CN108251398B - Zearalenone degrading enzyme, gene, preparation method and application thereof, and method for degrading zearalenone - Google Patents
Zearalenone degrading enzyme, gene, preparation method and application thereof, and method for degrading zearalenone Download PDFInfo
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Abstract
The invention relates to the field of microorganisms, and discloses a zearalenone degrading enzyme, a gene, a preparation method and application thereof, and a method for degrading zearalenone. Specifically, the invention provides a zearalenone degrading enzyme, which has an amino acid sequence shown in the following (a) and/or (b): (a) SEQ ID NO: 2; (b) SEQ ID NO: 2, and one or more of amino acid residues 1-262 and 264 in the amino acid sequence shown in the formula 2 still has the zearalenone degrading enzyme activity after being substituted, deleted or added. The zearalenone degrading enzyme provided by the invention can efficiently and quickly degrade ZEN, and has a good industrial application prospect.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a zearalenone degrading enzyme, a gene for coding the zearalenone degrading enzyme, a recombinant vector and a strain containing the gene, an additive, grain oil or feed containing the additive, a method for expressing the zearalenone degrading enzyme, application of the zearalenone degrading enzyme, the gene, the recombinant vector, the strain and the additive in degrading zearalenone, and a method for degrading the zearalenone.
Background
Zearalenone (ZEN), a mycotoxin produced by fusarium and released into the soil environment, is one of the fusarium toxins responsible for the widest range of contamination worldwide. In 1999, d.mello et al found that ZEN was able to reduce the survival rate of the embryo and the birth rate of the new born fetus in pregnant animals. The influence of ZEN on human body mainly includes tumor initiation, DNA contraction induction, chromosome aberration and the like. In addition, ZEN binds to 17 β -estradiol receptors, resulting in fat oxidation reactions, apoptosis and inhibition of protein and DNA synthesis, and may also inhibit biosynthesis of other macromolecules.
Zearalenone, one of the most widespread mycotoxins contaminating grain and oil or feed in the world, has detected the presence of ZEN in grains and agricultural by-products all over the world, e.g., europe, africa, north america, south america, and so on. ZEN can be through polluting crops such as cereal, and then get into human or animal internal, endangers human and animal's health, causes huge economic loss. Meanwhile, ZEN itself has problems of wide distribution, rapid propagation, heat resistance, high toxicity, long residual time and the like, and has attracted worldwide attention. Currently, most countries have very strict regulations on the content of ZEN in grain, oil or feed, for example, Australia regulations that the content of ZEN in grains cannot exceed 50 ng/g; italy specifies that the ZEN content in cereals and cereal products cannot exceed 100 ng/g; in France, the ZEN content of vegetable oils and cereals must be less than 200 ng/g.
At present, methods for removing ZEN toxin are mainly classified into physical methods, chemical methods and biological methods. Physical methods include mechanical separation treatment, high temperature inactivation, radiation treatment or adsorbents, etc.; the chemical method is to treat the toxin with acid-base solution or other compounds. However, the method of removing ZEN by physical and chemical treatment has limitations. For example, ZEN cannot be effectively passivated by heat treatment; although the ZEN content can be reduced to some extent by extrusion and treatment with an oxidizing agent, the use of extrusion methods and the use of oxidizing agents (such as ozone or hydrogen peroxide) in the preparation of feed and food products is limited by higher costs, loss of quality of treated samples, lower efficiency and specificity. In a word, the physical method has the defects of high cost, difficult operation, pollution to grain and oil, feed or environment caused by processed products and the like, and the chemical method can change the properties of the feed and generate harmful residues, so that the defects of food safety and the like exist, and the application of the two methods in actual production is limited.
The biological method mainly utilizes microorganisms or degradation products thereof to carry out toxin degradation, has the advantages of small influence on sensory properties, palatability and nutrient substances of raw materials and the like, and has the characteristics of safety, environmental protection and high efficiency, so that the research of removing ZEN in grain and oil or/feed by utilizing modern biotechnology has good application prospect. In the prior art, CN103937681A discloses a food-grade Aspergillus niger and application thereof in zearalenone degradation, the Aspergillus niger is co-cultured with ZEN with a final concentration of 2ppm for 48 hours under a proper condition (28 ℃, the inoculum size of a bacterial liquid is 2%), and the degradation rate of the ZEN is 89.56%. CN103981134A discloses a pseudomonas aeruginosa and application thereof in degrading zearalenone, the pseudomonas aeruginosa is co-cultured with ZEN with the final concentration of 2ppm for 72h under the appropriate condition (28 ℃), and the degradation rate of ZEN is 92.75%. CN103981133A discloses a bacillus amyloliquefaciens and application thereof in degradation of zearalenone, wherein the bacillus amyloliquefaciens is co-cultured with ZEN with the final concentration of 5ppm for 24 hours under a proper condition (28 ℃), and the degradation rate of ZEN is 95.99%. As can be seen, the existing methods for degrading ZEN by microorganisms have long degradation time, and the degradation rate is still to be improved.
In addition, the existing biological method for degrading ZEN is mostly carried out under mild conditions (such as 28 ℃ and pH of 7), however, no good solution exists under higher temperature load (such as the condition of transporting in a container or during feed granulation) or under severe acid-base conditions, which limits the application range of the biological method in degrading ZEN.
Therefore, there is a need to find a method for biodegrading ZEN toxin which is efficient and safe and can be used under higher temperature load or harsh acid-base conditions.
Disclosure of Invention
The present invention aims to overcome the above-mentioned drawbacks of the prior art and provide a zearalenone degrading enzyme, a gene encoding the zearalenone degrading enzyme, a recombinant vector and strain containing the gene, an additive, a method for expressing the zearalenone degrading enzyme, and the use of the zearalenone degrading enzyme, gene, recombinant vector, strain and additive in degrading zearalenone and a method for degrading zearalenone.
In order to achieve the above object, in a first aspect, the present invention provides a zearalenone degrading enzyme, wherein the zearalenone degrading enzyme has an amino acid sequence represented by the following (a) and/or (b):
(a) SEQ ID NO: 2;
(b) SEQ ID NO: 2, and one or more of amino acid residues 1-262 and 264 in the amino acid sequence shown in the formula 2 still has the zearalenone degrading enzyme activity after being substituted, deleted or added.
