CN102816745B

CN102816745B - Deoxynivalenol toxin degrading enzyme as well as encoding gene and application thereof

Info

Publication number: CN102816745B
Application number: CN201210335794.8A
Authority: CN
Inventors: 吴子丹; 伍松陵; 孙长坡; 任保中
Original assignee: Academy of State Administration of Grain
Current assignee: Academy of Sciences, State Bureau of Food and Materials Reserve
Priority date: 2012-09-11
Filing date: 2012-09-11
Publication date: 2014-02-05
Anticipated expiration: 2032-09-11
Also published as: CN102816745A

Abstract

The invention discloses a deoxynivalenol (DON) toxin degrading enzyme as well as an encoding gene and application thereof. The enzyme derives from fusarium oxysporum, which is the following protein (a) or (b): (a) a protein consisting of amino acid residue sequences of SEQ ID NO:1 in a sequence table; or (b) a protein which has activity for degrading deoxynivalenol, is derived from SEQ ID NO:1, and is obtained by subjecting the amino acid residue sequence SEQ ID NO:1 in the sequence table to substitution and/or deletion and/or addition of one or more amino acid residues. The deoxynivalenol toxin degrading enzyme has efficient degrading effect on DON.

Description

A kind of deoxynivalenol toxins degrading enzyme and encoding gene and application

Technical field

The invention belongs to enzyme engineering field, be specifically related to a kind of deoxynivalenol degrading enzyme and encoding gene thereof, and the application of deoxynivalenol degrading enzyme.

Background technology

Deoxynivalenol (Deoxynivalenol, DON), be the secondary metabolite producing while lacking nutritive substance by Fusarium, mainly from Fusarium graminearum and fusarium culmorum, and Fusarium graminearum is the important pathogen of wheat scab and maize kernel rot.DON is a kind of global cereal pollutent, and polluted water is in normal times first of Fusarium toxin, and this mycotoxins mainly betides wheat, barley, oat, rye and corn, and rare in paddy rice, Chinese sorghum and triticale.The pollution of deoxynivalenol not only causes significant damage, causes huge financial loss the mankind and animal health, and causes a large amount of international trade disputes.

At present, both at home and abroad the detoxicating method of DON mainly contains physical removal and absorption, chemical treatment etc.Physics, the chemical treatment method of contaminant toxin cereal are mainly contained: the processing of flushing and rinsing, thermal treatment, ionizing radiation, inorganic absorption, chemical reagent and ozone oxidation etc.But physical chemistry detoxification efficiency is the height of expection not, and changed the quality of food, easily caused the loss of nutritive substance, European Union does not allow applied chemistry method in food production to eliminate mycotoxins.

Because traditional physics and chemistry method has certain limitation, the pollution that being degraded to of microorganism or zymin successfully controlled mycotoxins provides new thinking.Biological degradation not only can efficiently be converted into toxin non-toxic products, Environmental Safety, and biological enzyme method specificity is strong, transformation efficiency is high.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of deoxynivalenol degrading enzyme and encoding gene thereof, this enzyme deoxynivalenol (DON) of can degrading.

For solving the problems of the technologies described above, the technical solution adopted in the present invention is:

Deoxynivalenol degrading enzyme provided by the present invention is named as Dasag, derives from Fusarium oxysporum (Fusarium oxysporum) ACCC No.36245(Chinese agriculture microbial strains preservation administrative center, is called for short ACCC)

Described deoxynivalenol degrading enzyme is following (a) or protein (b):

(a) protein that the amino acid residue sequence of the SEQ ID NO:1 in sequence table forms;

(b) SEQ ID NO:1 amino acid residue sequence in sequence table is there is to the protein being derived by SEQID NO:1 of degraded deoxynivalenol activity through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.

Wherein, the SEQ ID NO:1 in sequence table is comprised of 454 amino-acid residues.

