CN108251385A - Ochratoxin degrading enzyme, encoding gene, recombinant vector, cell, additive and its application - Google Patents
Ochratoxin degrading enzyme, encoding gene, recombinant vector, cell, additive and its application Download PDFInfo
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Abstract
The present invention relates to biotechnologies, and in particular to ochratoxin degrading enzyme, encoding gene, recombinant vector, cell, additive and its application are more particularly to ochratoxin degrading enzyme, which has SEQ ID NO：Sequence shown in 2 or its mutant, the encoding gene of the enzyme, carrier and cell containing the encoding gene, additive containing the enzyme and/or cell and/or its tunning, the application and the method for degradation ochratoxin/or other mycotoxins of the enzyme enzyme, the encoding gene, carrier, cell or additive in degradation ochratoxin and/or other mycotoxins.The present invention has the following advantages：It is environmental-friendly, it can efficiently temporarily degrade to ochratoxin, and do not generate any harmful side product.The enzyme can tolerate up to 80 DEG C of high-temperature catalytic, and the requirement to pH value is relatively low, and stability is good, additionally it is possible to fumonisin and a degree of degradation of T2 toxin.
Description
Technical field
The present invention relates to biotechnologies, and in particular, to a kind of ochratoxin degrading enzyme and its encoding gene contain
There are the recombinant vector and recombinant cell of the encoding gene, contain the degrading enzyme and/or the recombinant cell and/or its fermentation production
The additive of object, the degrading enzyme, the encoding gene of the degrading enzyme, the recombinant vector, the recombinant cell or described adds
Add application and a kind of degradation ochratoxin/of the agent in degradation ochratoxin and/or other mycotoxins or other are true
The method of verticillium toxin.
Background technology
Mycotoxin is a kind of secondary metabolites that Toxigenic fungi generates in the process in harm, mainly includes aspergillus flavus poison
Element, deoxynivalenol (also known as vomitoxin, DON), zearalenone (ZEN) and fumonisin (FUM), reddish brown song
Mould toxin and T-2 toxin etc..And ochratoxin therein is that another causes world's extensive concern after aflatoxin
Mycotoxin.It is a kind of important, contaminated food products mycotoxin.
Ochratoxin is mould by Aspergillus ochraceus (Aspergillusochraceus) and pure green cyan
(Penicilliumviridicatum) a kind of mould nephrotoxin generated, can be divided into A and B two types, the toxicity of A is larger.
Aspergillus ochraceus can generate the ochratoxin with toxic action concentration in a low temperature of 4 DEG C.Animal takes in 1ppm body weight doses
Ochratoxin A can be lethal at 5-6 days.Common lesion is tubular epithelial injury and the necrosis of intestinal lymphoid body of gland.Feeding
The daily ration of the ochratoxin of concentration containing 1ppm can cause animal polydipsia, frequent micturition, growth retardation and efficiency of feed utilization to reduce for 3 months；
Daily ration several weeks of feeding content down to 200ppb can detect injury of kidney.Other clinical symptoms also have diarrhea, apocleisis and dehydration.
Sometimes clinical symptoms unobvious, and be in the area of local epidemic disease in ochratoxin poisoning, animal is only considerable when butchering
The lesion observed is that kidney is pale, hard.
Marquardt can cause livestock and poultry to be poisoned when (1992) investigation shows that OTA contents are in 0.3-16mg/kg in feed,
The death rate is made to rise 2%-58%.Madsen etc. (1982) is reported, continuously feeds the feed of the g/kg of μ containing OTA200 4 months, to pig
Influence it is little, and when OTA contents are more than 1400 μ g/kg, significantly reduce the feed intake and the speed of growth of pig, amount of drinking water increases
Add.Huff etc. (1974) reports, the continuous feed for feeding the OTA containing 0.5-1.0mg/kg 3 weeks, on the weightenings of Broiler chicks without influence,
And the feed of the OTA containing 0.5mg/kg is continuously fed 6 weeks, can reduce the egg laying performance and feed conversion rate of laying hen.
Hult etc. (1976) reports that the allowance of OTA must not exceed 200 μ respectively in auspicious soil regulation pig and fowl mixed feed
G/kg and 1000 μ g/kg.Also related regulations are being worked out in the U.S..Other countries are there is not yet the allowance regulation in relation to OTA.It is domestic
GB 2761-2011 provide that the allowance of OTA in cereal, beans and its product must not exceed 5 μ g/kg.
At present for the removing of mycotoxin, at present both at home and abroad more universal method mainly have physical removal and absorption,
Chemical treatment etc..Adsorbent has also largely adsorbed the trace nutrient in feed and food while absorbing toxin, is sticked
Toxin after soil absorption, can not be decomposed, can cause secondary pollution.
Toxin efficiently can be converted into non-toxic products, Environmental Safety by biodegradation.Have become current mycotoxin to cut
Subtract treatment technology approach most promising in technology, it is not only safe and environment-friendly, efficient, but also utilize modern biotechnology
It conducts a research and meets very much the development trend of current China's energy-saving and emission-reduction.It is current to pollute grain as raw material hair by the use of mycotoxin
Ferment production ethyl alcohol is one of most important means, but by further dense in a large amount of by-product vinasse for being obtained in fermenting and producing of toxin
Contracting, substantially exceeds national limit standard and cannot utilize.
And at present for the method removal efficiency of the biological eliminating of ochratoxin is relatively low or degradation time
Long or thermal stability and ph stability are poor, therefore being capable of energy conservation and environmental protection and efficient degradation there is an urgent need for developing one kind
The method of ochratoxin.
Invention content
The purpose of the invention is to overcome the as above defect of the prior art, it is reddish brown to provide a kind of degradation that can efficiently in short-term
The enzyme of aspertoxin, and the enzyme has the thermal stability and resistance to acid and alkali improved.
To achieve these goals, in a first aspect, the present invention provides a kind of ochratoxin degrading enzyme, wherein, the drop
Solving enzyme has the amino acid sequence shown in (a) and/or (b) as follows：
(a)SEQ ID NO：Amino acid sequence shown in 2；
(b)SEQ ID NO：One in amino acid sequence shown in 2 in the 11st, 89,179 and 239 amino acids residues
Or several amino acid sequences still after replacing, lacking or add with ochratoxin degrading enzymatic activity.
Second aspect, the present invention also provides a kind of encoding gene of ochratoxin degrading enzyme, wherein, which has
Encode the nucleotide sequence of ochratoxin degrading enzyme as described above.
The third aspect, the present invention also provides a kind of recombinant vector, wherein, which contains base as described above
Cause.
Fourth aspect, the present invention also provides a kind of recombinant cell, wherein, which contains gene as described above
Or recombinant vector as described above.
5th aspect, the present invention also provides a kind of additive, wherein, which contains degrading enzyme as described above
And/or contain recombinant cell as described above and/or its tunning.