In a second aspect, the present invention also provides a gene encoding a zearalenone degrading enzyme, wherein the gene has a nucleotide sequence encoding the zearalenone degrading enzyme.
In a third aspect, the present invention also provides a recombinant vector, wherein the recombinant vector contains the above gene.
In a fourth aspect, the present invention also provides a strain, wherein the strain contains the above gene or the above recombinant vector.
In a fifth aspect, the present invention also provides an additive, wherein the additive comprises the zearalenone degrading enzyme described above, and/or comprises a fermentation product of the strain described above.
In a sixth aspect, the invention also provides a grain oil or feed, wherein the grain oil or feed contains the zearalenone or the additive.
In a seventh aspect, the present invention also provides a method for expressing zearalenone degrading enzyme, wherein the method comprises: introducing the recombinant vector into a host, and inducing the expression of a gene for encoding zearalenone degrading enzyme in the host; or inducing the strain to express zearalenone degrading enzyme.
In an eighth aspect, the invention also provides an application of the zearalenone degrading enzyme, the gene, the recombinant vector, the strain or the additive in degrading zearalenone.
In a ninth aspect, the present invention also provides a method for degrading zearalenone, the method comprising: contacting an enzymatic agent with a sample to be treated under conditions of an enzymatic degradation reaction; wherein, when the enzyme agent contains the zearalenone degrading enzyme or the additive, the conditions of the enzymatic degradation reaction include: the temperature is 20-55 ℃; the pH value is 3-9; alternatively, when the enzymatic agent comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1, the conditions of the enzymatic degradation reaction include: the temperature is 40-55 deg.C, and pH is 3-9.
The inventor discovers that the zearalenone degrading enzyme provided by the invention can degrade ZEN efficiently and rapidly in the research process, and has a good industrial application prospect. In addition, the zearalenone degrading enzyme provided by the invention also has the characteristics of acid and alkali resistance and high temperature resistance, so that the application range of the zearalenone degrading enzyme is further expanded.
In addition, the preferred embodiment of the invention uses saccharomyces cerevisiae, bacillus licheniformis and bacillus subtilis as host strains, and the fermentation products produced by the host strains are directly added into grain oil and/or feed, so that the influence on the palatability of the grain oil and/or feed is small; in the prior art, Escherichia coli is generally used as a host strain, and the fermentation product produced by the Escherichia coli is added into grain oil and/or feed, so that the palatability of the grain oil and/or feed is influenced. Therefore, the zearalenone degrading enzyme prepared by the method provided by the invention has wider application prospect, and is particularly applied to grain and oil and/or feed.
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 present invention provides a zearalenone degrading enzyme, wherein the zearalenone degrading enzyme has an amino acid sequence represented by the following (a) and/or (b):
(a) SEQ ID NO: 2;
(b) SEQ ID NO: 2, and one or more of amino acid residues 1-262 and 264 in the amino acid sequence shown in the formula 2 still has the zearalenone degrading enzyme activity after being substituted, deleted or added. Wherein, still having zearalenone degrading enzyme activity means that the percentage (relative activity) between the degradation rate of the ZEN by the protein derived from (a) and the degradation rate of (a) on ZEN is not less than 90% (or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100%) under the same assay conditions.
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.
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. For example, (b) can be obtained by attaching a tag (e.g., at least one of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc) to the amino terminus and/or the carboxyl terminus of (a) (i.e., (b) is an amino acid sequence of SEQ ID NO: 2 having a tag attached to the amino terminus and/or the carboxyl terminus). 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 preferred cases, the zearalenone degrading enzyme has the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or SEQ ID NO: 5, or, in SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or SEQ ID NO: 5 is linked at the amino terminus and/or the carboxy terminus to the amino acid sequence of the tag.
The zearalenone degrading enzyme can be obtained through artificial synthesis, or can be obtained through obtaining the coding gene and then performing biological expression.
In a second aspect, the present invention also provides a gene encoding a zearalenone degrading enzyme, wherein the gene has a nucleotide sequence encoding the zearalenone degrading enzyme.
It is well known in the art that 18 amino acids, of the 20 different amino acids that make up a protein, are each encoded by 2-6 codons, except for Met (ATG) or Trp (TGG), which are each encoded by a single codon (Sambrook et al, molecular cloning, Cold spring harbor laboratory Press, New York, USA, second edition, 1989, see appendix D page 950). That is, due to the degeneracy of genetic code, there is usually more than one codon determining one amino acid, and the substitution of the third nucleotide in the triplet codon will not change the composition of the amino acid, so that the nucleotide sequences of genes encoding the same protein may differ. From the amino acid sequences disclosed in the present invention and the amino acid sequences obtained from the amino acid sequences without the change in the enzyme activity, it is fully possible for those skilled in the art to derive the nucleotide sequences of the genes encoding them, which are obtained by biological methods (e.g., PCR method, mutation method) or chemical synthesis methods, based on the well-known codon tables, and therefore, the partial nucleotide sequences should be included in the scope of the present invention. Conversely, using the DNA sequences disclosed herein, amino acid sequences consistent with the activity of the enzymes of the invention may also be obtained by modifying the nucleic acid sequences provided herein by methods well known in the art, e.g., Sambrook et al (molecular cloning, Cold spring harbor laboratory Press, New York, U.S. Pat. No. 5, second edition, 1989).
Preferably, the gene has the sequence of SEQ ID NO: 7 (encoding zearalenone degrading enzyme represented by SEQ ID NO: 2), SEQ ID NO: 8 (encoding zearalenone degrading enzyme represented by SEQ ID NO: 3), SEQ ID NO: 9 (encoding zearalenone degrading enzyme represented by SEQ ID NO: 4) or SEQ ID NO: 10 (encoding zearalenone degrading enzyme represented by SEQ ID NO: 5).
As described above, the 5 'end and/or 3' end of the nucleotide sequence may be linked to a coding sequence for a tag commonly used in the art (e.g., at least one of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc), respectively.
The nucleotide sequence provided by the invention can be obtained by a Polymerase Chain Reaction (PCR) amplification method, a recombination method or an artificial synthesis method. For example, one skilled in the art can easily obtain templates and primers based on the nucleotide sequences provided by the present invention, and obtain the relevant sequences by PCR amplification.