The encoding gene of above-mentioned deoxynivalenol degrading enzyme also belongs to protection scope of the present invention.It can have one of following nucleotide sequence:

(a) nucleotide sequence shown in SEQ ID NO:2 in sequence table;

(b) polynucleotide of SEQ ID NO:1 protein sequence in code sequence list.

Wherein, the SEQ ID NO:2 in sequence table is by 1365 based compositions, and its encoding sequence is for holding the 1st to the 1365th bit base from 5 ', and coding has the protein of the amino acid residue sequence of SEQ ID NO:1 in sequence table.

The expression vector that contains gene of the present invention, clone, engineering bacteria and Host Strains all belong to protection scope of the present invention.

The present invention also provides a kind of method of expressing described deoxynivalenol degrading enzyme, be that the recombinant expression vector that contains above-mentioned deoxynivalenol degrading enzyme encoding gene is imported to host cell, express and obtain deoxynivalenol degrading enzyme.

Wherein, described host can be intestinal bacteria, yeast, mammalian cell, insect cell, Bacillus subtilus, genus bacillus or Bacterium lacticum etc., is preferably yeast.Described yeast is preferably Pasteur's moral pichia spp (Pichia pastoris), for example Pasteur's moral Pichia pastoris GS115.

For building the carrier that sets out of described recombination bacillus coli and recombinant yeast expression vector, can be the expression vector at above-mentioned host's expression alien gene, as can be at the pEB of expression in escherichia coli carrier, and the pPIC9K, the pPIC9 that in Pasteur's moral pichia spp (Pichia pastoris), express, pPIC3.5K etc.

Above-mentioned recombinant expression vector all can build according to a conventional method.

The present invention also provides the application of described deoxynivalenol degrading enzyme in degraded deoxynivalenol.

Advantage of the present invention: the deoxynivalenol degrading enzyme of expressing can be cut down mycotoxins deoxynivalenol in grain, plays effective effect to controlling grain contamination.

Accompanying drawing explanation

The SDS-PAGE figure of the expression product of Fig. 1 recombinant plasmid pPIC9K-Dasag,

Swimming lane 1, expression product; Swimming lane M, molecular weight of albumen standard,

Fig. 2 deoxynivalenol degrading enzyme ability is measured figure.

Embodiment

Experimental technique in following examples is ordinary method if no special instructions.

Embodiment 1 acquisition of deoxynivalenol degrading enzyme gene Dasag and the expression of deoxynivalenol degrading enzyme

The foundation of deoxynivalenol degrading enzyme gene plasmid library

(1) by reference culture Fusarium oxysporum ACCC No.36245(purchased from Chinese agriculture microbial strains preservation administrative center) access PDA liquid nutrient medium (potato 200g/L after activation, glucose 20g/L, agar 20g/L, pH nature), then by the same substratum of suspension access 100ml of bacterial strain, shaking table condition is 220 revs/min, 30 ℃, and 72 hours.After completing, cultivation uses 12000 revs/min of centrifugal collection thalline of whizzer.Extract RNA(Trizol method);

(2) take RNA as the synthetic cDNA sequence of template reverse transcription;

2, the acquisition of deoxynivalenol degrading enzyme gene

The cDNA sequence obtaining in step 1 of take is template, carries out PCR reaction under the guiding of primer 1 and primer 2, the sequence of amplification deoxynivalenol degrading enzyme gene.

Primer 1:5'GGAATTC cATATGaCTGCACTAAACGTTACAAAC3'(line part base is Nde I recognition site)

And primer 2: 5'CCC aAGCTTcTAGCCAATGAATTGCCCATAC3'(line part base is Hind III recognition site)

In PCR reaction, PCR reaction conditions is: 94 ℃, keep 5 minutes, then by following temperature variation program loop 30 times: be warming up to 94 ℃, keep 1 minute, be cooled to 54 ℃, keep 1 minute, be warming up to 68 ℃, keep 2 minutes; Then in 68 ℃, keep 10 minutes, finally, in 4 ℃ of insulations 10 minutes, finish amplified reaction.By agarose electrophoretic analysis, obtain the single band of about 1.4kb, after the PCR product after amplification detects, the amplified production of object band size is reclaimed to test kit with DNA gel and reclaim, and detect its concentration.Reclaim PCR product and connect pMD19-T Vector conversion e. coli jm109, obtain recombinant plasmid pMD19-Dasag order-checking and identify.This deoxynivalenol degrading enzyme gene DNA sequence dna is sequence table SEQ ID NO:2, and its corresponding aminoacid sequence is sequence table SEQ ID NO:1.