6th aspect, the present invention also provides degrading enzyme as described above, gene as described above, recombinations as described above
Carrier, recombinant cell as described above and/or its tunning or additive as described above degradation ochratoxin and/
Or the application in other mycotoxins.
7th aspect, the present invention also provides a kind of method of degrade ochratoxin/or other mycotoxins, wherein,
This method includes：Under conditions of enzyme degradation reaction, by degrading enzyme as described above, recombinant cell as described above and/or its
Tunning or additive as described above are contacted with pending sample.
The present invention has the advantage that：It is environmental-friendly, it efficiently can temporarily degrade to ochratoxin, and not
Generate any harmful side product.Ochratoxin degrading enzyme provided by the invention can tolerate up to 80 DEG C of high temperature, and to pH value
Requirement it is relatively low, have stability it is good.In addition, the ochratoxin degrading enzyme of the present invention also is able to fumonisin and T2 poison
The a degree of degradation of element.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Specific embodiment
The specific embodiment of the present invention is described in detail below.It is it should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood to comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It between the endpoint value of a range and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
In a first aspect, the present invention provides a kind of ochratoxin degrading enzyme, wherein, the degrading enzyme have following (a) and/
Or the amino acid sequence shown in (b)：
(a)SEQ ID NO：Amino acid sequence shown in 2；
(b)SEQ ID NO：One in amino acid sequence shown in 2 in the 11st, 89,179 and 239 amino acids residues
Or several amino acid sequences still after replacing, lacking or add with degrading enzymatic activity.
20 kinds of amino acid residues of constitutive protein matter, four classes are segmented into according to pendant polar：1st, nonpolar amino acid：
Alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine
(Phe), tryptophan (Trp) and proline (Pro)；2nd, the uncharged amino acid of polarity：Glycine (Gly), serine
(Ser), threonine (Thr), cysteine (Cys), aspartic acid (Asn), glutamine (Gln) and tyrosine (Tyr)；3rd, band
The amino acid of positive charge：Arginine (Arg), lysine (Lys) and histidine (His)；4th, negatively charged amino acid：Asparagus fern ammonia
Sour (Asp) and glutamic acid (Glu) (referring to " biochemistry " (second edition) first volume, Shen Tong, Wang Jingyan, the 82-83 pages, high religion
Educate publishing house, December nineteen ninety).If it happens the amino acid residue substitution of a classification is belonged in protein, such as is taken by Arg
Replace Ile for Lys or by Leu, effect of the residue played in protein domain (for example provides positive charge or formed and dredged
The effect of water bag structure) do not change, therefore the stereochemical structure of protein can't be had an impact, therefore still can be real
The function of existing albumen.The amino acid residue substitution for belonging to a classification can be happened at any one amino acid of above-mentioned enzyme
On resi-dues.
As previously mentioned, enzyme provided by the invention can also be modified or is mutated, derivative protein is obtained.Institute of the present invention
It states " derivative protein " to refer to the enzyme with above-mentioned amino acid sequence with the difference on amino acid sequence, it is possibility to have not shadow
It rings the difference on the modified forms of sequence or haves both at the same time.These albumen include natural or induction genetic variant.It is described
Induction variant can be obtained by various technologies, such as the random mutation of radiation or mutagens generation, can also be by such as fixed
The technology of point mutation method or other known molecular biology." the derivative protein " is further included with natural L-form amino acid
Residue analog (such as D types amino acid) and with it is non-naturally occurring or synthesis amino acid (such as beta-amino acids, γ-
Amino acid etc.) analog.
Modification (not changing primary structure usually, i.e., do not change amino acid sequence) form includes：In vivo or in vitro albumen
Chemical derivative form such as acetylation or carboxylated.Modification further include glycosylation, such as those in the synthesis and processing of albumen or
Albumen that is glycosylation modified and generating is carried out in further processing step.This modification can be by being exposed to progress sugar by albumen
The enzyme (glycosylase or deglycosylation enzyme of such as mammal) of base and complete.Modified forms are further included with phosphorylation amino
The sequence of sour residue (such as phosphotyrosine, phosphoserine, phosphothreonine).It further includes and is modified to improve its anti-egg
White hydrolysis property or the albumen for optimizing solubility property.And in the present invention, in asparagine (Asn), the silk of the degrading enzyme
Propylhomoserin (Ser) and threonine (Thr) residue carry out that N- and/or O- is glycosylation modified can further to improve dissolubility at any one
And thermal stability.
In order to facilitate purifying, the label that this field can also be used common (such as Poly-Arg, Poly-His, FLAG,
At least one of Strep-tag II and c-myc) modification is added to (a) or (b).The label does not interfere with the present invention
The activity of the enzyme of offer in actual application, can choose whether addition label according to demand.
In situations where it is preferred, the degrading enzyme has SEQ ID NO：Amino acid sequence shown in 2.
Second aspect, the present invention also provides a kind of gene of encoding Aspergillus ochraceus toxins degrading enzyme, wherein, which has
Encode the nucleotide sequence of above-mentioned degrading enzyme.
It is well known in the art that in 20 kinds of different amino acid of constitutive protein matter, except Met (ATG) or Trp (TGG) points
Not Wei single password coding it is outer, other 18 kinds of amino acid respectively by 2-6 codon coding (Sambrook etc., molecular cloning,
CSH Press, New York, the U.S., the second edition, 1989, see the Appendix D of page 950).I.e. due to the degeneracy of genetic codon
Property, determines the most more than one of codon of an amino acid, the displacement of third nucleotide in triplet codon, often not
The composition of amino acid can be changed, therefore the nucleotide sequence for encoding the gene of same protein can be different.Those skilled in the art according to
Well known password sublist, it is constant from amino acid sequence disclosed by the invention and by the enzymatic activity that the amino acid sequence obtains
Amino acid sequence, can derive the nucleotide sequence of the gene that can encode them completely, by biological method (such as
PCR method, mutation method) or chemical synthesis process obtain the nucleotide sequence, therefore the partial nucleotide sequence all should
It is included in the scope of the present invention.
In situations where it is preferred, the gene has SEQ ID NO：1、SEQ ID NO：3、SEQ ID NO：4 and SEQ
ID NO：Nucleotide sequence shown in 5.
As described above, correspondingly, the 5' ends and/or 3' ends of nucleotide sequence can also be connected with the common label in this field
The coded sequence of (such as at least one of Poly-Arg, Poly-His, FLAG, Strep-tag II and c-myc).
Nucleotide sequence provided by the invention can usually use PCR (PCR) amplification, recombination method or people
Work synthetic method obtains.For example, those skilled in the art can be easy to according to nucleotide sequence provided by the present invention
To template and primer, carry out amplification using PCR and obtain related sequence.
Once obtain related nucleotide sequence, it is possible to obtain related amino acid sequence with recombination method is large batch of.It is logical
Gained nucleotide sequence is often cloned into carrier, then is transferred in host, it is then thin from the host after proliferation by conventional method
The isolated related nucleotide sequence of born of the same parents.
