CN108251405B - Complex enzyme and additive, application thereof and method for removing mycotoxin - Google Patents

Abstract

The invention relates to the field of removal of mycotoxin, and discloses a complex enzyme and an additive, application of the complex enzyme and the additive, and a method for removing mycotoxin. The compound enzyme contains amidase and esterase, and the compound enzyme can remove mycotoxin, particularly fumonisin, ochratoxin and T2 toxin; additive containing the compound enzyme and application thereof in removing mycotoxin, in particular fumonisin, ochratoxin and T2 toxin, and a method for removing mycotoxin. Through the technical scheme, the amidase and the esterase are combined for use, ochratoxin A, fumonisin and T-2 toxin can be removed simultaneously, the removal efficiency is greatly improved compared with the efficiency of singly using one enzyme, and vomitoxin, aflatoxin and zearalenone toxin can be removed to a certain degree.

Description

Complex enzyme and additive, application thereof and method for removing mycotoxin

Technical Field

The invention relates to the field of removal of mycotoxins, in particular to a complex enzyme, an additive containing the complex enzyme, application of the complex enzyme and/or the additive in removal of mycotoxins, and a method for removing mycotoxins.

Background

Mycotoxins are secondary metabolites generated by toxic fungi in the harm process, and mainly comprise aflatoxin, deoxynivalenol (also called vomitoxin, DON), Zearalenone (ZEN), Fumonisin (FUM), ochratoxin, T-2 toxin and the like.

In recent years, due to frequent occurrence of extreme climate in China, the outbreak of gibberellic disease mainly harmful to wheat and corn is caused, pollution of Fumonisins (FUM), ochratoxins and T-2 generated by fungi is aggravated, and the wheat and corn with overproof pollution are continuously increased. The research on the safe utilization of the grain with toxin exceeding the standard not only can protect the grain-planting benefits of farmers in China, but also can prevent the grain from flowing into the grain market and maintain the national grain food quality safety is urgent.

For the removal of mycotoxin, the current common methods at home and abroad mainly comprise physical removal, adsorption, chemical treatment and the like. The adsorbent adsorbs a large amount of micronutrients in the feed and food while adsorbing mycotoxin, and the toxin adsorbed by the clay cannot be decomposed, so that secondary pollution is caused.

Currently, the method for producing ethanol by fermenting grain polluted by mycotoxin as a raw material is one of the most main means, but the toxin is further concentrated in a large amount of byproduct vinasse obtained by fermentation production, and the contents of fumonisins, ochratoxins and T-2 greatly exceed the national limit standard and cannot be utilized.

Biological removal can efficiently convert toxins into nontoxic products, and is environment-friendly and safe. The method has become the treatment technical approach with the most development prospect in the current mycotoxin reduction technology, is safe, environment-friendly and efficient, and is in line with the development trend of energy conservation and emission reduction in China by using modern biotechnology to develop research. However, in the biological removal aspect, it is difficult to simultaneously and efficiently remove a plurality of toxins, and when a compound enzyme agent is used, the removal efficiency of one toxin is often improved, while the removal efficiency of other toxins is reduced. When Fumonisins (FUM), ochratoxins and T-2 toxins are involved, the three toxins are much lower in content than vomitoxin, aflatoxins and zearalenone toxins, so that relatively few studies are made and it is more difficult to achieve efficient removal of them simultaneously.

Therefore, the development of a complex enzyme preparation which can save energy, protect environment and efficiently remove a plurality of mycotoxins, particularly can remove the mycotoxins simultaneously is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a compound complex enzyme preparation capable of simultaneously and efficiently removing a plurality of fungaltoxins, particularly fumonisin, ochratoxin and T2 toxin, an additive containing the complex enzyme preparation, application of the complex enzyme preparation and/or the additive in removing the fungaltoxins, particularly fumonisin, ochratoxin and T2 toxin, and a method for removing the fungaltoxins, particularly fumonisin, ochratoxin and T2 toxin.

