro
165 170 175
Asn Val His Ser Ser Gly Ala Ala Leu Ser Gln Thr Ala Gly His Gly
180 185 190
Asp Ile Phe Ala Leu Pro Ala Gly Glu Val Leu Gly Ser Tyr Gly Val
195 200 205
Met Asn Pro Arg Pro Gly Tyr Trp Gly Ala Gly Pro Leu Cys Ile Ala
210 215 220
Asp Gly Val Glu Glu Val Arg Arg Ala Val Arg Leu Gln Ile Met Arg
225 230 235 240
Gly Ala Lys Val Ile Lys Val Met Ala Ser Gly Gly Val Met Ser Arg
245 250 255
Asp Asp Asn Pro Asn Phe Ala Gln Phe Ser Pro Glu Glu Leu Lys Val
260 265 270
Ile Val Glu Glu Ala Ala Arg Gln Asn Arg Ile Val Ser Ala His Val
275 280 285
His Gly Lys Ala Gly Ile Met Ala Ala Ile Lys Ala Gly Cys Lys Ser
290 295 300
Leu Glu His Val Ser Tyr Ala Asp Glu Glu Val Trp Glu Leu Met Lys
305 310 315 320
Glu Lys Gly Ile Leu Tyr Val Ala Thr Arg Ser Val Ile Glu Ile Phe
325 330 335
Leu Ala Ser Asn Gly Glu Gly Leu Val Lys Glu Ser Trp Ala Lys Leu
340 345 350
Gln Ala Leu Ala Asp Ser His Leu Lys Ala Tyr Gln Gly Ala Ile Lys
355 360 365
Ala Gly Val Thr Ile Ala Leu Gly Thr Asp Thr Ala Pro Gly Gly Pro
370 375 380
Thr Ala Leu Glu Leu Gln Phe Ala Val Glu Arg Gly Gly Met Thr Pro
385 390 395 400
Leu Glu Ala Ile Lys Ala Ala Thr Ala Asn Ala Pro Leu Ser Val Gly
405 410 415
Pro Gln Ala Pro Leu Thr Gly Gln Leu Arg Glu Gly Tyr Glu Ala Asp
420 425 430
Val Ile Ala Leu Glu Glu Asn Pro Leu Glu Asp Ile Lys Val Phe Gln
435 440 445
Glu Pro Lys Ala Val Thr His Val Trp Lys Gly Gly Lys Leu Phe Lys
450 455 460
Gly Pro Gly Ile Gly Pro Trp Gly Glu Asp Ala Arg Asn Pro Phe Leu
465 470 475 480
<210> 3
<211> 1443
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 3
atggtccgcc gaattgcttc agctacacct cacgtgcaat cgcccatgtc gccattgggc 60
acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120
atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180
gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240
ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300
cccgtgctca tgcctggttt gtgggattgc gacatgcatt ttggcgggga tgacgattat 360
tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420
cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480
gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcattcg 540
tctggcgctg cactcagtca gacagctgga cacggcgata tcttcgctct tccagcaggc 600
gaagtactgg ggagttatgg agtaatgaac ccacgccctg ggtactgggg ggcagggccg 660
ctatgtatcg ccgatggcgt agaggaggtc cgacgagcag tgaggttgca gatctttcgc 720
ggtgcaaagg ttatcaaagt gatggcctct gggggtgtca tgtcgcgaga cgataatccc 780
aactttgcac agttctctcc agaagaactg aaggtgatag tggaagaggc ggctcgacag 840
aaccggatcg tttctgcaca tgtgcatggc aaggcgggga ttatggctgc tatcaaagca 900
ggctgcaaga gtctggagca tgtgtcttat gctgacgagg aggtctggga gctcatgaaa 960
gagaagggaa ttttgtatgt ggccacacgc tcggttattg aaatctttct ggctagtaat 1020
ggagaggggt tggtgaaaga gtcgtgggcc aagttgcagg cccttgccga ttcgcatttg 1080
aaagcttatc agggagctat taaggcgggt gttaccattg cgttgggaac ggataccgcc 1140
cccggtggtc ctaccgcact tgagttgcag tttgccgtcg agagaggagg tatgacgccg 1200
ttggaggcca tcaaagccgc aactgcgaac gctcccctgt cagttggtcc acaagcaccg 1260
ttgacgggtc agcttcgcga ggggtatgag gcagatgtga ttgcgttgga ggagaatcca 1320
ttggaggaca tcaaagtctt tcaggagccg aaggcagtta cccacgtctg gaagggaggg 1380
aaactgttca aaggtccagg tattggtccg tggggagaag atgcacgtaa tccttttctg 1440
tag 1443
<210> 4
<211> 1440
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 4
atggtccgcc gaattgcttc agctacacct gtgcaatcgc ccatgtcgcc attgggcaca 60
acatactgcg tccgtcctaa tcctgtttca ctgaatcttc aaagaagacc tctcgtgatc 120
gcatcaacag acgaggccaa ggtcactata atatatgccg gactattaat ccctggcgac 180
ggagaacctc tgcgcaatgc tgccctagtc atcagcgata agatcatcgc gttcgttgga 240
tccgaagccg acatccctaa ggactacctc cggtccacgc agtctactca tcgtgtcccc 300
gtgctcatgc ctggtttgtg ggattgcgac atgcattttg gcggggatga cgattattac 360