The structure of the recombinant expression vector that 3, contains deoxynivalenol degrading enzyme coding gene sequence

The PCR product two ends that obtain have Nde I and Hind III restriction endonuclease sites, with (Nde I) and (Hind III) restriction enzyme, PCR product and plasmid pPIC9K are carried out to double digestion reaction simultaneously, enzyme is cut system 50 μ L: object fragment or plasmid 20 μ L, 10 * K Buffer, 5 μ L, Nde I 2 μ L, Hind III 2 μ L, ddH ₂o 21 μ L, enzyme tangent condition is 37 ℃ of reaction 3h.Enzyme is cut product and is connected after post reclaims, and connection product transforms e. coli jm109, and through kalamycin resistance screening, the positive bacterium colony of picking is cultivated, and recombinant expression vector is cut evaluation, sequence verification through PCR evaluation, enzyme.Carrier after agarose electrophoresis.The object fragment reclaiming and carrier segments are quantitatively also in molar ratio for the ratio of 3:1 is carried out external connection with T4DNA ligase enzyme, ligation system 10 μ L: object fragment 5 μ L, pPIC9K carrier 2 μ L 10 * T4DNA connect damping fluid 1 μ L, T4DNA ligase enzyme (350U/ μ L) 1 μ L, ddH2O 1 μ L.16 ℃ of connections are spent the night, and connect product and transform e. coli jm109, and through kalamycin resistance screening, 37 ℃ of shaking culture 6-8h of picking colony, carry out respectively the enzyme of PCR evaluation and recombinant plasmid and cut evaluation.PPIC9K-Dasag after the recombinant expression vector called after obtaining.Ultrasonic disruption, centrifugal collection supernatant, gets 15 μ L supernatants and detects with SDS-PAGE electrophoresis.PPIC9K-Dasag is checked order, prove and merge that to connect into the DNA sequence dna of plasmid pPIC9K identical with sequence table SEQ ID NO:2, the recombinant expression vector pPIC9K-Dasag that structure contains deoxynivalenol degrading enzyme gene sequence is correct.

4, the expression of deoxynivalenol degrading enzyme in pichia spp

Recombinant expression vector pPIC9K-Dasag is made it after linearizing with Sal I digestion with restriction enzyme, adopt electric shock mode, linearizing carrier pPIC9K-Dasag is imported in Pichia pastoris GS115 (Beijing Mao Jian couple stars Science and Technology Ltd.), through selective medium, cultivate the high expression level bacterial strain of screening His+ and anti-G418.The single colony inoculation growing on picking selective medium is in 5ml YPD liquid nutrient medium (yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L) in, cultivate after 12-24 hour for 30 ℃, proceed to 500mlBMGY substratum (1% yeast extract paste, 2% peptone, yeast nitrogen (YNB) 1.34%, 100mM phosphoric acid buffer PH6.0, 4 * 10-5 vitamin H, 1% glycerine) in, continue to be cultured to the OD600=2-3 of bacterium liquid, centrifugal collection thalline, use again without carbon source BMMY substratum (1% yeast extract paste, 2% peptone, yeast nitrogen (YNB) 1.34%, 100M phosphoric acid buffer PH6.0, 4 * 10-5 vitamin H, 0.5% methyl alcohol) add 0.5% methyl alcohol to carry out inducing culture after being diluted to OD600=1, between incubation period, adding methyl alcohol to final concentration every 24 hours is 0.5%, be cultured to 118 hours and can stop cultivating.Centrifugal collection supernatant, gets 15 μ L supernatants and detects with SDS-PAGE electrophoresis.Result is as shown in Figure 1: swimming lane 1 is protein standard molecular weight (97KDa, 66KDa, 45KDa, 31KDa, 21KDa, 14KDa);

Swimming lane

2,3 is expression product, and arrow represents object band, and this shows the about 52.3KDa of molecular weight of the protein that recombinant bacterial strain is expressed under methanol induction, in the same size with the theoretical molecular going out from the amino acid section of pushing away (52.3kDa).