In order to achieve the above objects, in one aspect, the present invention provides a complex enzyme, wherein the complex enzyme comprises amidase and esterase, and the complex enzyme is capable of removing mycotoxins, particularly fumonisins, ochratoxins and T2 toxins.

In a second aspect, the invention also provides an additive, wherein the additive contains the complex enzyme.

In a third aspect, the invention provides the use of a complex enzyme as described above and/or an additive as described above for the removal of mycotoxins, particularly fumonisins, ochratoxins and T2 toxins.

In a fourth aspect, the present invention provides a method for removing mycotoxins, wherein the method comprises: the complex enzyme and/or the additive as described above is contacted with a sample contaminated with mycotoxins, preferably fumonisins, ochratoxins and T2 toxins, to remove the mycotoxins from the sample.

Through the technical scheme, the amidase and the esterase are combined for use, ochratoxin A, fumonisin and T-2 toxin can be removed simultaneously, the removal efficiency is greatly improved compared with the efficiency of singly using one enzyme, and vomitoxin, aflatoxin and zearalenone toxin can be removed to a certain degree.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the invention provides a complex enzyme, wherein the complex enzyme contains amidase and esterase, and the complex enzyme can remove mycotoxin.

According to the invention, the amidase and the esterase are matched for use, so that various mycotoxins can be removed, but when the mycotoxins relate to fumonisin, ochratoxin and T2 toxin, the removal efficiency is obviously improved, and compared with a single enzyme preparation, the removal efficiency of the complex enzyme preparation provided by the invention is greatly improved.

Although the object of the present invention can be achieved by using an amidase in combination with an esterase, the inventors of the present invention have found that the removal efficiency of mycotoxins, particularly fumonisin, ochratoxin and T2 toxin, is further improved by using an amidase having an amino acid sequence as follows. Specifically, the amidase has: (a) SEQ ID NO: 2 (encoded by the nucleotide sequence shown in SEQ ID NO: 1); (b) SEQ ID NO: 2, and one or more of amino acid residues 11, 89, 179 and 239 in the amino acid sequence shown in the sequence table 2 still has the amino acid sequence shown in SEQ ID NO: 2, preferably having an amino acid sequence represented by seq id NOs: 3. 4 and 5, or a pharmaceutically acceptable salt thereof. More preferably, the amino acid sequence of the amidase is SEQ ID NO: 2 or an amino acid represented by SEQ ID NOs: 3. 4 and 5, or a pharmaceutically acceptable salt thereof.

Although the object of the present invention can be achieved by using an amidase in combination with an esterase, the inventors of the present invention have found that the removal efficiency of mycotoxins, particularly fumonisins, ochratoxins and T2 toxin, is further improved by using an esterase having an amino acid sequence. Specifically, the esterase has an amino acid sequence coded by at least one nucleotide sequence shown in SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17. Preferably, the amino acid sequence of the esterase is an amino acid sequence encoded by at least one nucleotide sequence shown in SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17.

Among them, after knowing the preferred amino acid sequences of the amidases and esterases as above, the nucleotide sequences encoding the amidases and esterases can be easily obtained by a method of codon reverse extrapolation. Meanwhile, due to the degeneracy of the codon, a plurality of nucleotide sequences which encode the same amino acid sequence but have different nucleotide sequences can be obtained, and all of the nucleotide sequences are within the protection scope of the invention.

The 20 amino acid residues constituting a protein can be classified into four types according to the side chain polarity: 1. non-polar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro); 2. polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), aspartic acid (Asn), glutamine (Gln) and tyrosine (Tyr); 3. positively charged amino acids: arginine (Arg), lysine (Lys), and histidine (His); 4. negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "biochemistry" (second edition) on the book, shengdi, wang spec rock, pages 82-83, higher education press, 1990, 12 months). If the substitution of amino acid residues belonging to the same class, for example, substitution of Arg for Lys or Leu for Ile, occurs in the protein, the role of the residues in the protein domain (e.g., the role of providing positive charge or forming a hydrophobic pocket structure) is not changed, and thus the steric structure of the protein is not affected, and thus the function of the protein can still be achieved. The substitution of an amino acid residue in the same class may occur at any amino acid residue position of the above enzymes.