aacgattata catctggtct ggccactcat ccagcatcat caggtgctcg actagcccgt 420
ggttgctggg aagcattgca gaatgggtat acatcctacc gcgacctagc cggatacggg 480
tgcgaggtcg caaaggcgat caatgatggc actatcgttg gtccaaacgt gcattcgtct 540
ggcgctgcac tcagtcagac agctggacac ggcgatatct tcgctcttcc agcaggcgaa 600
gtactgggga gttatggagt aatgaaccca cgccctgggt actggggggc agggccgcta 660
tgtatcgccg atggcgtaga ggaggtccga cgagcagtga ggttgcagat ctttcgcggt 720
gcaaaggtta tcaaagtgat ggcctctggg ggtgtcatgt cgcgagacga taatcccaac 780
tttgcacagt tctctccaga agaactgaag gtgatagtgg aagaggcggc tcgacagaac 840
cggatcgttt ctgcacatgt gcatggcaag gcggggatta tggctgctat caaagcaggc 900
tgcaagagtc tggagcatgt gtcttatgct gacgaggagg tctgggagct catgaaagag 960
aagggaattt tgtatgtggc cacacgctcg gttattgaaa tctttctggc tagtaatgga 1020
gaggggttgg tgaaagagtc gtgggccaag ttgcaggccc ttgccgattc gcatttgaaa 1080
gcttatcagg gagctattaa ggcgggtgtt accattgcgt tgggaacgga taccgccccc 1140
ggtggtccta ccgcacttga gttgcagttt gccgtcgaga gaggaggtat gacgccgttg 1200
gaggccatca aagccgcaac tgcgaacgct cccctgtcag ttggtccaca agcaccgttg 1260
acgggtcagc ttcgcgaggg gtatgaggca gatgtgattg cgttggagga gaatccattg 1320
gaggacatca aagtctttca ggagccgaag gcagttaccc acgtctggaa gggagggaaa 1380
ctgttcaaag gtccaggtat tggtccgtgg ggagaagatg cacgtaatcc ttttctgtag 1440
<210> 5
<211> 1446
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 5
atggtccgcc gaattgcttc agctacacct cgcgtgcaat cgcccatgtc gccattgggc 60
acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120
atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180
gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240
ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300
cccgtgctca tgcctggttt gtgggattgc gacatgcatt ttggcgggga tgacgattat 360
tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420
cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480
gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcatcat 540
tcgtctggcg ctgcactcag tcagacagct ggacacggcg atatcttcgc tcttccagca 600
ggcgaagtac tggggagtta tggagtaatg aacccacgcc ctgggtactg gggggcaggg 660
ccgctatgta tcgccgatgg cgtagaggag gtccgacgag cagtgaggtt gcagatcatg 720
cgcggtgcaa aggttatcaa agtgatggcc tctgggggtg tcatgtcgcg agacgataat 780
cccaactttg cacagttctc tccagaagaa ctgaaggtga tagtggaaga ggcggctcga 840
cagaaccgga tcgtttctgc acatgtgcat ggcaaggcgg ggattatggc tgctatcaaa 900
gcaggctgca agagtctgga gcatgtgtct tatgctgacg aggaggtctg ggagctcatg 960
aaagagaagg gaattttgta tgtggccaca cgctcggtta ttgaaatctt tctggctagt 1020
aatggagagg ggttggtgaa agagtcgtgg gccaagttgc aggcccttgc cgattcgcat 1080
ttgaaagctt atcagggagc tattaaggcg ggtgttacca ttgcgttggg aacggatacc 1140
gcccccggtg gtcctaccgc acttgagttg cagtttgccg tcgagagagg aggtatgacg 1200
ccgttggagg ccatcaaagc cgcaactgcg aacgctcccc tgtcagttgg tccacaagca 1260
ccgttgacgg gtcagcttcg cgaggggtat gaggcagatg tgattgcgtt ggaggagaat 1320
ccattggagg acatcaaagt ctttcaggag ccgaaggcag ttacccacgt ctggaaggga 1380
gggaaactgt tcaaaggtcc aggtattggt ccgtggggag aagatgcacg taatcctttt 1440
ctgtag 1446
<210> 6
<211> 1179
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 6
atgcggaacg tcagcgacaa ggcgccgccc cacgagacgc tcaccgtagt cgtcgcggca 60
atgatcgttg gcacggccgc cttgatggtg cttggaatac agcccatcct tctcggcgcc 120
cttgtagagg aggggcgtat tcccgccgag gggttgggat cggcggcaac ggtggaaata 180
ctggcgatcg cggcgggaac atgcatcgga cccgttctta tgaagacggg atatctgcgg 240
gcgaaatgcg cggcactctg cttaatgctc gccgcaatca acttcggatt gacgttgccg 300
ggtttcgatt tgcccatcgt ggcttgccga gcggcagcgg gagccctgga aggtctttcg 360
ctcagcgcgg cgatcctgat catgactcat aatcggcggc cggaccggct gagcggaata 420
tttctgggcg cgcagacgat accgcaggta atatctgctt atttgctccc gacggagatt 480
attccgcgct gggggagcgc aggcggcttc acgatcctgg gcattctcgc ggcgatcgcc 540