The third aspect, the present invention also provides a kind of recombinant vector, wherein, which contains said gene.
In the present invention, the recombinant vector can contain said gene provided by the invention.It is used in recombinant vector
Various carriers known in the art, such as commercially available various plasmids, clay, bacteriophage and retrovirus can be selected in " carrier ",
Present invention preferably uses plasmid as carrier.The structure of recombinant vector, which may be used, to have cutting in vector multiple cloning site
The various endonucleases in site (such as pUC18, can use Sal I, BamH I, EcoR I etc.；It, can be with for pPICZ ɑ A
Use Nde I, Nhe I, EcoR I, Bam H, Hind III etc.；For PET30a, can use BamH I, Hind III, Nde I,
Xho I etc.) digestion acquisition linear plasmid is carried out, it connect, is recombinated with using the genetic fragment of identical nucleic acid inscribe cleavage
Plasmid.Present invention preferably employs Nde I and Xho I double digestion PET30a carriers and genetic fragment connected to it, linked enzymes
Connection, structure obtain recombinant vector.
Fourth aspect, the present invention also provides a kind of recombinant cell, wherein, the recombinant cell contain said gene or on
State recombinant vector.
In the present invention, the cell can be the cell containing gene of the present invention, can also be normal by this field
Above-mentioned recombinant vector is converted, transduceed or is transfected into host cell with the recombinant cell of acquisition, such as calcium chloride by the method for rule
Method, chemical conversion or electroporated, it is preferably electroporated.
In a kind of preferred embodiment of the present invention, the cell is spore.It should be understood that art used herein
Language " spore " refers to fungi or bacterial spore, endospore or outer spore.
Cell according to the present invention can be any suitable cell.It is highly preferred that be any suitable bacterium, fungi or
Plant cell.It is even furthermore preferable that the cell is selected from Escherichia coli, streptomyces (Streptomyces), Hansenula
(Hansenula), trichoderma (Trichoderma) (particularly trichoderma reesei (T.reesei)), bacillus
(Bacillus), lactobacillus (Lactobacillus), aspergillus (particularly aspergillus niger), plant cell and/or bacillus
The spore of category, trichoderma or aspergillus.
5th aspect, the present invention also provides a kind of additive, wherein, which contains degradation provided by the invention
Enzyme and/or contain above-mentioned recombinant cell and/or its tunning.
In situations where it is preferred, the additive is using degrading enzyme provided by the invention as active constituent.In the addition
In agent, the content of the degrading enzyme is 0.001-10g/kg, more preferably preferably 0.01-8g/kg, 0.1-5g/kg.In addition,
It can also be containing well known to a person skilled in the art solvent (such as glycerine, carbohydrate and protease inhibitors egg in the additive
White protective agent), agonist etc..
According to the present invention, also containing physiologically acceptable carrier in the feed and additive, wherein, the physiology
Upper acceptable carrier is selected from least one of following substance：Maltodextrin, lime stone (calcium carbonate), cyclodextrin, wheat,
Wheat bran or wheat component, rice or rice bran, sucrose, starch, Na2SO4, talcum powder and PVA and their mixture.
It will be apparent for a person skilled in the art that can these additives be added to pollution has the feed of mycotoxin
And/or grain and oil material is to reduce existing endotoxin level.
The Feed Material of the present invention can include：A) cereal, for example, granule cereal (such as wheat, barley, naked barley, oat with
And combination thereof) and/or big grain cereal such as maize or sorghum；B) by-product from cereal, for example, it is corn protein powder, dry
Vinasse and soluble matter (DDGS), wheat bran, sizing, wheat wheat-middlings, rice bran, rice husk, oat shell, palm kernel and citrus pulp；c)
Ensilage；D) protein derived from following source：Such as soybean, sunflower, peanut, lupin, pea, broad bean, cotton, card
Nola, fish meal, dry plasma albumen, meat and bone meal, potato protein, whey, copra, sesame；E) from plant and animal source
The oil ＆ fat of acquisition；F) minerals and vitamins.
The grain and oil of the present invention refer to the grains such as cereal, beans and oil plant and its general designation of processing finished product and semi-finished product, special
Do not refer to the edible product of the mankind.For example, the grain and oil can be the edible grain and oil production of the common mankind in this field
Product, specifically, the grain and oil can include in cereal and its agricultural and sideline product, oil ＆ fat product, drinks, milk and its product etc.
It is at least one.
It will be apparent to one skilled in the art that feed according to the present invention or additive can also include other groups
Divide such as stabilizer and/or incremental agent and/or enzyme.
Wherein, the enzyme can be selected from but be not limited to：Aflatoxin detoxifying enzymes, ochratoxin lactonase, volt horse
Toxin Carboxylesterase, fumonisin aminopherase, amino polyol amine oxidase, the hydrolysis of deoxynivalenol epoxides
Enzyme, carboxypeptidase, aspergillus niger aspartic protease PEPAa, PEPAb, PEPAc and PEPAd, elastoser, aminopeptidase, stomach
Protease or pepsin sample protease, trypsase or trypsin like proteases, bacterialprotease, be related to starch metabolism,
Fiber degradation, lipid-metabolism enzyme, be related to glycogen metabolism protein or enzyme, amylase, arabinase, arabinofuranose
Enzyme, catalase, cellulase, chitinase, renin, cutinase, deoxyribonuclease, epimerase, esterase, half
Lactoside enzyme, dextranase, glucan lyase, endoglucanase, glucoamylase, glucose oxidase, glucosidase,
Including β-glucosyl enzym, glycuronidase, hemicellulase, hexoxidase, hydrolase, invertase, isomerase, lipolytic enzyme,
Laccase, lyases, mannosidase, oxidizing ferment, oxidoreducing enzyme, pectate lyase, pectin acetyl esterases, pectin go to polymerize
Enzyme, pectinesterase, pectin decomposing enzyme, peroxidase, phenol oxidase, phytase, polygalacturonase, protease,
Sandlwood-galacturonic acid enzyme, ribalgilase, African hesperidium element, transferase, transport protein, Transglutaminases, zytase,
Hexoxidase, acid phosphatase and combination thereof.
Preferably, the method for preparing additive according to the present invention includes mixing step, which includes mixing such as
At least one of upper described degrading enzyme, recombinant cell and its tunning, it is optionally physiologically acceptable at least one
Carrier, solvent, agonist, stabilizer, incremental agent or enzyme are mixed together.
It is apparent to the skilled person that, as precautionary step, any feed and/or grain and oil can be added an additive to
In material.
For example, when the additive is used for the feed of cultivated animals, in the additive also containing bacillus licheniformis,
Bacillus subtilis, bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, lactoenterococcus, lactobacillus acidophilus, Lactobacillus casei,
German-style Lactobacillus lactate subspecies, lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, candida utili, baby's bifid bar
Bacterium, bifidobacterium longum, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, animal bifidobacteria, rice
Aspergillus, bacillus lentus, bacillus pumilus, lactobacillus cellobiosas, lactobacillus fermenti and Lactobacillus delbrueckii
At least one of subspecies.