As mentioned above, the enzymes provided by the present invention may also be modified or mutated to obtain the derived proteins. The "derived protein" of the present invention means a protein having a difference in amino acid sequence from the enzyme having the above amino acid sequence, and may have a difference in modified form which does not affect the sequence, or both. These proteins include natural or induced genetic variants. The induced variants may be obtained by various techniques, such as random mutagenesis by irradiation or mutagenic agents, etc., or by techniques such as site-directed mutagenesis or other known molecular biology techniques. The "derived proteins" also include analogs having residues of natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta-amino acids, gamma-amino acids, etc.).

Modifications (which do not generally alter primary structure, i.e., do not alter amino acid sequence) include: chemically derivatized forms of the protein such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those proteins that result from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility. In the present invention, however, the solubility and thermostability may be further improved by performing N-and/or O-glycosylation modification at any one of asparagine (Asn), serine (Ser) and threonine (Thr) residues of the complex enzyme.

For ease of purification, additional modifications of (a) or (b) may also be made using tags commonly used in the art, such as at least one of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc. The label does not influence the activity of the enzyme provided by the invention, and whether the label is added or not can be selected according to requirements in the practical application process.

In order to further improve the removing effect of the enzyme complex preparation on mycotoxin, the weight ratio of the amidase to the esterase is preferably 1: (0.01-100), more preferably 1: (0.05-20), more preferably 1: (0.1-10), more preferably 1: (0.2-5).

In the process of research, the inventor of the present invention found that, when the enzyme complex formulation of the present invention further contains phosphatidylserine and/or zinc element, the removal efficiency of mycotoxin can be further improved. Preferably, the content of the phosphatidylserine and/or zinc element is 1-30 wt%, and more preferably 5-10 wt%, based on the total weight of the complex enzyme preparation.

In the present invention, the amidase and esterase can be prepared by the existing methods, for example, by cloning a nucleotide sequence encoding the relevant amino acid sequence into a vector, transferring the vector into a host, and then separating the relevant nucleotide sequence from the proliferated host cells by the conventional method, or can be obtained commercially, and the phosphatidylserine and/or zinc element can be obtained commercially, or can be obtained by other normal methods.

According to the invention, in order to further improve the detoxification effect of the complex enzyme, the complex enzyme also contains one or more additional enzymes; the one or more additional enzymes may be selected from, but are not limited to: aflatoxin detoxication enzyme, ochratoxin lactonase, fumonisin carboxyesterase, fumonisin aminotransferase, aminopolyol amine oxidase, deoxynivalenol epoxide hydrolase, carboxypeptidase, Aspergillus niger aspartic protease PEPAa, PEPAb, PEPAc and PEPAd, elastase, aminopeptidase, pepsin or pepsin-like protease, trypsin or trypsin-like protease, bacterial protease, enzyme involved in starch metabolism, fiber removal, lipid metabolism, protein or enzyme involved in glycogen metabolism, amylase, arabinase, arabinofuranosidase, catalase, cellulase, chitinase, rennin, cutinase, deoxyribonuclease, epizyme, esterase, galactosidase, isomerase, glucanase, endoglucanase, glucoamylase, glucose oxidase, -glucosidases including beta-glucosidase, glucuronidase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, lipolytic enzyme, laccase, lyase, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectin depolymerase, pectin methylesterase, pectin lyase, peroxidase, phenoloxidase, phytase, polygalacturonase, protease, rhamnogalacturonase, ribonuclease, african sweet fruit element, transferase, transporter, transglutaminase, xylanase, hexose oxidase, acid phosphatase and combinations thereof.

In the present invention, for convenience of use, it is preferable that the components of the enzyme complex formulation are mixed and packaged together, or may be separately packaged and then mixed at the time of use.