gcgatcgcgg ctctgtgcct cgtcgatcgc gttgagctcg atccgacgac cgttaacgac 600
gacttgcagt ggtcacccgc ggcgatcgtc atttcgatgg cggcattcgt tcaattctcg 660
ggggtcggtg ccgcatggag ctatctggag cgactggctg cgcagcacgg attttcggga 720
gaaacgatcg gtatcgccat ttccgggagt ttgctttgcc aggtaggcgg ggcttggctg 780
gccgcttgga tcggtgggcg ggtcggatat cgcttcgcct taatcgctgg gagcctgctt 840
caggcgggca acgtgatcgc attggcggtg gccgatcagc caagctggtt tatttccgct 900
tcctgtgctt tcggcctgtt ctggttggcg atgcagccct tccaaatccg cttcgcgatc 960
gcgatagata acagccggca gcttgctgta ctgctgacgc cgatcgccct cgtcgggttg 1020
agcgcggggc ccttgttgct ctctcgcttt gccggggcga ccgacttgcg ctggatcttt 1080
gtggggagtt cgaccttgtt gctggccagc gcgcttctgt atctttgcgc ttctctgttt 1140
caaccgcgcg gaaaggtgat cgctgaaacg gtggacgta 1179
<210> 7
<211> 651
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 7
atgacatcgc aggtcaagct tcgtagcgcg gcaaagcggc cgcgcagtcc taaaagcgag 60
cgaggtcttg ctcgttacga gtccttgctt gatgcgaccg acaggctgtt ggtcgatcta 120
gaccccgatc aggtcggtct ctatcagatt gcagaggaag cgggtgcctc accgtcgtcc 180
gtctatcatt tctttccgac caaggaagtg gctcatctcg ctctgatgcg ccgctatctg 240
gaggggctcc ggaatctcga cgcgatggaa gtcgacatcg gccagctcga aagctggcag 300
gacctgatga agttggatca gatcagggcg cgagactatt ataatagcca cccgcccgcc 360
ctcaagcttc tgttcggcgg atatggcggg gtcgaggcca gaaagcttga cgagcgatac 420
tccgaggaaa tcgtgagctc catgtatggc agatacaacg gcattttcca tatgccgcaa 480
atggagaatg aggctctcat gttcacgatc tgcttcgcaa ttctcgacgc ggtatgggcc 540
gtctcctttc gccggttcgg tgaaattacg tcggattttc ttcgggaggg gcaagcggct 600
tgcattgcct attgccgaca ctatctgccc gagcgaacgc catcagcgtg a 651
<210> 8
<211> 945
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 8
gtggccagca agttcaactg tgagttactc gatctgcgat catttgttgc ggtgtatgaa 60
acgcgaagtt ttagccacgc cgcgcggctt ctgaatcaat cgcagcccgc gctcagccgg 120
agaatccagc gcctcgagag tctcgtgggc ggtccgttgt tcgagcggac cagtcggtcg 180
cttgccgaaa cggcgctcgg caaagagttg ctcccggtcg cccaccgagc gttggaactt 240
gtcgatacgt cgctgtttgc gtcgcccaat gtccgggagt tccgctggac agacatcacg 300
attgcctgtg tacagaccgc cgccttccat gttctcccgc gagctgcgcg cttgtacatg 360
gatcaaaatc cgagggtccg actccgcatc cttgacgtgc cggcggtcga ggctgcggac 420
ctggttgcga gcggcgaggc ggagttcggc atcagcattg agagcctgtt gccatcaagc 480
ctgcggttcg atgcgctcca cgaggacccg ttcggcctgg catgccaccg aagccatccg 540
ctggcgtcgc tcgagatcct tgaatggacg caattgaaag gtgaaagcct gatcgccgtt 600
caccgtgcga gccggaaccg cacgttgctc gatgccgaac tcgcgcgcaa caatatcgcg 660
ctggaatggc ggtatgaggt cgcgcatctg acgacggcgc tgggattgat cgatgcgcaa 720
ttgggtgtcg ctgttatgcc ccgcatggtt atgccccgct cgggtcggtc ggaggtcgtc 780
tggcgccccg tcgtcgcgcc ggtcgtccaa cgcacgatcg gcatcgttca gcgccgcacc 840
ggctcgatgc accctgccgc acagcaattg cttgcgcggc tccgcgcggc ctggtcgtcc 900
gccaatctgg gcgacatcgc gtctcgcgaa gatggggcat cgtga 945
<210> 9
<211> 1623
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 9
gtgaaagagc accaatgccg tggcggccgg gcgtcccccg ctgcgcccgc cacgtggctt 60
gcgcggatca gcgtttcccg gggggcctcc gccatcgcct ggaccttcat gcttggcgca 120
actgccattc ccgtggctgc gcaaactgac gatccgaagc tcgttcgtca tacccagtcg 180
ggcgccgtcg agggcgtcga gggcgacgtc gagacttttt tgggaatacc cttcgcggct 240
ccgccggtcg gcgacctgcg atggcggccg ccggctccgc cgagggcgtg ggcgggcacc 300
agggacggcc gccgctttgc gcccgattgc atcgggaacg agcggcttag agaggggagc 360
cgggctgccg ggacgagcga agactgcctc tatctgaata tctggtctcc caaacaggtc 420
ggtaaggggg ggctccccgt catgatctgg gtttacggcg gtgggtttag cggcggttct 480
ggcgcggtgc catattatga cggctctgcg ctcgcgcaga agggcgtggt ggtcgtcacg 540
ttcaactatc gcgccgggat tctgggcttt cttgcccatc cggcgctttc aaaggaaagt 600
ccgaatggcg tgtcgggcaa ctatggtctt ctcgacatgc tcgcggcgtt caaatgggtt 660
cagaacaaca taagggagtt cggcggagac ccgaaccgtg tcacggtctt tggcgagtcc 720
gccggcgcga gcgcgctcgg actgctcctg acctcgccgc tcagtgagag cgccttcaat 780