Once the nucleotide sequence of interest is obtained, the amino acid sequence of interest can be obtained in large quantities by recombinant methods. The nucleotide sequence obtained is usually cloned into a vector and transferred into a host, and the relevant nucleotide sequence is isolated from the propagated host cells by a conventional method.
In addition, the nucleotide sequence can be synthesized by a known artificial chemical synthesis method.
In a third aspect, the present invention also provides a recombinant vector, wherein the recombinant vector contains the above gene.
In the present invention, the recombinant vector may contain the above-mentioned gene provided by the present invention. As the "vector" used in the recombinant vector, various vectors known in the art, such as various commercially available plasmids, cosmids, phages, retroviruses and the like can be used, and plasmids are preferably used as the vector in the present invention. The recombinant vector can be constructed by digesting a linear plasmid with various endonucleases capable of cleaving at the multiple cloning site of the vector (for example, SalI, BamH I, EcoRI and the like can be used for pUC 18; NdeI, NheI, EcoRI, BamH I, Hind III and the like can be used for pPICZ alpha A; BamH I, Hind III, XhoI and the like can be used for PET30 a) and ligating the linear plasmid with a gene fragment cleaved with the same endonuclease to obtain a recombinant plasmid. The invention preferably adopts Nde I and Xho I double enzyme digestion PET30a vector and gene fragment connected with the vector, and the recombinant vector is constructed by ligase connection.
In a fourth aspect, the present invention also provides a strain, wherein the strain contains the above gene or the above recombinant vector.
In the present invention, the strain may be a strain containing the gene of the present invention, or a recombinant strain obtained by transforming, transducing or transfecting the above recombinant vector into a host by a method conventional in the art, such as calcium chloride method, chemical transformation or electroporation transformation, preferably electroporation transformation.
According to the invention, the strain may be a bacterium and/or a fungus; preferably, the strain is at least one of a coccus, a bacillus, a spirillum, a yeast and a mold; more preferably, the strain is at least one of saccharomyces cerevisiae, 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, saccharomyces cerevisiae, rhodopseudomonas palustris, bifidobacterium infantis, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, bifidobacterium animalis, aspergillus niger, aspergillus oryzae, bacillus lentus, bacillus pumilus, lactobacillus cellobiosus, lactobacillus fermentum, lactobacillus delbrueckii subsp bulgaricus, propionibacterium propionate, lactobacillus buchneri, lactobacillus paracasei, bacillus coagulans and bacillus brevis; further preferably, the strain is at least one of Bacillus licheniformis (Bacillus lincheniformis), Bacillus subtilis (Bacillus subtilis) and Saccharomyces cerevisiae (Saccharomyces cerevisiae).
In a fifth aspect, the present invention also provides an additive, wherein the additive comprises the zearalenone degrading enzyme provided by the present invention, and/or comprises a fermentation product of the above strain.
In a preferred case, the additive comprises the zearalenone degrading enzyme provided by the present invention as an active ingredient. Based on the total weight of the additive, the content of the zearalenone degrading enzyme is 10-90 wt%.
In the present invention, 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.
In the present invention, the additive may further comprise the strain as described above, and will not be described herein.
In the present invention, the additive is preferably used as a grain and oil and/or feed additive.
In a sixth aspect, the invention also provides a grain oil or feed, wherein the grain oil or feed contains the additive. The content of the zearalenone degrading enzyme is 1-10ppm, preferably 2-8ppm, and more preferably 4-6ppm based on the total weight of the grain oil or feed. In the present invention, "ppm" means "μ g/mL" when the grain or feed is liquid; when the grain or feed is a solid, "ppm" means "μ g/g".
In the present invention, the term "grain and oil" refers to a general term for grains, beans and other grains and oils, and finished products and semi-finished products thereof, and particularly to products that can be eaten by humans. 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.
In the present invention, the term "feed" refers to the general term of food for animals raised in agriculture or animal husbandry. For example, the feed may be a food commonly used in the art for feeding animals, and in particular, the feed may include: 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.
In the present invention, the grain or feed may further comprise a physiologically acceptable carrier, wherein the physiologically acceptable carrier is at least one selected from the group consisting of: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat bran or wheat component, rice or rice bran, sucrose, starch, Na2SO4And talc and mixtures thereof.
In a seventh aspect, the present invention also provides a method for expressing zearalenone degrading enzyme, wherein the method comprises: introducing the recombinant vector into a host, and inducing the expression of a gene for encoding zearalenone degrading enzyme in the host; or inducing the strain to express zearalenone degrading enzyme.
According to the invention, the host may be a bacterium and/or a fungus; preferably, the host is at least one of a coccus, bacillus, helicobacter, yeast and mold; more preferably, the host is at least one of saccharomyces cerevisiae, 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, saccharomyces cerevisiae, rhodopseudomonas palustris, bifidobacterium infantis, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, bifidobacterium animalis, aspergillus niger, aspergillus oryzae, bacillus lentus, bacillus pumilus, lactobacillus cellobiosus, lactobacillus fermentum, lactobacillus delbrueckii subsp bulgaricus, propionibacterium propionate, lactobacillus buchneri, lactobacillus paracasei, bacillus coagulans, and bacillus brevis; further preferably, the host is at least one of bacillus licheniformis, bacillus subtilis and saccharomyces cerevisiae.
In the present invention, the above-mentioned recombinant vector can be introduced into a host in a manner conventionally used in the art, for example, calcium chloride method, chemical transformation or electroporation transformation, preferably electroporation transformation.
In the present invention, the conditions for inducing expression may include: the temperature is 30-40 deg.C, pH is 6-8, and the time is 12-72 h. Preferably, the conditions for inducing expression comprise: the temperature is 35-40 deg.C, pH is 6.5-7.5, and the time is 24-72 h.
In an eighth aspect, the invention also provides an application of the zearalenone degrading enzyme, the gene, the recombinant vector, the strain or the additive in degrading zearalenone, preferably an application in degrading zearalenone in grain oil and/or feed.