For example, when the additive is used for ensilage or ox feed, also containing production propionic acid propionic acid in the additive
At least one of bacillus, lactobacillus buchneri and Lactobacillus paracasei.
For example, when the additive be used for poultry, growing-finishing pig and aquiculture animal feed when, the feed or
Also contain bacillus coagulans and/or Brevibacillus laterosporus in additive.
Based on description as above, the present invention also provides a kind of grain and oil or feed, wherein, the grain and oil or feed contain above-mentioned
Additive.
6th aspect, the present invention also provides degrading enzyme as described above, gene as described above, recombinations as described above
Carrier, recombinant cell as described above and/or its tunning or additive as described above degradation ochratoxin and/
Or the application in other mycotoxins.
According to the present invention, other described mycotoxins are preferably fumonisin and T2 toxin.
7th aspect, the present invention also provides a kind of method of degrade ochratoxin/or other mycotoxins, wherein,
This method includes：Under conditions of enzyme degradation reaction, by degrading enzyme as described above, recombinant cell as described above and/or its
Tunning or additive as described above are contacted with pending sample.
According to the present invention, the condition of the contact can include：Temperature is 20-80 DEG C, pH value 2-9；Preferably, temperature
It is 30-80 DEG C, pH value 4-8；It is highly preferred that temperature is 30-70 DEG C, pH value 6-8.
In the present invention, the time of the enzyme degradation reaction can be 5-60min, preferably 10-30min.According to this hair
Bright, other described mycotoxins are preferably fumonisin and T2 toxin.
According to the present invention, the pending sample can be grain and oil and/or feed.The grain and oil and/or feed are such as
On have been carried out being discussed in detail, details are not described herein again.
The present invention will be described in detail by way of examples below.
In following embodiment and comparative example, ochratoxin A, fumonisin FB1 and T2 toxin standard items are purchased from Sigma
Company；
Fumonisin FB1 is examined by high performance liquid chromatography according to the method in GB 5009.240-2016 standards
It surveys, vomitoxin is detected according to the method in GB/T 30956-2014 standards, according to GB/T 23501-2009 standards
In method T2 toxin is detected；
Toxin degradation rate %=(before reaction in sample after quality-reaction of toxin in sample toxin quality)/reaction before
Quality × 100% of toxin in sample.
Embodiment 1
The present embodiment is used to illustrate degrading enzyme provided by the invention and its preparation method and application.
(1) acquisition of gene
(precious bioengineering (Dalian) Co., Ltd of commission, similarly hereinafter) following nucleotide is synthesized by artificial chemistry synthetic method
Segment；In SEQ ID NO：Addition Nde I restriction enzyme sites, 3 ' ends after 5 ' end initiation codon ATG of the nucleotide sequence shown in 1
Add Xho I restriction enzyme sites and terminator codon TAG.
(2) structure of recombinant plasmid
(there is His labels, purchase to PET30a plasmids using restriction enzyme Nde I and Xho I (being purchased from NEB companies)
In Invitrogen companies of the U.S.) double digestion is carried out, in 37 DEG C of water-bath digestion 4h, digestion system (50 μ L) is as follows：
By digestion products into after row agarose gel electrophoresis, purifying recycling.Then, (it is purchased from Takara using T4 ligases
Company) reaction is attached, 4h is connected at room temperature, obtains recombinant plasmid.The linked system (10 μ L) used is as follows：
(3) acquisition of recombinant bacterial strain
Turn DH5 α competence using the recombinant plasmid electricity obtained by step (2) on Bio-Rad Gene Pulse electroporations
Cell (is purchased from Takara companies), and electric conversion condition is：Voltage 1500V, 25 μ F of capacitance, 200 Ω of resistance, transformation time is with DNA
The difference of sample and change, and provided automatically by instrument, usually in the range of 3.5-4s.Then, the cell after conversion is coated with
It is screened on the LB solid plates containing kanamycins, is inverted culture 2 days in 37 DEG C, picking positive bacterium colony is inoculated in LB liquid
In body culture medium, to obtain recombinant bacterial strain.
Then make with method known in this field to extract plasmid, then entrust precious bioengineering (Dalian) limited public affairs
Department is sequenced, the results show that gene as above is successfully transformed into Escherichia coli, shows the recombinant bacterial strain of the present invention
It builds successfully.
(4) preparation of enzyme
Obtained recombinant bacterial strain is inoculated in LB fluid nutrient mediums, and (beef extract 5g, peptone 10g, sodium chloride 5g, moisturizing is extremely
1000mL, 115 DEG C of sterilizing 20min are spare, pH 7) in, 3 days are cultivated in 37 DEG C to OD600Between 2 and 6, bacterium is collected by centrifugation in value
Body, with phosphate buffer (PBS, 135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, 8mM K2HPO4, pH 7.2) and solution
After suspension, ultrasonic disruption, centrifuging and taking supernatant obtains crude enzyme liquid.
Crude enzyme liquid is placed in the ammonium sulfate powder for, being slowly added to grind on ice, it is stirring while adding, it is added to ammonium sulfate saturation
Until.Left and right for 24 hours is stood at 4 DEG C, 12000r/min centrifugation 50min abandon supernatant, dissolved and precipitated with a small amount of PBS (pH 7.2).It will
The precipitation dialysis of PBS dissolvings, removes ammonium sulfate, is resuspended in buffer solution (pH 7.4,50mM NaCl, imidazoles containing 10mM).According to
The recombinase given expression to contains His labels, affinitive layer purification is carried out using Ni columns, after 1mL/min balances Ni columns, with flow
0.5mL/min is directly by the crude enzyme liquid loading of resuspension；It is continuing with buffer solution (pH 7.4,50mM NaCl, imidazoles containing 10mM)
1mL/min elutes unadsorbed or absorption non-specific foreign protein；With buffer solution (pH 7.4,50mM NaCl, 500mM imidazoles)
Destination protein is collected in elution, to obtain the enzyme solution of purifying.
(5) influence of temperature and pH to enzymatic activity
The enzyme solution and suitable ochratoxin A standard items that appropriate step (4) obtains are taken, using physiological saline to match
It makes in obtained mixed liquor, a concentration of 100ng/ml of enzyme, a concentration of 1000ppb of ochratoxin A.Reaction temperature is
Respectively 20 DEG C, 37 DEG C, 60 DEG C and 80 DEG C (pH value 7), the pH value of reaction is respectively 2,3,5,7 and 9 (37 DEG C of temperature), reaction
20 μ L reaction products carry out the residual of high performance liquid chromatography detection ochratoxin A after 30min, and calculate degradation rate.As a result such as
Shown in table 1 and 2.
Embodiment 2
The present embodiment is used to illustrate degrading enzyme provided by the invention and its preparation method and application.
Degrading enzyme, and the influence of measuring temperature and pH to enzymatic activity are prepared according to the method for embodiment 1, the difference is that
Use SEQ ID NO：3 replace SEQ ID NO：1.