caggcgatac tgcaaagtcc gggtctggcc aggccgctcg ccacgctttc tgaaagcgaa 840
gcgaatgggc tggagctggg agccgatatt tctgctctac ggcgtgccga tgcgggcgaa 900
ttgacgaaga tcgcgcaatc gcgaataccc atgtcgcgcc agttcaccaa gccgcggccg 960
atgggtccga ttctggacgg ctatgttttg cgcacccttg acgtcgatgc cttcgccaag 1020
ggggccttcc gcaagatacc cgttctggtc ggcggaaacg ccgacgaagg gcgcgctttt 1080
acggatcgcc tgccggtcaa aacggtcctt gaatatcgag cctatctcac agaacaattt 1140
ggtgacgagg cggacgcatg ggagcgttgt tatcccgcga actccgacgc cgacgtcccc 1200
gccgccgttg cccgtctttt tggggatagt cagttcaaca acgggatcga gctgctctcg 1260
gcagccttcg cgaaatggcg aacgccgctt tggagatatc gctttacggg cattccagga 1320
gccggccgtc gccccgccac gcatggagac gaaattccct atgtcttcgc aaatctgggg 1380
ccgtcgtccg tatctatgtt tgggtcgctc gaaggcggcg ccggggcgtc ggacatcaaa 1440
cttgcgaccg aaatgtccgc ggcctgggtg agcttcgcgg tgcacggggt ccccgatcag 1500
ggcacgaaat cgcactggcc gcgcttcgag cggcgagggg agatcatgac ttttggttcg 1560
caggttggct ctggggaagg tcttggagtt tcgccgagca aagcctgcca accctcaaaa 1620
tag 1623
<210> 10
<211> 1503
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 10
ttggagtttc gccgagcaaa gcctgccaac cctcaaaata gcgcccggcc tgtgcgtgct 60
tcagcacgcc gtcccgcttt gcgggcgacg ggctgtgccc tctgcctaga aggaagtaag 120
ttgcgctacg acgtcgcgat aattggaggt ggcaacgctg cattgacggc agccgtgacg 180
gcgcgtgaag cgggggcctc ggttcttgtg atcgagcatg cgccgcgcgc catgcgcggc 240
ggcaacagtc gtcacacacg caatatgcgt acgatgcacg aacgtcccct gtcgccgttg 300
accggtgaat attcggcgga cgaatattgg aatgatcttg tccgcgtcac gggggggcgc 360
accgacgaag aactcgcgcg gctcgttatc cgcaacacca ccgacgctat tcccttcatg 420
acgcgctgcg gtgtgcgttt ccagccctcg ctgtcgggca cgctgagttt atcgcgaacc 480
aacgcattct tccttggcgg cgggaaggcg cttgtaaacg catattacgc cacggccgaa 540
cggctaggcg tcgatattct ctatgattct gaggtgaccg agatcaacct tcagcaaggc 600
gtcgtgcagc gtctgcaatt gcgcagccgg ggattccctg tcgaagtgga agccaaggct 660
gccatcgcct cgtccggagg attccaggca aatcttgact ggctctcaag cgcatggggg 720
cctgctgcgg cgaacttcat cgtacggggc acgccatatg cgactggcac ggtgctcaag 780
aacctgttgg agcaaggcgt cgcctcggtg ggagatccaa cccaatgcca tgctgtcgcg 840
atcgatgggc gagcgcccaa atacgacggc ggcatcgtca cacgactgga ctgcgttccc 900
ttctcgatcg tcgtcaacaa ggacgccttg cgcttctacg atgaaggcga agatgtgtgg 960
ccgaagcgtt acgccatatg gggtcgcttg gtggcacagc agcctgatca gatcgctttc 1020
agcataatcg atcggcaggc cgaagacctc ttcatgccgt cagtgttccc ccccgtgcaa 1080
gcggacacga tcgcgggtct ggccgagaaa ctcggtctga atcccgtaac cctggaacgc 1140
acggtggccg aattcaacgc cgcatgcgtg cccggcgaat tcggcggcca agatctcgac 1200
gacctccaca ccgagggaat cgaaccaaag aaatccaact gggcccgacc gattattgtg 1260
cccccgttca gcgcctatcc tctccggccc gggatcacct tcacctatct cggcgtcaag 1320
gtagacagcc gtgcgcgggt catcatggag acaggtgagc cgacaaaaaa cctgtttgct 1380
tcgggggaaa taatggcggg cagcattctc ggccaaggtt atctcgctgg atttggaatg 1440
gcgattggta ccgtattcgg ccgcatcgcg ggttgggagg ccgcacgtca tgcaggattt 1500
tga 1503
<210> 11
<211> 1173
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 11
atgcaggatt ttgatctcgt aaaaatgctg tctgacttgc cgtcggcgcc ggagctggaa 60
gccaggcgcg ttatggaggt gtgcaacgcg tgccgctatt gcgaagggtt ctgcgcggta 120
tttcctgcaa tgaccttgca gcgtcatttc gccagcggcg atctcagcca cctcgccaat 180
ctctgccact cgtgccaagg ttgctattac gcctgccaat acgcccctcc gcatgagttc 240
ggaataaacg ttccaaaggc gctgtcggag ttgcggctcg agagctacga gcagcatgct 300
tggccccggc cggtcgccgc tctctatcgc aagaatgcgc tcatcatttc catcttgtcg 360
gcggcatgca taaccggcgt ccttctgctt gccgccatct tcaacgggga tgcacttttc 420
gcgaaacacg catcggtgcc cggcggcggg ttttacaacg ttattcctta tcaggcgatg 480
attgccgtcg cggcgaccac atttctttat tccgcgctgg cgctggcgat cagtctcgtt 540
cgcttttcgc ggacgatcgg tctgggaatt aaggttcttt atcagcacgt gccggttctt 600
cgggcgctac gcgatgcggc gactctgcga tatctcggcg gcagcgacgg cgaggggtgt 660