Preferably, the zearalenone is present in the grain oil and/or feed in an amount of at least 1ppm, preferably at least 10ppm, more preferably at least 20ppm, even more preferably at least 50ppm, most preferably at least 200 ppm.
In a ninth aspect, the present invention also provides a method for degrading zearalenone, the method comprising: contacting an enzymatic agent with a sample to be treated under conditions of an enzymatic degradation reaction; wherein, when the enzyme agent contains the zearalenone degrading enzyme or the additive, the conditions of the enzymatic degradation reaction include: the temperature is 20-55 ℃, preferably 30-40 ℃; the pH value is 3-9; preferably 6-8.
In the present invention, the sample to be treated may be grain oil and/or feed.
Preferably, the zearalenone is present in the grain and/or feed in an amount of at least 1ppm, preferably at least 10ppm, more preferably at least 20ppm, even more preferably at least 50ppm, most preferably at least 200 ppm.
In the present invention, when the enzymatic agent contains SEQ ID NO: 1, the conditions of the enzymatic degradation reaction include: the temperature is 40-55 deg.C, and pH is 3-9.
In the present invention, the zearalenone degrading enzyme is used in an amount of 1 to 10ppm, preferably 2 to 8ppm, more preferably 4 to 6ppm, based on the total weight of the sample to be treated. In the present invention, "ppm" means "μ g/mL" when the sample to be treated is a liquid; when the sample to be treated is a solid, "ppm" means "μ g/g".
In the present invention, the time of the enzymatic degradation reaction may be 0.5 to 48 hours, preferably 1 to 24 hours, more preferably 1 to 12 hours, and still more preferably 1 to 6 hours.
In the present invention, the form of the enzyme agent is not particularly limited as long as it is ensured that zearalenone in the sample to be treated can be degraded after the enzyme agent is added, and for example, the form of the enzyme agent may be liquid or may be dried powder after freeze-drying.
The present invention will be described in detail below by way of examples.
In the following examples and comparative examples, ZEN standards were purchased from Sigma company; "Room temperature" means "25 ℃";
preparing an LB liquid culture medium: 5g of beef extract, 10g of peptone and 5g of sodium chloride, supplementing water to 1000mL, sterilizing at 115 ℃ for 20min for later use, and keeping the pH value at 7;
the ZEN content was determined according to the method of GB/T28716-,
the degradation rate (%) of ZEN is (mass of ZEN in sample before reaction-mass of ZEN in sample after reaction)/mass of ZEN in sample before reaction × 100%.
Example 1
This example illustrates zearalenone degrading enzymes, methods of preparation and uses thereof provided by the present invention.
(1) Obtaining of genes
The following nucleotide sequences were synthesized by artificial chemical synthesis (consignment of Biotechnology, Inc., Dalian, the same below): in the amino acid sequence as set forth in SEQ ID NO: 7, protective bases CGC and NdeI enzyme cutting sites are added at the 5 'end of the nucleotide sequence shown in the specification, and protective bases CCG and XhoI enzyme cutting sites are added at the 3' end of the nucleotide sequence shown in the specification so as to obtain a corresponding gene fragment.
(2) Construction of recombinant plasmid
The gene fragment obtained in step (1) and the PET30a plasmid (with His tag, available from Invitrogen, usa) were double-digested with restriction enzymes NdeI and XhoI (available from NEB) respectively, and digested in a water bath at 37 ℃ for 4h, with the following digestion system (50 μ L):
and (3) carrying out agarose gel electrophoresis on the enzyme digestion product, and purifying and recovering the target fragment (plasmid enzyme digestion fragment and gene enzyme digestion fragment respectively). 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:
and then carrying out PCR verification on the obtained recombinant plasmid, and carrying out sequencing verification on a PCR product. The upstream and downstream primers used in PCR are shown in SEQ ID NO: 11 and SEQ ID NO: 12, the PCR amplification conditions were: 1min at 98 ℃; circulating for 30 times at 98 deg.C for 15s and 68 deg.C for 7 min; 3min at 72 ℃; keeping at 4 ℃.
The PCR products were then subjected to gel electrophoresis, which showed the resulting fragments to be of the expected size, and sequencing showed the PCR products to contain SEQ ID NO: 7, which indicates that the recombinant plasmid is successfully constructed.
(3) Obtaining of recombinant strains
BL21(DE3) PlysS E.coli strain (purchased from Beijing Huayuyo Biotech Co., Ltd., cat. No. NRR01330, the same below) was electrotransformed 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 chloramphenicol (Cam) for screening, inverted cultured at 37 ℃ for 2 days, and positive colonies were picked up and inoculated in an LB liquid medium to obtain a recombinant strain.
(4) Preparation of enzymes
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, collecting bacterial suspension, ice-cooling for 30min, centrifuging at 4 deg.C and 10000rpm for 20min, centrifuging to collect thallus, and adding phosphate buffer solution (PBS, 135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4，8mM K2HPO4pH 7.2), ultrasonically crushing, centrifuging at 10000rpm for 20min at 4 ℃, and collecting 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 (pH 7.4, 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 (pH 7.4, 50mM NaCl containing 10mM imidazole) to elute unadsorbed or adsorbed non-specific hybrid protein; the target protein was collected by elution with a buffer (pH 7.4, 50mM NaCl, 500mM imidazole) to obtain a purified enzyme solution.
(5) Enzyme Activity detection
Putting 980 mu L of the enzyme solution obtained in the step (4) into a 1.5mL centrifuge tube, adding 20 mu L of ZEN standard solution, and uniformly mixing to obtain a mixed solution, wherein the final concentration of the enzyme in the mixed solution is 5ppm, and the final concentration of the ZEN standard solution is 50 ppm.
Influence of reaction time on enzyme Activity
And reacting the mixed solution at 37 ℃ and pH 7, and detecting the residue of ZEN by high performance liquid chromatography by taking 20 mu L of a reacted sample at the time of reacting for 30min, 1h, 2h, 4h and 6h respectively. The results are shown in Table 1.
The results in table 1 show that the degradation rate of ZEN was 90% or more in the reaction time of 1 hour. Therefore, 1h was used as the reaction time in the following experiment.