Embodiment 3
The present embodiment is used to illustrate degrading enzyme provided by the invention and its preparation method and application.
Degrading enzyme, and the influence of measuring temperature and pH to enzymatic activity are prepared according to the method for embodiment 1, the difference is that
Use SEQ ID NO：4 replace SEQ ID NO：1.
Embodiment 4
The present embodiment is used to illustrate degrading enzyme provided by the invention and its preparation method and application.
Degrading enzyme, and the influence of measuring temperature and pH to enzymatic activity are prepared according to the method for embodiment 1, the difference is that
Use SEQ ID NO：5 replace SEQ ID NO：1.
Comparative example 1
This comparative example is blank control
Degrading enzyme is prepared according to the method for embodiment 1, the difference is that the empty vectors PET30a using non-transgene
Plasmid replaces recombinant plasmid used in embodiment 1.
Comparative example 2
The present embodiment is used to illustrate degrading enzyme provided by the invention and its preparation method and application.
Degrading enzyme, and the influence of measuring temperature and pH to enzymatic activity are prepared according to the method for embodiment 1, the difference is that
Use SEQ ID NO：6 (the amide enzyme sequences from CN103209597A) replace SEQ ID NO：1.
Table 1
Table 2
By the result of above example it is found that ochratoxin degrading enzyme provided by the invention can efficiently degrade it is reddish brown
Aspertoxin A, and the high stability of the enzyme, suitable for industrialized production.Also, ochratoxin drop provided by the invention
Solving enzyme also has acidproof and heat safe characteristic, this further expands the range of its application.
In addition, experimental result is shown, SEQ ID NO provided by the invention：11,89,179 and 239 amino acids residue in 1
It is substituted, lacks or adds (SEQ ID NO：3-5) equally can effectively it degrade afterwards to ochratoxin A, in addition, through
It is experimentally confirmed that ochratoxin degrading enzyme provided by the invention is ochratoxin A to the degradation efficiency of ochratoxin B
90%, the degradation efficiency for fumonisin and T2 toxin is respectively the 80% and 75% of ochratoxin A.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, it can be combined by any suitable means.In order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
SEQUENCE LISTING
<110>Cofco Nutrition And Health Research Institute Co., Ltd.
Cofco Group Co., Ltd.
<120>Ochratoxin degrading enzyme, encoding gene, recombinant vector, cell, additive and its application
<130> I40391COF
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1443
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 1
atggtccgcc gaattgcttc agctacacct cgcgtgcaat cgcccatgtc gccattgggc 60
acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120
atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180
gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240
ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300
cccgtgctca tgcctggttt gtgggattgc gacatgcatt ttggcgggga tgacgattat 360
tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420
cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480
gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcattcg 540
tctggcgctg cactcagtca gacagctgga cacggcgata tcttcgctct tccagcaggc 600
gaagtactgg ggagttatgg agtaatgaac ccacgccctg ggtactgggg ggcagggccg 660
ctatgtatcg ccgatggcgt agaggaggtc cgacgagcag tgaggttgca gatcatgcgc 720
ggtgcaaagg ttatcaaagt gatggcctct gggggtgtca tgtcgcgaga cgataatccc 780
aactttgcac agttctctcc agaagaactg aaggtgatag tggaagaggc ggctcgacag 840
aaccggatcg tttctgcaca tgtgcatggc aaggcgggga ttatggctgc tatcaaagca 900
ggctgcaaga gtctggagca tgtgtcttat gctgacgagg aggtctggga gctcatgaaa 960
gagaagggaa ttttgtatgt ggccacacgc tcggttattg aaatctttct ggctagtaat 1020
ggagaggggt tggtgaaaga gtcgtgggcc aagttgcagg cccttgccga ttcgcatttg 1080
aaagcttatc agggagctat taaggcgggt gttaccattg cgttgggaac ggataccgcc 1140
cccggtggtc ctaccgcact tgagttgcag tttgccgtcg agagaggagg tatgacgccg 1200
ttggaggcca tcaaagccgc aactgcgaac gctcccctgt cagttggtcc acaagcaccg 1260
ttgacgggtc agcttcgcga ggggtatgag gcagatgtga ttgcgttgga ggagaatcca 1320
ttggaggaca tcaaagtctt tcaggagccg aaggcagtta cccacgtctg gaagggaggg 1380
aaactgttca aaggtccagg tattggtccg tggggagaag atgcacgtaa tccttttctg 1440
tag 1443
<210> 2
<211> 480
<212> PRT
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 2
Met Val Arg Arg Ile Ala Ser Ala Thr Pro Arg Val Gln Ser Pro Met
1 5 10 15
Ser Pro Leu Gly Thr Thr Tyr Cys Val Arg Pro Asn Pro Val Ser Leu
20 25 30
Asn Leu Gln Arg Arg Pro Leu Val Ile Ala Ser Thr Asp Glu Ala Lys
35 40 45
Val Thr Ile Ile Tyr Ala Gly Leu Leu Ile Pro Gly Asp Gly Glu Pro
50 55 60
Leu Arg Asn Ala Ala Leu Val Ile Ser Asp Lys Ile Ile Ala Phe Val
65 70 75 80
Gly Ser Glu Ala Asp Ile Pro Lys Asp Tyr Leu Arg Ser Thr Gln Ser
85 90 95
Thr His Arg Val Pro Val Leu Met Pro Gly Leu Trp Asp Cys Asp Met
100 105 110
His Phe Gly Gly Asp Asp Asp Tyr Tyr Asn Asp Tyr Thr Ser Gly Leu
115 120 125
Ala Thr His Pro Ala Ser Ser Gly Ala Arg Leu Ala Arg Gly Cys Trp
130 135 140
Glu Ala Leu Gln Asn Gly Tyr Thr Ser Tyr Arg Asp Leu Ala Gly Tyr
145 150 155 160
Gly Cys Glu Val Ala Lys Ala Ile Asn Asp Gly Thr Ile Val Gly Pro
165 170 175
Asn Val His Ser Ser Gly Ala Ala Leu Ser Gln Thr Ala Gly His Gly
180 185 190
Asp Ile Phe Ala Leu Pro Ala Gly Glu Val Leu Gly Ser Tyr Gly Val
195 200 205
Met Asn Pro Arg Pro Gly Tyr Trp Gly Ala Gly Pro Leu Cys Ile Ala
210 215 220
Asp Gly Val Glu Glu Val Arg Arg Ala Val Arg Leu Gln Ile Met Arg
225 230 235 240
Gly Ala Lys Val Ile Lys Val Met Ala Ser Gly Gly Val Met Ser Arg
245 250 255
Asp Asp Asn Pro Asn Phe Ala Gln Phe Ser Pro Glu Glu Leu Lys Val
260 265 270
Ile Val Glu Glu Ala Ala Arg Gln Asn Arg Ile Val Ser Ala His Val
275 280 285
His Gly Lys Ala Gly Ile Met Ala Ala Ile Lys Ala Gly Cys Lys Ser
290 295 300
Leu Glu His Val Ser Tyr Ala Asp Glu Glu Val Trp Glu Leu Met Lys
305 310 315 320