aacgacgcgg acgagacatt ttcgacgacc cggcgaaaat ttcatcacgc ccttgcctat 720
ggcttcggac tttgtttcgc ggccacagcc acgggcacga tctacgatca tatgttcggc 780
tggccggcgc cctatgcgct tttcagcttg ccggtcgtcc tagggaccgt tggggggatc 840
ggaatggtcg tgggcgcgat cggcctactc tggctcaagc tggccggcga agacgctcct 900
cgatcaccgg cactgcttgg gccggatgtt gccctgttgg tgcttctgct tgccatagcg 960
gcaacgggcc tcctcctttt agcggtccgc agcaccgaag tcatgggcgt cgcgctcgcc 1020
gtccatctcg gcgtcgtctt ggccttcttt ttggtgatgc catacagcaa atttgtccac 1080
ggtatcttca ggctcacggc tctcgtgcgc catcatgctg accgcgaggc aagtaatggc 1140
ttcgcctcca gccctcccac gaaaaagggt taa 1173
<210> 12
<211> 1296
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 12
atggaacata tgaagtccgt tcgcgatcgc agtagcgtca tgcagatcgt gagagtggcg 60
agtggcaact gtctcgagca atatgatttc ttcgtttacg gcttctatgc ggcatatatt 120
gcgagaagct tttttccgac cggcgataac gcgacatcgc tcatgctttc attggccact 180
tttggcgctg gtttcctcat gaggcccttg ggggcgattt ttctcgggtc ctacatcgat 240
cgcgtcgggc gtcggaaagg cctgatcgtg acactcgcga tcatggccgt cggaaccctc 300
accattgcga tgactccaag ctatgaggca attggattac tcgcaccggt tatcgtgctc 360
gtcgggcgac ttttgcaggg tttttccgct ggagcagagt cgggtggcgt ctcagtgtac 420
ttggcggaaa ttgcgtcgcc caaatcgaga ggcttcttca cctcgtggca gtctgccagc 480
cagcaggtgg ccgtcatgat cgccgccgcg atcggtcttg cgctgcaatc aacgctttca 540
ccggagcaaa tgaacgactg gggatggcgg gtgcccttgt tgatcggatg cttgattatc 600
cccgtgatac tctggctgcg ccggtctctc ccggaaacga aagcctatct ccacatggag 660
cacaaggcgc attcgatcgg cgaatccctc cgcgaattgc aacagagctg ggggctgatc 720
ttgacgggca tggcgatgtc gatcctcacg acgaccacct tttacatgat taccgcctat 780
acgccgacat ttggcgagaa agcactcgga ctgagcccgc aagatgtcct gctggttacc 840
atcatggtcg gcgtgtcgaa cttcctgtgg cttccgatcg ggggtgctct ctcggatcgt 900
atcggtagaa ccccgatcct actggtcgtg ccggtcaccg ttctcgccat cgcctttccc 960
ctgatgagct ggctcgtcgc ggcaccgaca ttcggagcgc ttgcagctgt tctgctgact 1020
ttctccgcat gctttggact ctataatggg gcgctcatcg cgagactcac cgagattatg 1080
cctcccgcca ttagaaccct tggcttctcg ctggcgttca gtctcgcgac ctcgctgttc 1140
ggcggcttca ccccattggt aagtacggcg ctaatccacg cgacgggcag caattccgcg 1200
cctgcaatct ggctctgttt tgcggctttc atcagcttcg tcggtgtggc cgcatcgacc 1260
cggctgagcc ggccaatcgc cgaaggcgcc agatag 1296
<210> 13
<211> 1071
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 13
atgagagcag tagtttaccg aaatggcgaa cttgtcctgg gggcctatgc tgatccgata 60
cccgccgccg ggcaggtgct cgtcaagacc agagcatgcg gcatctgcgg atctgacctt 120
catttttgcg atcatgcgca ggcgtttacg aaccttgcat cgcgggcggg tatcgcctct 180
atggaagttg atttgtgtcg agacatcgtt ctggggcatg aattctgtgg cgagattatg 240
gagttcgggc cctctgcgga tcgtcgcttc aaacccggac agcttgtgtg ctcgctgccg 300
ctggcgatcg gtccgaccgg agcgcggacg attggctact cggatgagta tcccggcggg 360
ctcggcgaat atatggtcct cacggaagcg ctcttgctgc ctgttccgaa cggccttccg 420
gcgacctgcg cggcgttgac ggagccgatg gcggtgggat ggcatgccgt cgagatcgcg 480
caggttcaac cacatcacat ccctgtggtg atcgggtgcg gaccggtcgg gttggcagtc 540
gtcgctgccc tgaaacataa gcaagttgct ccgattattg cgtcggatcc atcgcccgat 600
cggcgtgctc ttgctctgcg gatgggcgcc gacgccgttg tcgatccgcg cgaagaatca 660
ccctttcgcc aggccgagaa gatcgcacgc ccggtcggac aaggtggggc cctgtccagc 720
tcattgctgt caaagtctca aatgatattc gaatgcgtag gggtgccggg catgcttcgg 780
catgcgatgg acggcgcgtc cgacgggtcc gagatcatgg tcgttggcgc atgcatgcag 840
ccggacgcga tcgagcccat gatcgggatg tttaaagcgc tcacgatcaa attctcgcga 900
acttacacgg gtgaggaatt cgccgcggtg cttcacatga taggtgaggg cgcactcgac 960
gtatctccgc tcgttaccga tgtgattggc ctgtccgatg tcccgtccgc gtttgaggct 1020
ctacggagtc caggcgccca agcaaaagtg attgtggacc cttggcgctg a 1071
<210> 14
<211> 1269
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 14
atggcgaacg gaacaaggca gaaagatctc agagaacgcg ccgaacgggt cattccgggc 60