Influence of temperature on enzyme Activity
After 6 parts of the above-mentioned mixed solution were reacted at 20 ℃, 30 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃ respectively for 1 hour at a pH of 7, 20. mu.L of the reacted sample was subjected to HPLC to detect the residue of ZEN. The results are shown in Table 2.
Influence of pH on enzyme Activity
7 parts of the mixed solution are respectively reacted for 1h at 37 ℃ and under the conditions that the pH value is 2, 3, 4, 5, 6, 8 and 9, and 20 mu L of a sample after the reaction is taken to carry out high performance liquid chromatography to detect the residue of ZEN. The results are shown in Table 3.
Example 2
This example illustrates zearalenone degrading enzymes, methods of preparation and uses thereof provided by the present invention.
A zearalenone degrading enzyme was prepared according to the method of example 1, except that the zearalenone degrading enzyme synthesized by an artificial chemical synthesis method as shown in SEQ ID NO: 8 instead of the nucleotide sequence shown in SEQ ID NO in step (1) of example 1: 7 to obtain a gene fragment.
The results of the effect of the reaction time on the enzyme activity are shown in Table 1, the results of the effect of the temperature on the enzyme activity are shown in Table 2, and the results of the effect of the pH on the enzyme activity are shown in Table 3.
Example 3
This example illustrates zearalenone degrading enzymes, methods of preparation and uses thereof provided by the present invention.
A zearalenone degrading enzyme was prepared according to the method of example 1, except that the zearalenone degrading enzyme synthesized by an artificial chemical synthesis method as shown in SEQ ID NO: 9 instead of SEQ ID NO: 7 to obtain a gene fragment.
The results of the effect of the reaction time on the enzyme activity are shown in Table 1, the results of the effect of the temperature on the enzyme activity are shown in Table 2, and the results of the effect of the pH on the enzyme activity are shown in Table 3.
Example 4
This example illustrates zearalenone degrading enzymes, methods of preparation and uses thereof provided by the present invention.
A zearalenone degrading enzyme was prepared according to the method of example 1, except that the zearalenone degrading enzyme synthesized by an artificial chemical synthesis method as shown in SEQ ID NO: 10 instead of SEQ ID NO: 7 to obtain a gene fragment.
The results of the effect of the reaction time on the enzyme activity are shown in Table 1, the results of the effect of the temperature on the enzyme activity are shown in Table 2, and the results of the effect of the pH on the enzyme activity are shown in Table 3.
Example 5
This example illustrates zearalenone degrading enzymes, methods of preparation and uses thereof provided by the present invention.
A zearalenone degrading enzyme was prepared according to the method of example 1, except that the zearalenone degrading enzyme synthesized by an artificial chemical synthesis method as shown in SEQ ID NO: 6 instead of the nucleotide sequence shown in SEQ ID NO in step (1) of example 1: 7 to obtain a gene fragment.
The results of the effect of the reaction time on the enzyme activity are shown in Table 1, the results of the effect of the temperature on the enzyme activity are shown in Table 2, and the results of the effect of the pH on the enzyme activity are shown in Table 3.
Example 6
This example illustrates zearalenone degrading enzymes, methods of preparation and uses thereof provided by the present invention.
A zearalenone degrading enzyme was prepared according to the method of example 1, except that the zearalenone degrading enzyme synthesized by an artificial chemical synthesis method as shown in SEQ ID NO: 13 instead of SEQ ID NO: 7 to obtain a gene fragment.
The results of the effect of the reaction time on the enzyme activity are shown in Table 1, the results of the effect of the temperature on the enzyme activity are shown in Table 2, and the results of the effect of the pH on the enzyme activity are shown in Table 3.
Example 7
This example illustrates zearalenone degrading enzymes, methods of preparation and uses thereof provided by the present invention.
A zearalenone degrading enzyme was prepared according to the method of example 1, except that SEQ ID NO: 7 was cloned into a yeast expression vector pYES2 (purchased from ThermoFisher Scientific, cat # V82520), and the resulting recombinant vector was transformed into INVSc1 s.cerevisiae (purchased from ThermoFisher Scientific, cat # C81000) to obtain a recombinant strain.
The results of the effect of the reaction time on the enzyme activity are shown in Table 1, the results of the effect of the temperature on the enzyme activity are shown in Table 2, and the results of the effect of the pH on the enzyme activity are shown in Table 3.
Example 8
This example illustrates zearalenone degrading enzymes, methods of preparation and uses thereof provided by the present invention.
Zearalenone degrading enzyme was prepared according to the method of example 1, except that the strong promoter P43, SEQ ID NO: 7 was cloned into the vector pHY300PLK (purchased from NTCC type culture Collection-Biovector plasmid vector species cell Gene Collection), and the resulting recombinant vector was transformed into Bacillus licheniformis ATCC14580 (Bacillus licheniformis ATCC14580 purchased from ATCC (American type culture Collection)), to obtain a recombinant strain.
The results of the effect of the reaction time on the enzyme activity are shown in Table 1, the results of the effect of the temperature on the enzyme activity are shown in Table 2, and the results of the effect of the pH on the enzyme activity are shown in Table 3.
Example 9
A zearalenone degrading enzyme was prepared according to the method of example 1, except that SEQ ID NO: 7 to the expression vector pHT43, and then transforming the obtained recombinant vector into Bacillus subtilis WB800N (both the expression vector pHT43 and the Bacillus subtilis WB800N are purchased from NTCC type culture Collection-Biovector plasmid vector species cell gene Collection) to obtain a recombinant strain.
The results of the effect of the reaction time on the enzyme activity are shown in Table 1, the results of the effect of the temperature on the enzyme activity are shown in Table 2, and the results of the effect of the pH on the enzyme activity are shown in Table 3.
Comparative example 1
A zearalenone degrading enzyme was prepared according to the method of example 1, except that the plasmid of the empty vector PET30a into which no gene was transferred was used instead of the recombinant plasmid used in example 1.
TABLE 1
TABLE 2
TABLE 3
Test example 1
The crude enzyme solutions obtained in examples 1-6 were mixed with corn flour contaminated with 50ppm ZEN (pH 6) and reacted at 40 ℃ for 6 hours with a final enzyme concentration of 5ppm by measuring the enzyme content in the crude enzyme solution based on the dry weight of the corn flour, and the ZEN degradation rates were measured to be 95.6%, 96.3%, 96.1%, 97.2%, 91.5% and 92.3%, respectively. However, the addition of the crude enzyme solution has a certain effect on the palatability of the cereal (i.e., the palatability of the corn flour changes after the crude enzyme solution is mixed in).