The invention also provides a preparation method of the enzyme composite preparation, which comprises the following steps: the components are uniformly mixed according to the weight ratio.

According to the invention, the fumonisins may include their respective toxin types, e.g. FA1, FA2, FB1, FB2, FB3, FB4, FC1, FC2, FC3, FC4 and FP1, of which the most preferred is fumonisin FB 1. The ochratoxins may also include their respective toxin types, for example ochratoxin a and ochratoxin B.

In a second aspect, the invention also discloses an additive, wherein the additive contains the complex enzyme.

In a preferable case, the additive takes the complex enzyme provided by the invention as an active ingredient. In the additive, the content of the complex enzyme is 0.001-10g/kg, preferably 0.01-8g/kg, and more preferably 0.1-5 g/kg. The additive may further contain a solvent (e.g., a protein protecting agent such as glycerol, a saccharide, and a protease inhibitor), an agonist, and the like, which are known to those skilled in the art.

According to the invention, the additive also comprises a physiologically acceptable carrier, wherein the physiologically acceptable carrier is selected from at least one of the following substances: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat bran or wheat component, rice or rice bran, sucrose, starch, Na₂SO₄Talc and PVA and mixtures thereof.

It will be apparent to those skilled in the art that these additives may be added to feed and/or grain and oil material contaminated with mycotoxins to reduce the level of toxins present.

The feed material of the invention may comprise: a) cereals, for example, small grain cereals (such as wheat, barley, rye, oats, and combinations thereof) and/or large grain cereals such as maize or sorghum; b) by-products from cereals, such as corn gluten meal, distillers dried grains with solubles (DDGS), wheat bran, wheat middlings, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) ensiling the feed; d) proteins from the following sources: such as soy, sunflower, peanut, lupin, pea, broad bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; e) oils and fats obtained from plant and animal sources; f) minerals and vitamins.

The grain and oil of the present invention refers to the general term of grains and oil materials such as grains, beans, etc. and the finished products and semi-finished products thereof, especially to the products which can be eaten by human beings. For example, the grain oil may be a grain oil product that is edible to humans and is common in the art, and specifically, the grain oil may include at least one of grains and agricultural byproducts thereof, oil and fat products, wines, milks and products thereof, and the like.

It will be apparent to the skilled person that the feed or additive according to the invention may further comprise other components such as stabilisers and/or extenders and/or enzymes.

Preferably, the process for preparing the additive according to the invention comprises a mixing step comprising mixing a complex enzyme as described above, optionally together with at least one physiologically acceptable carrier, solvent, agonist, stabilizer, extender or enzyme.

It will be apparent to the skilled person that as a preventive step, additives may be added to any feed and/or grain and oil material.

For example, when the additive is used in the feed for farmed animals, the additive may further comprise at least one of Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, enterococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp.

For example, when the additive is used for silage or cattle feed, the additive further contains at least one of propionibacterium propionicum, lactobacillus buchneri and lactobacillus paracasei.

For example, when the additive is used in the feed of meat poultry, growing-finishing pigs and aquaculture animals, the feed or additive also contains bacillus coagulans and/or brevibacillus laterosporus.

Based on the above description, the invention also provides a grain oil or feed, wherein the grain oil or feed contains the additive.

In a third aspect, the invention also provides the use of a complex enzyme as described above and/or an additive as described above for the removal of mycotoxins, in particular fumonisins, ochratoxins and T2 toxins.

In a fourth aspect, the present invention also provides a method for removing mycotoxins, wherein the method comprises: the complex enzyme and/or additive as described above is contacted with a sample contaminated with mycotoxins, preferably fumonisins, ochratoxins and T2 toxins, to remove the mycotoxins from the sample.

According to the invention, the contacting is preferably carried out under conditions in which the respective enzyme components of the complex enzyme are capable of exerting an enzymatic activity. The term "conditions for exerting an enzymatic activity" means conditions such as temperature and pH for exerting an enzymatic activity.