gggatgtacg gccacgagtc gacacggttg ctgccgccag aattccccca gttcttcagg 120
cgcgcgctgg gggcacgaat ttgggacgcc gacgagcagc cctatatcga ctatatgtgc 180
gcgtatgggc caaatttgct cggttaccgg caatccgaaa tcgaagccgc ggctgatgcg 240
cagcgacttc tcggcgacac catgaccggt ccttcggaga tcatggtcaa cctcgccgaa 300
gcctttgtgg gcatggtccg tcatgcggat tgggcgatgt tctgcaaaaa tggcagcgat 360
gccacctcaa cggcgatggt tctcgcgcgt gcccatacgg ggcgcaaaac catattatgc 420
gccaaaggcg cctatcatgg cgcttccccg tggaacactc cgcatactgc cgggattctc 480
gcttccgatc gcgtgcatgt cgcatattat acctataacg acgcccaaag cttatcggac 540
gcgttcaagg cgcacgatgg cgatattgcg gctgtctttg ccacaccttt ccgacacgaa 600
gtatttgagg accaggccct cgcccagctt gagttcgcgc gcaccgctcg aaaatgttgt 660
gacgagaccg gtgcgcttct ggtcgttgac gatgtgcgcg caggtttccg ggtggcgcgc 720
gattgcagct ggacgcattt gggtatcgaa cccgatctca gttgctgggg aaaatgcttt 780
gcgaatggct atccgatctc cgccctgctg ggctcgaaca aggcgcgcga tgcggcgcgg 840
gatatatttg tgaccggctc cttctggttc tctgcggtac cgatggcggc cgcgatcgaa 900
accctcagga tcattcgaga gacgccttat ctcgaaacgc tgatcgccag cggcgccgcc 960
ctgcgggcag gcctggaggc acagtctcag cgccatggtc ttgagttgaa gcagacgggc 1020
ccggcgcaga tgccgcaaat attctttgcg gacgatcccg attttcggat cggctatgcg 1080
tgggccgcgg cgtgcctgaa gggcggcgtc tatgttcatc cctatcacaa tatgtttctc 1140
tctgcggccc atacagttga cgatgtaacg gagaccctcg aggcgacgga tcgcgcgttc 1200
agcgcggtcc tcagagattt tgcgtctctc cagcctcatc ccattttaat gcaactcgcc 1260
ggtgcttga 1269
<210> 15
<211> 2835
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 15
atgtatcgga agttcagaat cgaaaagccc ggcaaggcaa atagtttgct cggcgcagta 60
gcgctcggca ccctcgcatt tcctgtctct gccagtgctc aggatagcga tcccgcatcg 120
ataggtcagc cggacgaagc ggacacggac cggggaacga gcgaaatcgt cgtgaccggc 180
agccgcctcc agaacggctt caattcgccg acgccggtta cagccgtatc cagcgagcag 240
ttgaaggagg catctccgac caaccttgcc gacgcactca accagctgcc cgtgttcaac 300
gacagcttga agacctccaa ccctggcacg acacccggaa cggggaacag cggtcagaac 360
ctgctcaaca tgcgcggcct cgggtcaaac cggaacctcg tcctgctgaa cggcaaccgt 420
ttcgtcgcga ccaatttcac aggctcggtc gatatcaacg tgctgccgca ggcgttggtc 480
aagcgcgtcg atgtcgtgac gggcggcgcc tcggccgcct acggttccga tgccgtttcg 540
ggcgtcatca acttcgtgct cgacgaagat ctggaaggca tcagggccga gctccagtcg 600
ggtgtttcaa cccgcggcga cctcccgtcc tacggcggtt cgatcgcctt cggcacttcg 660
tttgccgacg accggttgca cttgctcggc agcttcgaat attttcgaca ggacggaatc 720
cgggccgatg aagcaacggg tcgccgctgg ttcgacatcg ccgccggcca atatcccgtg 780
cccggcgcta cgacaggcgt cacggtcgtg cccgatattc gcagttctcg cggatcctac 840
ggcggacttg tcacgtccgg ccctctgaaa ggcatcgcgt ttttgcccgg aggagtccta 900
gggaccttcg actacgggaa ttttacgagc tcgtcgttcc agagcggcgg cgatggaccg 960
cgcgtgaata tcggcttcgc cccggatcag cttcgctaca acgcgttcct acgcgccgca 1020
tatgatgtgt ccgacactgt gcaggtgtat gcggagggca cctatgctta ttcccacacc 1080
aacctgggtg cattcgtaat atcgcatgtc ggtggctcga ataatttccg gatcttccgt 1140
gataacgcct tccttccggc tccactcgcg acgctcatgg acagaaatgc ccaggcttcg 1200
atcgttgtcg gtcgcttctc aagcgacttt cccttggtcg aaatcgagaa tttcgcaaag 1260
gtctaccgcg gcgctgccgg cttccgggca gacattggca atggctggaa actcgatggc 1320
tcggcctcct ttggccttac ggacctcgag cttcgtgaaa acaatctcac catcaaccgc 1380
aatctctacg ccgccgtcga tgcggtccgc gatcccgcgg gcaatatcgt ctgccgttca 1440
acactggccg gcctcgacca agattgcgtg ccgctcaatc tcttcggcac aggctcgccg 1500
agcgcgtcgg ccatcgacta tgtcaccgct gatggcgtcg ctcagctgag gcttgagcaa 1560
tatgtggcgg gactcacgat ttccggcgac ctcggcgata gcctgtcgtt cggcgcgggc 1620
ccggtctcgg tcgccgctgg tatcgaatat cgcaaggaga aggcccggca ggaaaccgac 1680
gcgatatcgc aggcgacgac ctcgatcacg ggaatcaggg gggctccggc ggcgcaggca 1740
ggtcggcctg gaggcttcaa tctctacaac ccacttccct tctcgggaag ctatgacatc 1800
aaggaaggtt ttgtcgaaat cggcgtcccg attctgaagg acagcgcgct gggacgttcg 1860