In addition, the crude enzyme solutions obtained in examples 1-6 were mixed with corn flour contaminated with 20ppm ZEN (pH 6), and the final enzyme concentration in the mixture was controlled to 5ppm by measuring the enzyme content in the crude enzyme solution based on the dry weight of the corn flour, and reacted at 40 ℃ for 6 hours, and the ZEN degradation rates were measured to be 97.5%, 97.9%, 98.1%, 99.2%, 94.6% and 95.4%, respectively. However, the addition of the crude enzyme solution has a certain effect on the palatability of the cereal (i.e., the palatability of the corn flour changes after the crude enzyme solution is mixed in).
In addition, the crude enzyme solutions obtained in examples 1-6 were mixed with corn flour contaminated with 10ppm of ZEN (pH 6), and the final enzyme concentration in the mixture was controlled to 5ppm by measuring the enzyme content in the crude enzyme solution based on the dry weight of the corn flour, and reacted at 40 ℃ for 6 hours, whereby the ZEN degradation rates were measured to be 100%, 97.6% and 98.4%, respectively. However, the addition of the crude enzyme solution has a certain effect on the palatability of the cereal (i.e., the palatability of the corn flour changes after the crude enzyme solution is mixed in).
Test example 2
The procedure of test example 1 was followed, except that the crude enzyme solutions obtained in examples 7 to 9 were used in place of the crude enzyme solution used in test example 1, respectively. The degradation rates of ZEN were determined to be 93.8%, 94.9% and 95.1%, respectively. Moreover, the addition of the above crude enzyme solution did not have any effect on the palatability of the grain.
Compared with the results of the comparative examples 1 to 3, the zearalenone degrading enzyme provided by the invention can degrade ZEN efficiently and rapidly, and has a good industrial application prospect. In addition, the zearalenone degrading enzyme provided by the invention also has the characteristics of acid and alkali resistance and high temperature resistance, so that the application range of the zearalenone degrading enzyme is further expanded.
In addition, as can be seen from a comparison of the results of test example 1 and test example 2, test example 2 used saccharomyces cerevisiae, bacillus licheniformis, and bacillus subtilis as host strains, respectively, and added zearalenone degrading enzyme produced therefrom or fermentation product thereof to grain oil and/or feed, the effect on palatability of grain oil and/or feed was small; in test example 1, the use of E.coli as a host strain, and the addition of the fermentation product produced by it to grain and/or feed, affected the palatability of the grain and/or feed. Therefore, the zearalenone degrading enzyme prepared by the method provided by the preferred embodiment of the invention has wider application prospect, and is particularly applied to grain, oil and/or feed.
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> zearalenone degrading enzyme, gene, preparation method and application thereof, and zearalenone degrading enzyme
Method (2)
<130> I40381COF
<160> 13
<170> PatentIn version 3.3
<210> 1
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<213> Clonostachys rosea
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Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
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Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Lys Tyr Val
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Val Glu Thr Thr Gln Lys His Leu
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Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
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Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
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Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
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Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
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Ser Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
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Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
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<210> 6
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<212> DNA
<213> Artificial Sequence
<220>
<223> The sequence is synthesized
<400> 6
atgcgcactc gcagcacaat ctcgaccccg aatggcatca cctggtacta tgagcaggag 60
ggtactggac ccgacgttgt cctcgtcccc gatggcctcg gagaatgcca gatgtttgac 120
agctccgtgt cgcaaattgc tgcccaaggc tttcgggtca ccacgtttga catgcccgga 180
atgtcccggt ctgcgaaggc accacccgag acctacactg aggtcacggc ccagaagctg 240
gcttcctatg tcatctccat cctggatgct cttgacatca agcacgctac tgtctggggc 300
tgcagctcag gagcttccac cgtcgtggcg ctgttgctcg gttaccccga caggatacgc 360
aacgccatgt gccacgaact gccaacaaag ctactggacc acctttcaaa caccgctgtg 420
ctcgaagacg aggaaatctc aaagatcctg gccaatgtaa tgttgaacga cgtgtctgga 480
ggctcggagg cgtggcaagc catgggggac gaggtgcacg cgagactgca caagaactac 540
ccggtttggg ctcgaggata ccctcgcact attcctccct cagctccggt taaggatctg 600
gaggctctgc gtgggaagcc cctggactgg actgtcggcg ctgcgacacc aaccgagtct 660
ttctttgaca acattgttac cgctaccaag gctggtgtca acattgggtt gcttccaggg 720
atgcatttcc cttatgtttc ccacccggac gttttcgcta aatatgttgt ggaaactacg 780
cagaagcatc tttga 795
<210> 7
<211> 795
<212> DNA
<213> Artificial Sequence
<220>
<223> The sequence is synthesized
<400> 7
atgcgcactc gcagcacaat ctcgaccccg aatggcatca cctggtacta tgagcaggag 60