Glu Lys Gly Ile Leu Tyr Val Ala Thr Arg Ser Val Ile Glu Ile Phe
325 330 335
Leu Ala Ser Asn Gly Glu Gly Leu Val Lys Glu Ser Trp Ala Lys Leu
340 345 350
Gln Ala Leu Ala Asp Ser His Leu Lys Ala Tyr Gln Gly Ala Ile Lys
355 360 365
Ala Gly Val Thr Ile Ala Leu Gly Thr Asp Thr Ala Pro Gly Gly Pro
370 375 380
Thr Ala Leu Glu Leu Gln Phe Ala Val Glu Arg Gly Gly Met Thr Pro
385 390 395 400
Leu Glu Ala Ile Lys Ala Ala Thr Ala Asn Ala Pro Leu Ser Val Gly
405 410 415
Pro Gln Ala Pro Leu Thr Gly Gln Leu Arg Glu Gly Tyr Glu Ala Asp
420 425 430
Val Ile Ala Leu Glu Glu Asn Pro Leu Glu Asp Ile Lys Val Phe Gln
435 440 445
Glu Pro Lys Ala Val Thr His Val Trp Lys Gly Gly Lys Leu Phe Lys
450 455 460
Gly Pro Gly Ile Gly Pro Trp Gly Glu Asp Ala Arg Asn Pro Phe Leu
465 470 475 480
<210> 3
<211> 1443
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 3
atggtccgcc gaattgcttc agctacacct cacgtgcaat cgcccatgtc gccattgggc 60
acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120
atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180
gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240
ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300
cccgtgctca tgcctggttt gtgggattgc gacatgcatt ttggcgggga tgacgattat 360
tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420
cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480
gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcattcg 540
tctggcgctg cactcagtca gacagctgga cacggcgata tcttcgctct tccagcaggc 600
gaagtactgg ggagttatgg agtaatgaac ccacgccctg ggtactgggg ggcagggccg 660
ctatgtatcg ccgatggcgt agaggaggtc cgacgagcag tgaggttgca gatctttcgc 720
ggtgcaaagg ttatcaaagt gatggcctct gggggtgtca tgtcgcgaga cgataatccc 780
aactttgcac agttctctcc agaagaactg aaggtgatag tggaagaggc ggctcgacag 840
aaccggatcg tttctgcaca tgtgcatggc aaggcgggga ttatggctgc tatcaaagca 900
ggctgcaaga gtctggagca tgtgtcttat gctgacgagg aggtctggga gctcatgaaa 960
gagaagggaa ttttgtatgt ggccacacgc tcggttattg aaatctttct ggctagtaat 1020
ggagaggggt tggtgaaaga gtcgtgggcc aagttgcagg cccttgccga ttcgcatttg 1080
aaagcttatc agggagctat taaggcgggt gttaccattg cgttgggaac ggataccgcc 1140
cccggtggtc ctaccgcact tgagttgcag tttgccgtcg agagaggagg tatgacgccg 1200
ttggaggcca tcaaagccgc aactgcgaac gctcccctgt cagttggtcc acaagcaccg 1260
ttgacgggtc agcttcgcga ggggtatgag gcagatgtga ttgcgttgga ggagaatcca 1320
ttggaggaca tcaaagtctt tcaggagccg aaggcagtta cccacgtctg gaagggaggg 1380
aaactgttca aaggtccagg tattggtccg tggggagaag atgcacgtaa tccttttctg 1440
tag 1443
<210> 4
<211> 1440
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 4
atggtccgcc gaattgcttc agctacacct gtgcaatcgc ccatgtcgcc attgggcaca 60
acatactgcg tccgtcctaa tcctgtttca ctgaatcttc aaagaagacc tctcgtgatc 120
gcatcaacag acgaggccaa ggtcactata atatatgccg gactattaat ccctggcgac 180
ggagaacctc tgcgcaatgc tgccctagtc atcagcgata agatcatcgc gttcgttgga 240
tccgaagccg acatccctaa ggactacctc cggtccacgc agtctactca tcgtgtcccc 300
gtgctcatgc ctggtttgtg ggattgcgac atgcattttg gcggggatga cgattattac 360
aacgattata catctggtct ggccactcat ccagcatcat caggtgctcg actagcccgt 420
ggttgctggg aagcattgca gaatgggtat acatcctacc gcgacctagc cggatacggg 480
tgcgaggtcg caaaggcgat caatgatggc actatcgttg gtccaaacgt gcattcgtct 540
ggcgctgcac tcagtcagac agctggacac ggcgatatct tcgctcttcc agcaggcgaa 600
gtactgggga gttatggagt aatgaaccca cgccctgggt actggggggc agggccgcta 660
tgtatcgccg atggcgtaga ggaggtccga cgagcagtga ggttgcagat ctttcgcggt 720
gcaaaggtta tcaaagtgat ggcctctggg ggtgtcatgt cgcgagacga taatcccaac 780
tttgcacagt tctctccaga agaactgaag gtgatagtgg aagaggcggc tcgacagaac 840
cggatcgttt ctgcacatgt gcatggcaag gcggggatta tggctgctat caaagcaggc 900
tgcaagagtc tggagcatgt gtcttatgct gacgaggagg tctgggagct catgaaagag 960
aagggaattt tgtatgtggc cacacgctcg gttattgaaa tctttctggc tagtaatgga 1020
gaggggttgg tgaaagagtc gtgggccaag ttgcaggccc ttgccgattc gcatttgaaa 1080
gcttatcagg gagctattaa ggcgggtgtt accattgcgt tgggaacgga taccgccccc 1140
ggtggtccta ccgcacttga gttgcagttt gccgtcgaga gaggaggtat gacgccgttg 1200
gaggccatca aagccgcaac tgcgaacgct cccctgtcag ttggtccaca agcaccgttg 1260
acgggtcagc ttcgcgaggg gtatgaggca gatgtgattg cgttggagga gaatccattg 1320
gaggacatca aagtctttca ggagccgaag gcagttaccc acgtctggaa gggagggaaa 1380
ctgttcaaag gtccaggtat tggtccgtgg ggagaagatg cacgtaatcc ttttctgtag 1440
<210> 5
<211> 1446
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 5
atggtccgcc gaattgcttc agctacacct cgcgtgcaat cgcccatgtc gccattgggc 60
acaacatact gcgtccgtcc taatcctgtt tcactgaatc ttcaaagaag acctctcgtg 120
atcgcatcaa cagacgaggc caaggtcact ataatatatg ccggactatt aatccctggc 180
gacggagaac ctctgcgcaa tgctgcccta gtcatcagcg ataagatcat cgcgttcgtt 240
ggatccgaag ccgacatccc taaggactac ctccggtcca cgcagtctac tcatcgtgtc 300
cccgtgctca tgcctggttt gtgggattgc gacatgcatt ttggcgggga tgacgattat 360
tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420
cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480
gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcatcat 540
tcgtctggcg ctgcactcag tcagacagct ggacacggcg atatcttcgc tcttccagca 600
ggcgaagtac tggggagtta tggagtaatg aacccacgcc ctgggtactg gggggcaggg 660
ccgctatgta tcgccgatgg cgtagaggag gtccgacgag cagtgaggtt gcagatcatg 720
cgcggtgcaa aggttatcaa agtgatggcc tctgggggtg tcatgtcgcg agacgataat 780
cccaactttg cacagttctc tccagaagaa ctgaaggtga tagtggaaga ggcggctcga 840
cagaaccgga tcgtttctgc acatgtgcat ggcaaggcgg ggattatggc tgctatcaaa 900
gcaggctgca agagtctgga gcatgtgtct tatgctgacg aggaggtctg ggagctcatg 960
aaagagaagg gaattttgta tgtggccaca cgctcggtta ttgaaatctt tctggctagt 1020
aatggagagg ggttggtgaa agagtcgtgg gccaagttgc aggcccttgc cgattcgcat 1080
ttgaaagctt atcagggagc tattaaggcg ggtgttacca ttgcgttggg aacggatacc 1140
gcccccggtg gtcctaccgc acttgagttg cagtttgccg tcgagagagg aggtatgacg 1200
ccgttggagg ccatcaaagc cgcaactgcg aacgctcccc tgtcagttgg tccacaagca 1260
ccgttgacgg gtcagcttcg cgaggggtat gaggcagatg tgattgcgtt ggaggagaat 1320
ccattggagg acatcaaagt ctttcaggag ccgaaggcag ttacccacgt ctggaaggga 1380
gggaaactgt tcaaaggtcc aggtattggt ccgtggggag aagatgcacg taatcctttt 1440
ctgtag 1446
<210> 6
<211> 1443
<212> DNA
<213> Artificial
<220>
<223> The sequence is synthesized.