ctgaacttaa acggcgccgt ccgatatgcc gattacagcc agtccggtgg agtaacaacc 1920
tggaagctgg gcggagaata tgagccgatc gacggcctca ggttccgcgc gacccgttcg 1980
cgagatatcc gcgggccaag ccttgtcgag ctattcgacc ccggccgtca ggcgacgctc 2040
aattcaattt atggcggaca ggctgtgcag acgcggttct ttaccgccgg caacgcggat 2100
ttgcgccctg aaaaggcgga cgtccttaca ttcggcgcgg tgctacgccc cgccttcgtg 2160
ccggggtttc agttttcggt cgatcgctat gtggtgaagg tgaagggcgc gatcgatttc 2220
ctccttcccc agcaggaaat cgacgcgtgc gatgcaggaa acaccttctt ctgcgacctc 2280
ataacggaga atccggacgg caccatcaca gtgacgggtc ccaatctcaa cctggctgtc 2340
cagaaagcgg cgggaattga cttcgaggcc tattactcac gccccgtcgg cggcggcacg 2400
ttcagtcttc gtgcgctggc aacgcaccat acctctgcct atcgcatcgc gaccggctcg 2460
gcgcccatcc gttcgctcgg acaaccggac acgccaaaat ggtcggccaa cttccaggcg 2520
cgatattcga ccgacgattg ggcgcttctc gtgcagcagc gcttcatcgc agcatcggtg 2580
ttcaatgccg acaatgtgga gggcgtcgat acgaatttga accacgctcc ggcggtttgg 2640
tacaccgacg cgacattgac cttcgacatc gcggcttttg gccagaagca gcagctgttt 2700
ctatcggtca ataatttgtt cgaccgagat ccgccaatag cgacgaacga ccccagcagt 2760
ttttccagcc cgaccagctc tgcctatgat ccggtcggcc gctattttaa tgtcggggtc 2820
cgtttccgga tctga 2835
<210> 16
<211> 551
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 16
atgcgcctca cgggcggaga attattggca cgatgtttgg ccgtcgaagg cgtccggtat 60
gtcttcggcc tcatgtcgcc ggaggtggat ccgctcctgg ctgcgctcga agacaatggg 120
atattgttcg tcccggtgcg gcacgaggcc gccgcagcct atatggccga gggcatttac 180
aagaccaccg gacaggtcgc cgcgattgtc acgaatccgg gtcccggtac ggcaaacctt 240
ctgcctggag tcgtgacggc acgccacgaa ggggttccct tcgtcgcaat aacgtcccag 300
catcaacttg gtgtcgttta tccctgcacg ccaaaaacct ttcagggaca agaccagatc 360
gacctctttc gacccgcggt taaatggggc gcacccatct tcgcctggaa ccggattgtc 420
gaaatcaccc atatggcgtt ccgggaaatg tgggccggca ggccgggacc cgttcagttg 480
gaaatcccga rgtctgtgat gtatgktgtg ggcgaacgag gaccacggta gaagtttacr 540
gatcgccgac a 551
<210> 17
<211> 1272
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 17
atggaattga gccgccaacg agaccaggcc ttgagggagc gcgcccaagc ggtgatcccg 60
ggcgggatgt acggtcacga gtcgacctat ctgatgcccg agggcacgcc acagttcttc 120
agtcgcggca aaggcgcccg actttgggac gccgacggca acgagtatgt cgattacatg 180
tgcgcctatg gccccaacct gctgggttac ggcttcgaac ccgtcgaagc ggccgccgca 240
gcccagcaag cccggggcga taccctgacc gggccgtcgg aggtgatggt gcagttggcg 300
gaagacttcg tcgcgcaaat cagccacgcg gactgggcca tgttctgcaa gaacggcaca 360
gacgccacct caatggcgat ggtcatcgcg cgcgcacaca ccggccggaa gacgatcctc 420
tgcgcgaaag gcgcctatca tggggccgcg ccttggtgca cgccgatcct ggccggaacg 480
ctaccggagg atcgcgcctt tgtagtctac tacgactaca atgacgccca aagcctcgtc 540
gacgccttcg aggcccatca ggacgacgtc gcggcgatct tcgccacccc tcaccgtcac 600
gaggtgttca gcgaccagat cgatcctgat ccggaatatg cggccagcgt gcgggcgctc 660
tgcgacaaga gcggcgccct gctcgtcgtc gacgaagttc gagccgggtt caggatcgcg 720
cgcgactgca gctgggccaa gatcggcgtc gctccggatc tgagcacctg gggcaagtgc 780
ttcgccaacg gctatccgat ctcggcggtc ctagggggcg aaaaggtgcg cagcgcggca 840
aaggccgtct acgtcaccgg ctcgttctgg ttctcggcca cgcccatggc cgcagccgtc 900
gaaaccctga agcaaatccg cgagaccgac tatctcgagc ggatcaacgc ggccgggacc 960
cgcctgcgcg agggcctgca gcagcaggct gctcacaacg gctttacgtt gcgccaaacg 1020
gggcccgtct ccatgcccca agtcctcttc gaggaagatc ccgattttcg ggtcggctac 1080
ggctgggttc gcgaatgcct gaagcgaggg gtgtacttca gcccctacca taacatgttc 1140
ctgtcggcgg cccatagcga ggcggacctg gccaagaccc ttgcggctac cggcgacgcc 1200
ttcgtcgagc tacgcgccaa gcttccgagc ctagaaatcc accaacccct cctcgccctg 1260
agagcggcct aa 1272
Claims (10)
1. a kind of complex enzyme, which is characterized in that the complex enzyme contains amidase and esterase, which can remove fungi poison
Element, particularly fumonisin, ochratoxin and T2 toxin.