ggtactggac ccgacgttgt cctcgtcccc gatggcctcg gagaatgcca gatgtttgac 120
agctccgtgt cgcaaattgc tgcccaaggc tttcgggtca ccacgtttga catgcccgga 180
atgtcccggt ctgcgaaggc accacccgag acctacactg aggtcacggc ccagaagctg 240
gcttcctatg tcatctccat cctggatgct cttgacatca agcacgctac tgtctggggc 300
tgcagctcag gagcttccac cgtcgtggcg ctgttgctcg gttaccccga caggatacgc 360
aacgccatgt gccacgaact gccaacaaag ctactggacc acctttcaaa caccgctgtg 420
ctcgaagacg aggaaatctc aaagatcctg gccaatgtaa tgttgaacga cgtgtctgga 480
ggctcggagg cgtggcaagc catgggggac gaggtgcacg cgagactgca caagaactac 540
ccggtttggg ctcgaggata ccctcgcact attcctccct cagctccggt taaggatctg 600
gaggctctgc gtgggaagcc cctggactgg actgtcggcg ctgcgacacc aaccgagtct 660
ttctttgaca acattgttac cgctaccaag gctggtgtca acattgggtt gcttccaggg 720
atgcatttcc cttatgtttc ccacccggac gttttcgcta aatatgttgt ggaaactacg 780
cagaagtacc tttga 795
<210> 8
<211> 795
<212> DNA
<213> Artificial Sequence
<220>
<223> The sequence is synthesized
<400> 8
atgcgcactc gcagcacaat ctcgaccccg aatggcatca cctggtacta tgagcaggag 60
ggtactggac ccgacgttgt cctcgtcccc gatggcctcg gagaatgcca gatgtttgac 120
agctccgtgt cgcaaattgc tgcccaaggc tttcgggtca ccacgtttga catgcccgga 180
atgtcccggt ctgcgaaggc accacccgag acctacactg aggtcacggc ccagaagctg 240
gcttcctatg tcatctccat cctggatgct cttgacatca agcacgctac tgtctggggc 300
tgcagctcag gagcttccac cgtcgtggcg ctgttgctcg gttaccccga caggatacgc 360
aacgccatgt gccacgaact gccaacaaag ctactggacc acctttcaaa caccgctgtg 420
ctcgaagacg aggaaatctc aaagatcctg gccaatgtaa tgttgaacga cgtgtctgga 480
ggctcggagg cgtggcaagc catgggggac gaggtgcacg cgagactgca caagaactac 540
ccggtttggg ctcgaggata ccctcgcact attcctccct cagctccggt taaggatctg 600
gaggctctgc gtgggaagcc cctggactgg actgtcggcg ctgcgacacc aaccgagtct 660
ttctttgaca acattgttac cgctaccaag gctggtgtca acattgggtt gcttccaggg 720
atgcatttcc cttatgtttc ccacccggac gttttcgcta aatatgttgt ggaatttacg 780
cagaagtacc tttga 795
<210> 9
<211> 795
<212> DNA
<213> Artificial Sequence
<220>
<223> The sequence is synthesized
<400> 9
atgcgcactc gcagcacaat ctcgaccccg aatggcatca cctggtacta tgagcaggag 60
ggtactggac ccgacgttgt cctcgtcccc gatggcctcg gagaatgcca gatgtttgac 120
cgctccgtgt cgcaaattgc tgcccaaggc tttcgggtca ccacgtttga catgcccgga 180
atgtcccggt ctgcgaaggc accacccgag acctacactg aggtcacggc ccagaagctg 240
gcttcctatg tcatctccat cctggatgct cttgacatca agcacgctac tgtctggggc 300
tgcagctcag gagcttccac cgtcgtggcg ctgttgctcg gttaccccga caggatatgc 360
aacgccatgt gccacgaact gccaacaaag ctactggacc acctttcaaa caccgctgtg 420
ctcgaagacg aggaaatctc aaagatcctg gccaatgtaa tgttgaacga cgtgtctgga 480
ggctcggagg cgtggcaagc catgggggac gaggtgcacg cgagactgca caagaactac 540
ccggtttggg ctcgaggata ccctcgcact attcctccct cagctccggt taaggatctg 600
gaggctctgc gtgggaagcc cctggactgg actgtcggcg ctgcgacacc aaccgagtct 660
ttctttgaca acattgttac cgctaccaag gctggtgtca acattgggtt gcttccaggg 720
atgcatttcc cttatgtttc ccacccggac gttttcgcta aatatgttgt ggaaactacg 780
cagaagtacc tttga 795
<210> 10
<211> 795
<212> DNA
<213> Artificial Sequence
<220>
<223> The sequence is synthesized
<400> 10
atgcgcatcc gcagcacaat ctcgaccccg aatggcatca cctggtacta tgagcaggag 60
ggtactggac ccgacgttgt cctcgtcccc gatggcctcg gagaatgcca gatgtttgac 120
agctccgtgt cgcaaattgc tgctagcggc tttcgggtca ccacgtttga catgcccgga 180
atgtcccggt ctgcgaaggc accacccgag acctacactg aggtcacggc ccagaagctg 240
gcttcctatg tcatctccat cctggatgct cttgacatca agcacgctac tgtctggggc 300
tgcagctcag gagcttccac cgtcgtggcg ctgttgctcg gttaccccga caggatacgc 360
aacgccatgt gccacgaact gccaacaaag ctactggacc acctttcaaa caccgctgtg 420
ctcgaagacg aggaaatctc aaagatcctg gccaatgtaa tgttgaacga cgtgtctgga 480
ggctcggagg cgtggcaagc catgggggac gaggtgcacg cgagactgca caagaactac 540
ccggtttggg ctcgaggata ccctcgcact attcctccct cagctccggt taaggatctg 600
gaggctctgc gtgggaagcc cctggactgg actgtcggcg ctgcgacacc aaccgagtct 660
ttctttgaca acattgttac cgctaccaag gctggtgtca acattgggtt gcttccaggg 720
atgcatttcc cttatgtttc ccacccggac gttttcgcta aatatgttgt ggaaactacg 780
cagaagtacc tttga 795
<210> 11
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> The sequence is synthesized
<400> 11
cggagaatgc cagatgtt 18
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> The sequence is synthesized
<400> 12
cagacagtag cgtgcttgat 20
<210> 13
<211> 795
<212> DNA
<213> Artificial Sequence
<220>
<223> The sequence is synthesized
<400> 13
atgcgcactc gcagcacaat ctcgaccccg aatggcatca cctggtacta tgagcaggag 60
ggtactggac ccgacgttgt cctcgtcccc gatggcctcg gagaatgcca gatgtttgac 120
agctccgtgt cgcaaattgc tgcccaaggc tttcgggtca ccacgtttga catgcccgga 180
atgtcccggt ctgcgaaggc accacccgag acctacactg aggtcacggc ccagaagctg 240
gcttcctatg tcatctccat cctggatgct cttgacatca agcacgctac tgtctggggc 300
tgcagctcag gagcttccac cgtcgtggcg ctgttgctcg gttaccccga caggatatgc 360
aacgccatgt gccacgaact gccaacaaag ctactggacc acctttcaaa caccgctgtg 420
ctcgaagacg aggaaatctc aaagatcctg gccaatgtaa tgttgaacga cgtgtctgga 480
ggctcggagg cgtggcaagc catgggggac gaggtgcacg cgagactgca caagaactac 540
ccggtttggg ctcgaggata ccctcgcact attcctccct cagctccggt taaggatctg 600
gaggctctgc gtgggaagcc cctggactgg actgtcggcg ctgcgacacc aaccgagtct 660
ttctttgaca acattgttac cgctaccaag gctggtgtca acattgggtt gcttccaggg 720
atgcatttcc cttatgtttc ccacccggac gttttcgcta aatatgttgt ggaaactacg 780
cagaagtacc tttga 795
Claims (30)
1. A zearalenone degrading enzyme characterized in that the amino acid sequence of the zearalenone degrading enzyme is an amino acid sequence represented by the following (a) or (b):
(a) SEQ ID NO: 2;
(b) SEQ ID NO: 3. SEQ ID NO: 4 or SEQ ID NO: 5.