<400> 6
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 cacatgcatt ttggcgggga tgacgattat 360
tacaacgatt atacatctgg tctggccact catccagcat catcaggtgc tcgactagcc 420
cgtggttgct gggaagcatt gcagaatggg tatacatcct accgcgacct agccggatac 480
gggtgcgagg tcgcaaaggc gatcaatgat ggcactatcg ttggtccaaa cgtgcattcg 540
tctggcgctg cactcagtca gacagctgga cacggcgata tcttcgctct tccagcaggc 600
gaagtactgg ggagttatgg agtaatgaac ccacgccctg ggtactgggg ggcagggccg 660
ctatgtatcg ccgatggcgt agaggaggtc cgacgagcag tgaggttgca gatcatgcgc 720
ggtgcaaagg ttatcaaagt gatggcctct gggggtgtca tgtcgcgaga cgataatccc 780
aactttgcac agttctctcc agaagaactg aaggtgatag tggaagaggc ggctcgacag 840
aaccggatcg tttctgcaca tgtgcatggc aaggcgggga ttatggctgc tatcaaagca 900
ggctgcaaga gtctggagca tgtgtcttat gctgacgagg aggtctggga gctcatgaaa 960
gagaagggaa ttttgtatgt ggccacacgc tcggttattg aaatctttct ggctagtaat 1020
ggagaggggt tggtgaaaga gtcgtgggcc aagttgcagg cccttgccga ttcgcatttg 1080
aaagcttatc agggagctat taaggcgggt gttaccattg cgttgggaac ggataccgcc 1140
cccggtggtc ctaccgcact tgagttgcag tttgccgtcg agagaggagg tatgacgccg 1200
ttggaggcca tcaaagccgc aactgcgaac gctcccctgt cagttggtcc acaagcaccg 1260
ttgacgggtc agcttcgcga ggggtatgag gcagatgtga ttgcgttgga ggagaatcca 1320
ttggaggaca tcaaagtctt tcaggagccg aaggcagtta cccacgtctg gaagggaggg 1380
aaactgttca aaggtccagg tattggtccg tggggagaag atgcacgtaa tccttttctg 1440
tag 1443
Claims (10)
1. a kind of ochratoxin degrading enzyme, which is characterized in that the degrading enzyme has the amino acid shown in following (a) and/or (b)
Sequence：
(a)SEQ ID NO：Amino acid sequence shown in 2；
(b)SEQ ID NO：One or several in amino acid sequence shown in 2 in the 11st, 89,179 and 239 amino acids residues
A amino acid sequence still after replacing, lacking or add with ochratoxin degrading enzymatic activity；
Preferably, the amino acid sequence of the degrading enzyme such as SEQ ID NO：Shown in 2.
2. a kind of encoding gene of ochratoxin degrading enzyme, which is characterized in that the gene is described in claim 1 with encoding
The nucleotide sequence of ochratoxin degrading enzyme；
Preferably, the gene has SEQ ID NO：1、SEQ ID NO：3、SEQ ID NO：4 and SEQ ID NO：Shown in 5
Nucleotide sequence.
3. a kind of recombinant vector, which is characterized in that the recombinant vector contains the gene described in claim 2.
4. a kind of recombinant cell, which is characterized in that the recombinant cell contains gene or the claim 3 described in claim 2
The recombinant vector；
Preferably, institute's recombinant cell is selected from Escherichia coli, streptomyces, Hansenula, trichoderma, bacillus, newborn bar
It is one or more in the spore of Pseudomonas, aspergillus, plant cell and/or bacillus, trichoderma and aspergillus.
5. a kind of additive, which is characterized in that the additive contains degrading enzyme described in claim 1 and/or wanted containing having the right
Seek the recombinant cell and/or its tunning described in 4.
6. additive according to claim 5, wherein, the additive is also containing bacillus licheniformis, bacillus subtilis
Bacterium, bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, lactoenterococcus, lactobacillus acidophilus, Lactobacillus casei, German-style lactobacillus
Lactic acid subspecies, lactobacillus plantarum, Pediococcus acidilactici, Pediococcus pentosaceus, candida utili, bifidobacterium infantis, long bifid bar
Bacterium, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, animal bifidobacteria, aspergillus oryzae, slow bud
One kind in spore bacillus, bacillus pumilus, lactobacillus cellobiosas, lactobacillus fermenti and lactobacillus delbruockii subspecies bulgaricus
It is or a variety of；
Preferably, the additive is used for ensilage or ox feed, and the additive is also containing production propionibacterium acide-propionici, Bu Shi
At least one of lactobacillus and Lactobacillus paracasei；Or
The additive is used for the feed of poultry, growing-finishing pig and aquiculture animal, and the additive is also containing condensation bud
Spore bacillus and/or Brevibacillus laterosporus.