According to the present invention, the mycotoxin contaminated sample may comprise any sample containing mycotoxins, e.g. grain oil and/or feed, etc. Preferably, the complex enzyme preparation is used in an amount of at least 0.1ppm, preferably 0.1-15ppm, more preferably 1-10ppm, and even more preferably 4-8ppm, based on the total weight of the mycotoxin-contaminated sample. In the present invention, "ppm" means "μ g/mL" when the mycotoxin-contaminated sample is a liquid; when the vomitoxin-contaminated sample is a solid, "ppm" means "μ g/g".

In the following examples and comparative examples, ochratoxin a, fumonisin FB1 and T2 toxin standards were purchased from sigma; phosphatidylserine was purchased from shanghai xin sail biotechnology limited.

Detecting fumonisins FB1 by high performance liquid chromatography according to a method in GB 5009.240-2016 standard, detecting vomitoxins by a method in GB/T30956-2014 standard, and detecting T2 toxins by a method in GB/T23501-2009 standard;

the toxin degradation rate = (mass of toxin in sample before reaction-mass of toxin in sample after reaction)/mass of toxin in sample before reaction × 100%.

Examples

The present invention will be described in detail below by way of examples, but the present invention is not limited thereto. In the following preparation examples, examples and comparative examples, each material was commercially available unless otherwise specified, and each method was a conventional method in the art.

Preparation example 1

This preparation example is intended to illustrate the preparation of the amidase provided by the present invention

(1) Obtaining of genes

The following nucleotide fragments were synthesized by an artificial chemical synthesis method (entrusted to treasure bioengineering (Dalian) Co., Ltd., the same shall apply hereinafter); in SEQ ID NO: 1, adding Nde I enzyme cutting site after the 5 'end initiation codon ATG, and adding Xho I enzyme cutting site and termination codon TAG at the 3' end.

(2) Construction of recombinant plasmid

The PET30a plasmid (His-tagged, available from Invitrogen USA) was double digested with restriction enzymes Nde I and Xho I (available from NEB) in a 37 ℃ water bath for 4h, with the following restriction enzyme systems (50. mu.L):

and (4) carrying out agarose gel electrophoresis on the enzyme digestion product, and then purifying and recovering. Then, ligation was performed using T4 ligase (purchased from Takara) for 4 hours at room temperature to obtain a recombinant plasmid. The ligation system (10. mu.L) used was as follows:

(3) obtaining of recombinant strains

DH 5. alpha. competent cells (purchased from Takara) were electroporated on a Bio-Rad Gene Pulse electrotransfer apparatus using the recombinant plasmid obtained in step (2) under the following conditions: the voltage 1500V, the capacitance 25. mu.F, the resistance 200. omega. and the conversion time vary from one DNA sample to another and are automatically given by the instrument, generally in the range of 3.5-4 s. Then, the transformed cells were spread on an LB solid plate containing kanamycin for screening, inverted culture was performed at 37 ℃ for 2 days, and positive colonies were picked and inoculated in an LB liquid medium to obtain a recombinant strain.

Then, the plasmid was extracted by a method known in the art, and sequencing was performed by Taobao bioengineering (Dalian) Co., Ltd. to show that the above gene was successfully transformed into E.coli, indicating that the recombinant strain of the present invention was successfully constructed.

(4) Preparation of amidase

The obtained recombinant strain was inoculated into LB liquid medium (beef extract 5g, peptone 10g, sodium chloride 5g, water supplemented to 1000mL, sterilized at 115 ℃ for 20min, pH 7), cultured at 37 ℃ for 3 days to OD₆₀₀The value is 2-6, the cells are collected by centrifugation and washed with phosphate buffer (PBS, 135mM NaCl, 2.7mM KCl, 1.5mM KH)₂PO₄，8mM K₂HPO₄pH 7.2), ultrasonic crushing, centrifuging and taking supernatant to obtain crude enzyme solution.