2. A gene encoding a zearalenone degrading enzyme characterized in that the nucleotide sequence of the gene is a nucleotide sequence encoding the zearalenone degrading enzyme of claim 1.
3. The gene of claim 2, wherein the nucleotide sequence of the gene is SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 9, or a nucleotide sequence shown in the specification.
4. A recombinant vector comprising the gene of claim 2.
5. The recombinant vector according to claim 4, wherein the recombinant vector contains the gene of claim 3.
6. A strain comprising the gene of claim 2.
7. The strain of claim 6, wherein the strain contains the gene of claim 3.
8. The strain of claim 6, wherein the strain contains the recombinant vector of claim 4.
9. The strain of any one of claims 6 to 8, wherein the strain is Bacillus licheniformis (Bacillus licheniformis)Bacillus lincheniformis) Bacillus subtilis preparation (B)Bacillus subtilis) And Saccharomyces cerevisiae (Saccharomyces cerevisiae) At least one of (1).
10. An additive comprising the zearalenone degrading enzyme of claim 1.
11. A grain oil or feed, which comprises the zearalenone degrading enzyme of claim 1.
12. A method of expressing a zearalenone degrading enzyme, the method comprising:
introducing the recombinant vector of claim 4 into a host, and inducing expression of a gene encoding a zearalenone degrading enzyme in said host.
13. A method of expressing a zearalenone degrading enzyme, the method comprising: inducing the strain of any one of claims 6-9 to express a zearalenone degrading enzyme.
14. The method of claim 12 or 13, wherein the host is at least one of bacillus licheniformis, bacillus subtilis, and saccharomyces cerevisiae.
15. Use of the zearalenone degrading enzyme of claim 1, the gene of claim 3, the recombinant vector of claim 4 or the strain of claim 6 for degrading zearalenone.
16. Use of the additive of claim 10 for the degradation of zearalenone.
17. Use of the zearalenone degrading enzyme of claim 1, the gene of claim 3, the recombinant vector of claim 4 or the strain of claim 6 for degrading zearalenone in grain oil and/or feed.
18. Use of the additive according to claim 10 for degrading zearalenone in grain, oil and/or feed.
19. Use according to claim 17 or 18, wherein the zearalenone is present in an amount of at least 1ppm in the grain and/or feed.
20. The use according to claim 19, wherein the zearalenone is present in an amount of at least 10ppm in the grain and/or feed.
21. The use according to claim 20, wherein the zearalenone is present in an amount of at least 20ppm in the grain and/or feed.
22. The use according to claim 21, wherein the zearalenone is present in the grain and/or feed in an amount of at least 50 ppm.
23. A method of degrading zearalenone, the method comprising: contacting an enzymatic agent with a sample to be treated under conditions of an enzymatic degradation reaction;
the enzymatic agent comprising the zearalenone degrading enzyme of claim 1, wherein the conditions of the enzymatic degradation reaction include: the temperature is 20-55 deg.C, and pH is 3-9.
24. The method of claim 23, wherein the conditions of the enzymatic degradation reaction comprise: the temperature is 30-40 ℃; the pH value is 3-9.
25. The method of claim 24, wherein the conditions of the enzymatic degradation reaction comprise: the temperature is 30-40 ℃; the pH value is 6-8.
26. The method according to any one of claims 23 to 25, wherein the sample to be treated is a grain oil and/or a feed.
27. The method of claim 26, wherein the zearalenone is present in an amount of at least 1ppm in the sample to be treated.
28. The method of claim 27, wherein the zearalenone is present in an amount of at least 10ppm in the sample to be treated.
29. The method of claim 28, wherein the zearalenone is present in an amount of at least 20ppm in the sample to be treated.
30. The method of claim 29, wherein the zearalenone is present in an amount of at least 50ppm in the sample to be treated.
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| CN110511914B (en) * | 2019-09-02 | 2021-07-20 | 中国农业科学院北京畜牧兽医研究所 | Zearalenone hydrolase ZH9572 as well as encoding gene and application thereof |
| CN110527677B (en) * | 2019-09-02 | 2021-07-20 | 中国农业科学院北京畜牧兽医研究所 | Zearalenone hydrolase mutant ZHDM2 and coding gene and application thereof |
| CN110819608B (en) * | 2019-10-29 | 2022-03-15 | 湖北大学 | Hydrolysis method of zearalenone and derivatives thereof |
| CN112430611A (en) * | 2020-11-30 | 2021-03-02 | 华南理工大学 | Optimized zearalenone degrading enzyme ZHD-P encoding gene, recombinant thallus, surface display system and application |
| CN112961846B (en) * | 2021-02-05 | 2022-03-01 | 青岛根源生物技术集团有限公司 | Zearalenone degrading enzyme mutant with improved enzyme activity and coding gene and application thereof |
| CN113355304B (en) * | 2021-05-19 | 2022-02-18 | 中国农业科学院生物技术研究所 | Protein CpoC with zearalenone degrading enzyme activity and gene and application thereof |
| CN113755468B (en) * | 2021-09-08 | 2023-06-30 | 暨南大学 | Zearalenone hydrolase with improved resistance to trypsin |
| CN117987332B (en) * | 2024-04-07 | 2024-06-14 | 广东省农业科学院农业质量标准与监测技术研究所 | Lactobacillus paracasei and application thereof |
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