7. additive according to claim 5 or 6, wherein, the additive is also physiologically subjected to containing at least one
Carrier；The physiologically acceptable carrier is selected from maltodextrin, lime stone, cyclodextrin, wheat, wheat bran or wheat group
Point, rice or rice bran, sucrose, starch, Na2SO4, it is one or more in talcum powder and PVA；
Preferably, the additive also contains one or more other enzymes；One or more other enzymes, it is preferred that
It is selected from：Aflatoxin detoxifying enzymes, zearalenone lactonase, fumonisin Carboxylesterase, fumonisin aminopherase,
Amino polyol amine oxidase, deoxynivalenol epoxide hydrolase, carboxypeptidase, aspergillus niger aspartic protease
PEPAa, PEPAb, PEPAc and PEPAd, elastoser, aminopeptidase, pepsin or pepsin sample protease, pancreas egg
White enzyme or trypsin like proteases, bacterialprotease, be related to starch metabolism, fiber degradation, lipid-metabolism enzyme, be related to glycogen
Protein or enzyme, amylase, arabinase, arabinofuranosidase, catalase, cellulase, the chitin of metabolism
Enzyme, renin, cutinase, deoxyribonuclease, epimerase, esterase ,-galactosidase, dextranase, glucan cracking
Enzyme, endoglucanase, glucoamylase, glucose oxidase, glucosidase, including β-glucosyl enzym, glycuronidase, half
Cellulase, hexoxidase, hydrolase, invertase, isomerase, lipolytic enzyme, laccase, lyases, mannosidase, oxidation
Enzyme, oxidoreducing enzyme, pectate lyase, pectin acetyl esterases, pectin remove polymerase, pectinesterase, pectin decomposing enzyme, peroxide
Compound enzyme, phenol oxidase, phytase, polygalacturonase, protease, sandlwood-galacturonic acid enzyme, ribalgilase,
One kind in African hesperidium element, transferase, transport protein, Transglutaminases, zytase, hexoxidase and acid phosphatase
It is or a variety of.
8. according to the additive described in any one of claim 5-7, wherein, the additive is with 0.001-10g/kg additives
Level contain the albumen of gene code described in degrading enzyme described in claim 1 and/or claim 2.
9. the recombinant vector described in gene, claim 3 described in degrading enzyme described in claim 1, claim 2, right
It is required that the additive in recombinant cell and/or its tunning or claim 5-8 described in 4 described in any one is being degraded
Application in ochratoxin and/or other mycotoxins.
A kind of 10. method of degrade ochratoxin/or other mycotoxins, which is characterized in that this method includes：It degrades in enzyme
It, will be in the recombinant cell described in degrading enzyme described in claim 1, claim 4 or claim 5-8 under conditions of reaction
Additive described in any one is contacted with pending sample；The pending sample is preferably grain and oil and/or feed.
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| CN201611247161.6A CN108251385B (en) | 2016-12-29 | 2016-12-29 | Ochratoxin degrading enzyme, encoding gene, recombinant vector, cell, additive and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234196A (en) * | 2018-09-28 | 2019-01-18 | 上海市农业科学院 | A kind of mixed bacterial and its crude enzyme preparation of the fumonisin B1 that degrades |
| CN109557306A (en) * | 2019-01-09 | 2019-04-02 | 中粮集团有限公司 | A kind of ochratoxin A degrading enzyme and its application and the immuno-chromatographic test paper strip for detecting ochratoxin A |
| CN110172426A (en) * | 2019-05-30 | 2019-08-27 | 华中农业大学 | There is the bacillus subtilis of high-efficiency detoxicating to T-2 toxin |
| CN110499254A (en) * | 2019-07-22 | 2019-11-26 | 吉林大学 | A kind of salt resistance alkali Aspergillus ochraceus bacterial strain W1 and its microbial inoculum and application |
| CN110495549A (en) * | 2019-07-05 | 2019-11-26 | 中国农业大学 | A kind of 1,4 beta-glucanase and its application in inhibition Aspergillus ochraceus |
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| CN116144533A (en) * | 2022-11-29 | 2023-05-23 | 国家粮食和物资储备局科学研究院 | Xi Leshi Brevibacillus brevis for degrading OTA and inhibiting fungal growth generated by OTA, OTA degrading enzyme and application thereof |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109234196A (en) * | 2018-09-28 | 2019-01-18 | 上海市农业科学院 | A kind of mixed bacterial and its crude enzyme preparation of the fumonisin B1 that degrades |
| WO2020073845A1 (en) * | 2018-10-09 | 2020-04-16 | 天津科技大学 | Fumonisin degrading enzyme fumdps and gene and use thereof |
| CN111394342B (en) * | 2019-01-03 | 2021-03-16 | 安徽农业大学 | Amidase, and coding gene, recombinant vector, recombinant bacterium and application thereof |
| CN111394342A (en) * | 2019-01-03 | 2020-07-10 | 安徽农业大学 | Amidase, and coding gene, recombinant vector, recombinant bacterium and application thereof |
| CN109557306A (en) * | 2019-01-09 | 2019-04-02 | 中粮集团有限公司 | A kind of ochratoxin A degrading enzyme and its application and the immuno-chromatographic test paper strip for detecting ochratoxin A |
| CN109557306B (en) * | 2019-01-09 | 2022-03-04 | 中粮集团有限公司 | Ochratoxin A degrading enzyme, application thereof and immunochromatography test strip for detecting ochratoxin A |
| CN110172426A (en) * | 2019-05-30 | 2019-08-27 | 华中农业大学 | There is the bacillus subtilis of high-efficiency detoxicating to T-2 toxin |
| CN110495549B (en) * | 2019-07-05 | 2022-04-22 | 中国农业大学 | Beta-glucanase and application thereof in inhibition of aspergillus ochraceus |
| CN110495549A (en) * | 2019-07-05 | 2019-11-26 | 中国农业大学 | A kind of 1,4 beta-glucanase and its application in inhibition Aspergillus ochraceus |
| CN110499254A (en) * | 2019-07-22 | 2019-11-26 | 吉林大学 | A kind of salt resistance alkali Aspergillus ochraceus bacterial strain W1 and its microbial inoculum and application |
| CN110499254B (en) * | 2019-07-22 | 2022-05-06 | 吉林大学 | Saline-alkali-resistant aspergillus ochraceus strain W1, and microbial inoculum and application thereof |
| CN115281316A (en) * | 2022-06-24 | 2022-11-04 | 黄淮学院 | Composite fermentation microbial inoculum for degrading mycotoxin in meat product and preparation method of high-digestibility fermented dried chicken |
| CN116144533A (en) * | 2022-11-29 | 2023-05-23 | 国家粮食和物资储备局科学研究院 | Xi Leshi Brevibacillus brevis for degrading OTA and inhibiting fungal growth generated by OTA, OTA degrading enzyme and application thereof |
| CN116144533B (en) * | 2022-11-29 | 2023-09-05 | 国家粮食和物资储备局科学研究院 | Xi Leshi Brevibacillus brevis for degrading OTA and inhibiting fungal growth generated by OTA, OTA degrading enzyme and application thereof |
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