The crude enzyme solution was placed on ice, and the ground ammonium sulfate powder was slowly added thereto with stirring until saturation with ammonium sulfate. Standing at 4 deg.C for about 24h, centrifuging at 12000r/min for 50min, discarding supernatant, and dissolving precipitate with small amount of PBS (pH 7.2). The PBS-solubilized pellet was dialyzed, ammonium sulfate removed, and resuspended in buffer (pH7.4, 50mM NaCl, containing 10mM imidazole). According to the fact that expressed recombinase contains His labels, an Ni column is used for affinity chromatography purification, after the Ni column is balanced by 1mL/min, the resuspended crude enzyme liquid is directly loaded at the flow rate of 0.5 mL/min; continuing to use 1mL/min buffer (pH7.4, 50mM NaCl, containing 10mM imidazole) to elute unadsorbed or adsorbed non-specific hybrid protein; the target protein was collected by elution with a buffer (pH7.4, 50mM NaCl, 500mM imidazole) to obtain a purified amidase solution, and freeze-dried to obtain amidase dry powder X1.

Preparation examples 2 to 4

Preparation examples 2 to 4 are illustrative of the preparation of amidase provided by the present invention

The preparation of amidases X2-X4 was carried out according to the method of preparation example 1, except that preparation examples 2-4 used SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: 5 substitution of SEQ ID NO: 1.

preparation examples 5 to 16

Preparation examples 5 to 16 are illustrative of the preparation of the esterase provided by the present invention

Preparation of esterase Z1-Z12 was carried out according to the method of preparation 1, respectively, except that preparations 5 to 16 used 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 substitution of SEQ ID NO: 1.

examples 1 to 14 and comparative examples 1 to 3

Used for explaining the removal effect of the complex enzyme provided by the invention on fumonisins FB1, ochratoxin A and T2 toxin.

The compound enzyme preparation is prepared by using normal saline according to the proportion of each example and comparative example in the table 1, so that the concentration of the compound enzyme in the obtained mixed solution is 100ng/ml, and the concentrations of fumonisins FB1, ochratoxins A and T2 toxin are 1000ppb respectively. The reaction temperature is 37 ℃ respectively, the reaction pH value is 5 respectively, 20 mu L of reaction products after 30min of reaction are used for detecting the residues of fumonisins FB1, ochratoxin A and T2 toxin, and the degradation rate is calculated. The results are shown in Table 2.

TABLE 1

	Amidases	Esterase	Phosphatidylserine	Zinc
					Example 1	X1 50ng/ml	Z1 50ng/ml	5ng/ml	---
Example 2	X2 80ng/ml	Z2 20ng/ml	---	8ng/ml
					Example 3	X3 20ng/ml	Z3 80ng/ml	5ng/ml	5ng/ml
Example 4	X1 50ng/ml	Z1 50ng/ml	---	---
					Example 5	X1 50ng/ml	Z4 50ng/ml	5ng/ml	---
Example 6	X1 50ng/ml	Z5 50ng/ml	5ng/ml	---
					Example 7	X1 50ng/ml	Z6 50ng/ml	5ng/ml	---
Example 8	X1 50ng/ml	Z7 50ng/ml	5ng/ml	---
					Example 9	X1 50ng/ml	Z8 50ng/ml	5ng/ml	---
Example 10	X1 50ng/ml	Z9 50ng/ml	5ng/ml	---
					Example 11	X1 50ng/ml	Z10 50ng/ml	5ng/ml	---
Example 12	X1 50ng/ml	Z11 50ng/ml	5ng/ml	---
					Example 13	X1 50ng/ml	Z12 50ng/ml	5ng/ml	---
Example 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

As can be seen from the data in table 2 above, the combination of amidase and esterase enables efficient removal of fumonisins and ochratoxins, and also enables removal of most of the T2 toxin. And when the complex enzyme is added with the phosphatidylserine and/or the zinc, the removal efficiency of the toxin is further improved.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

SEQUENCE LISTING

<110> Zhongliang Nutrition and health research institute Co., Ltd

COFCO Ltd.