5, the purifying of deoxynivalenol degrading enzyme expression product

The deoxynivalenol degrading enzyme that host expresses during for intestinal bacteria is used GE company histidine mark affinity column purifying target protein.Concrete method is carried out with reference to the specification sheets of the histidine-tagged fusion protein purification experiment flow of HisTrap FF; And the deoxynivalenol degrading enzyme of expressing in pichia spp can direct secretion in the supernatant of nutrient solution, be directly used in enzyme assay.

The activity of embodiment 2 deoxynivalenol degrading enzymes detects

Sample preparation: the pichia yeast ASAG2 bacterial strain (experimental group) of restructuring and wild-type pichia yeast GS115 bacterial strain (control group, empty plasmid is integrated bacterial strain) abduction delivering.After 5 days, get supernatant liquor 800 μ L and put in 1.5mL centrifuge tube, then add the DON of 200 μ L 100ppm, make final concentration reach 20ppm.Add rear 30 ℃ of reaction 0h-5h aftertreatment.Get the sample 10 μ L that handle well and enter the residual of high performance liquid chromatography detection DON.Detection method is carried out according to GB/T 23504-2009 method.High performance liquid chromatography testing conditions: Agilent Eclipse XDB-C18,150mm * 4.6mm (5 μ m) chromatographic column, moving phase: water/methyl alcohol=80%/20%, UV-detector detects, 30 ℃ of column temperatures, flow velocity 1.0ml/min, sample size 10 μ L.By high performance liquid chromatography, detected and learnt after the supernatant liquor liquid of abduction delivering in Tuner and DON process, the high degradation rate of DON is 30%(Fig. 2).Can show, the degrading enzyme that Dasag gene cloning and expression goes out has the activity of degraded DON.

Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here cannot give all embodiments exhaustive.Every still row in protection scope of the present invention of apparent variation that technical scheme of the present invention extends out or change that belong to.

Sequence table

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Claims

1. a deoxynivalenol degrading enzyme is the protein that the amino acid residue sequence of the SEQ ID NO:1 in sequence table forms.

2. the encoding gene of deoxynivalenol degrading enzyme claimed in claim 1.

3. the encoding gene of deoxynivalenol degrading enzyme according to claim 2, is characterized in that, the encoding gene of described deoxynivalenol degrading enzyme is the nucleotide sequence shown in SEQ ID NO:2 in sequence table.

4. the expression vector, transgenic cell line or the Host Strains that contain the encoding gene of deoxynivalenol degrading enzyme described in claim 2 or 3.

5. the application of deoxynivalenol degrading enzyme in degraded deoxynivalenol described in claim 1.

6. the application of deoxynivalenol degrading enzyme encoding gene in degraded deoxynivalenol described in claim 2 or 3.

7. a method of expressing deoxynivalenol degrading enzyme claimed in claim 1, be that the recombinant expression vector that contains the deoxynivalenol degrading enzyme encoding gene described in claim 2 or 3 is imported to host cell, express and obtain deoxynivalenol degrading enzyme.

8. method according to claim 7, is characterized in that: described host is intestinal bacteria, yeast, mammalian cell, insect cell, genus bacillus or Bacterium lacticum; For building the carrier that sets out of described recombinant expression vector, be pEB, pPIC9K, pPIC9, pPIC3.5k.

9. method according to claim 8, is characterized in that: described host is Pasteur's moral pichia spp.

10. method according to claim 9, is characterized in that: described host is Pasteur's moral Pichia pastoris GS115.