2. complex enzyme according to claim 1, wherein, the amidase has：
(a)SEQ ID NO：Amino acid sequence shown in 2；
(b)SEQ ID NO：One or several in amino acid sequence shown in 2 in the 11st, 89,179 and 239 amino acids residues
It is a that still there is SEQ ID NO after replacing, lacking or add：The amino acid sequence of amino acid sequence activity shown in 2；It is preferred that
With by SEQ ID NOs：3rd, the nucleotide sequence coded amino acid sequence shown in 4 and 5.
3. complex enzyme according to claim 1, wherein, the esterase have SEQ ID NOs: 6,7,8,9,10,11,
12nd, the amino acid sequence encoded described in nucleotide sequence shown at least one of 13,14,15,16 and 17.
4. complex enzyme according to claim 1, wherein, the weight ratio of the amidase and esterase is 1：0.01-100.
5. complex enzyme according to claim 1, wherein, the complex enzyme also contains phosphatidylserine and/or Zn-ef ficiency.
6. complex enzyme according to claim 1, wherein, the complex enzyme also contains one or more other enzymes；It is described
One or more other enzymes are selected from：Aflatoxin detoxifying enzymes, zearalenone lactonase, fumonisin Carboxylesterase,
It is fumonisin aminopherase, amino polyol amine oxidase, deoxynivalenol epoxide hydrolase, carboxypeptidase, black
Aspergillus aspartic protease PEPAa, PEPAb, PEPAc and PEPAd, elastoser, aminopeptidase, pepsin or stomach egg
White enzyme sample protease, trypsase or trypsin like proteases, bacterialprotease are related to starch metabolism, fiber degradation, lipid
The enzyme of metabolism, the protein for being related to glycogen metabolism or enzyme, amylase, arabinase, arabinofuranosidase, hydrogen peroxide
Enzyme, cellulase, chitinase, renin, cutinase, deoxyribonuclease, epimerase, esterase, galactosidase, Portugal
Dextranase, glucan lyase, endoglucanase, glucoamylase, glucose oxidase ,-glucosidase, including β-glucose
Glycosides enzyme, glycuronidase, hemicellulase, hexoxidase, hydrolase, invertase, isomerase, lipolytic enzyme, laccase, cracking
Enzyme, mannosidase, oxidizing ferment, oxidoreducing enzyme, pectate lyase, pectin acetyl esterases, pectin remove polymerase, pectinesterase
Enzyme, pectin decomposing enzyme, peroxidase, phenol oxidase, phytase, polygalacturonase, protease, sandlwood-galactolipin
Aldehydic acid enzyme, ribalgilase, African hesperidium element, transferase, transport protein, Transglutaminases, zytase, hexoxidase,
Acid phosphatase and combination thereof.
7. a kind of additive, which is characterized in that the additive contains the complex enzyme described in any one in claim 1-6；
Preferably, on the basis of the total weight of additive, the dosage of the complex enzyme is 1-15ppm.
8. additive according to claim 7, wherein, the additive is also containing bacillus licheniformis, bacillus subtilis
Bacterium, bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, lactoenterococcus, lactobacillus acidophilus, Lactobacillus casei, German-style lactobacillus
Lactic acid subspecies, lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, candida utili, bifidobacterium infantis, long bifid bar
Bacterium, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, animal bifidobacteria, aspergillus oryzae, slow bud
In spore bacillus, bacillus pumilus, lactobacillus cellobiosas, lactobacillus fermenti and lactobacillus delbruockii subspecies bulgaricus at least
It is a kind of；
Preferably, the additive is used for ensilage or ox feed, and the additive is also containing production propionibacterium acide-propionici, Bu Shi
At least one of lactobacillus and Lactobacillus paracasei；Or
The additive is used for the feed of poultry, growing-finishing pig and aquiculture animal, and the additive is also containing condensation bud
Spore bacillus and/or Brevibacillus laterosporus.
9. the complex enzyme described in any one and/or the additive described in claim 7 or 8 are true in removing in claim 1-6
Application in verticillium toxin particularly fumonisin, ochratoxin and T2 toxin.
A kind of 10. method for removing mycotoxin, which is characterized in that this method includes：By any one institute in claim 1-6
The additive described in complex enzyme and/or claim 7 or 8 stated is contacted with the sample that mycotoxin pollutes, to remove in sample
Mycotoxin, the mycotoxin is preferably fumonisin, ochratoxin and T2 toxin.
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| CN113774040A (en) * | 2020-11-24 | 2021-12-10 | 中国科学院上海营养与健康研究所 | B-family fumonisin degrading enzyme, construction method and application thereof |
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| CN114642239A (en) * | 2022-03-25 | 2022-06-21 | 播恩集团股份有限公司 | Method for improving DDGS nutritional quality and application thereof |
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