<120> complex enzyme and additive, application thereof and method for removing mycotoxin

<130>I40389COF

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aaccggatcg tttctgcaca tgtgcatggc aaggcgggga ttatggctgc tatcaaagca 900

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tag 1443

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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 AsnGly 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 AlaAsp 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 GluAsp 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 (11)

1. The compound enzyme is characterized by comprising amidase and esterase, and the compound enzyme can remove mycotoxin, wherein the mycotoxin is fumonisin, ochratoxin and T2 toxin;

wherein the amino acid sequence of the amidase is the amino acid sequence shown in SEQ ID NO. 2 or the amino acid sequence coded by the nucleotide sequences shown in SEQ ID NOS: 3, 4 and 5;

wherein the amino acid sequence of the esterase is the amino acid sequence coded by at least one nucleotide sequence shown in SEQ ID NOs 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17.

2. The complex enzyme according to claim 1, wherein the weight ratio of amidase to esterase is 1: 0.01-100.

3. The complex enzyme of claim 1, wherein the complex enzyme further comprises phosphatidylserine and/or zinc.

4. The complex enzyme of claim 1, wherein the complex enzyme further comprises one or more additional enzymes;

the one or more additional enzymes are selected from one or more of oxidoreductases, transferases, hydrolases, lyases, and isomerases.

5. The complex enzyme of claim 4, wherein the one or more additional enzymes are selected from the group consisting of: aflatoxin detoxication enzyme, zearalenone lactonase, fumonisin carboxyl esterase, fumonisin aminotransferase, aminopolyol amine oxidase, deoxynivalenol epoxide hydrolase, carboxypeptidase, aspergillus niger aspartic protease PEPAa, aspergillus niger aspartic protease PEPAb, aspergillus niger aspartic protease PEPAc, aspergillus niger aspartic protease PEPAd, elastase, aminopeptidase, pepsin, trypsin, amylase, arabinase, catalase, cellulase, chitinase, rennin, cutinase, deoxyribonuclease, galactosidase, glucan lyase, glucose oxidase, glucuronidase, hemicellulase, hexose oxidase, laccase, mannosidase, pectate lyase, pectin acetylesterase, pectin depolymerase, pectin methylesterase, pectinolytic enzyme, pectinase, aminopolyol amine oxidase, deoxynivalenol epoxide hydrolase, carboxypeptidase, PEPAa, pep, cellulase, one or more of phytase, polygalacturonase, rhamnogalacturonase, ribonuclease, transglutaminase and acid phosphatase.

6. An additive, which is characterized in that the additive contains the compound enzyme of any one of claims 1 to 5.

7. The additive of claim 6, wherein the amount of the complex enzyme is 1-15ppm based on the total weight of the additive.

8. The supplement of claim 7, wherein the supplement further comprises at least one of bacillus licheniformis, bacillus subtilis, bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, enterococcus lactis, lactobacillus acidophilus, lactobacillus casei, lactobacillus delbrueckii subsp lactis, lactobacillus plantarum, pediococcus acidilactici, pediococcus pentosaceus, candida utilis, bifidobacterium infantis, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, bifidobacterium animalis, aspergillus oryzae, bacillus lentus, bacillus pumilus, lactobacillus cellobiosus, lactobacillus fermentum, and lactobacillus delbrueckii subsp bulgaricus.

9. The supplement of claim 8, wherein the supplement is for silage, the supplement further comprising at least one of propionibacterium propionicum, lactobacillus buchneri, and lactobacillus paracasei; or

The additive is used for feed of meat poultry, growing-finishing pigs and aquaculture animals, and also contains bacillus coagulans and/or brevibacillus laterosporus.

10. Use of a complex enzyme according to any one of claims 1 to 5 and/or an additive according to any one of claims 6 to 9 for the removal of fumonisins, ochratoxins and T2 toxins.

11. A method for removing mycotoxins, comprising: contacting the complex enzyme of any one of claims 1-5 and/or the additive of any one of claims 6-9 with a sample contaminated with mycotoxins to remove mycotoxins from the sample;

wherein the mycotoxins are fumonisins, ochratoxins and T2 toxins.