CN110408662A - A method of AFQ1 and epi-AFQ1 is prepared using microbial enzyme oxidation catalysis AFB1 - Google Patents
A method of AFQ1 and epi-AFQ1 is prepared using microbial enzyme oxidation catalysis AFB1 Download PDFInfo
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Abstract
The present invention provides a kind of method for preparing aflatoxin Q 1 (AFQ1) and its epimer (epi-AFQ1) using microbial enzyme oxidation catalysis aflatoxin B1 (AFB1).The preparation method simple process and low cost of AFQ1 and epi-AFQ1 mycotoxin standard items provided by the invention, it is easy to accomplish prepare with scale and production.
Description
Technical field
The invention belongs to Biochemical Engineering technical field, relate generally to a kind of prepare aflatoxin using microbial enzyme
The method and its application of Q1 (AFQ1) and its epimer (epi-AFQ1).
Background technique
Aflatoxin Q 1 (AFQ1) is one of the metabolite of animal's liver metabolism aflatoxin B1 (AFB1).Animal
Liver microsomes P450 enzyme system (CYP1A2, CYP2A6, CYP2B7, CYP3A3, CYP3A4) has certain metabolism to AFBl,
Main path is hydroxylation, demethylation and epoxidation reaction.AFBl is generated AFBO after P450 enzyme system epoxidation, with DNA adduction
Modificator gene is mutated afterwards.Research shows that the activity that different P450 activation AFBl generates AFBO is different, in low concentration of substrate,
The epoxidation that CYP1A2 is mainly responsible for AFBl generates AFBO.In high concentration of substrate, in addition to CYP1A2, CYP3A4 can also play one
Fixed epoxidation, oxidation AFBl generate AFBO.However in people's liver research shows that the most important function of CYP3A4 is
AFQl, which is generated, by oxidation AFBl is detoxified it (Gallagher etc., 1994).People only studied to this stage at present, closed
No any research report how is further metabolized to AFQ1 in vivo in animal body.It is ground simultaneously about the toxicity of AFQ1
Study carefully also very limited.In fact, for understand AFQ1 to the toxicity of body, AFQ1 further metabolic process in animal body and
What end product of metabolism is, these researchs are very necessary.If carrying out these researchs needs to use AFQ1 standard items certainly.
Be about the preparation of AFQ1 standard items and production correlative study and report at present it is very limited, can not find it is complete in detail
Most production method.Tracing it to its cause mainly has following two points: 1) AFQ1 is only an intermediate supersession of animal's liver metabolism AFB1
Product, low output hardly result in sterling；2) not yet find that other biological approaches can be generated and chemical synthesis AFQ1.Due to upper
It states reason and limits the production of AFQ1 standard items, cause its price extremely expensive, also limit the progress of research work.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of aspergillus flavus with short production cycle, at low cost
The method of toxin Q1 (AFQ1) and its epimer (epi-AFQ1).
To achieve the goals above, the invention adopts the following technical scheme:
It is a kind of to prepare aflatoxin Q 1 (AFQ1) and its epimer (epi- using microbial enzyme oxidation catalysis AFB1
AFQ1 method), comprising the following steps:
(1) preparation of microbial enzyme: the conversion of -80 DEG C of preservations is taken to have the recombination bacillus coli of PET-31b-CotA plasmid
Rosseta (DE3) is inoculated in 5mL LB liquid medium and activates overnight, and being transferred to 500mL liquid amount in 1:100 ratio is
In the triangular flask of 300mL, 180r/min, culture is 0.6 to OD600 under the conditions of 37 DEG C, adds final concentration of 0.1mM IPTG and lures
Lead destination protein expression.Fermentation liquid is collected, 4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant；With the phosphate buffer weight of pH7.0
Outstanding thallus, 4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant, repeated washing thallus is three times.Then somatic cells are resuspended in 1mg/
In the binding buffer of mL, ultrasonication, 4 DEG C, 12000rpm is centrifuged 10min, collects supernatant and filters.Use nickel ion
Affinity column (Ni2+- NTA) purification of recombinant proteins, microbial enzyme is made, which can aoxidize AFB1.
(2) bioconversion of AFB1: AFB1 is dissolved into the mother liquor that 20 μ g/mL are made into dimethyl sulfoxide, by following 5mL
Reaction system is tested: 4.3mL sodium phosphate buffer (0.1M, pH8.0), 0.2mL CotA albumen (100 μ g), 0.5mL
AFB1 solution.Not add the system of microbial enzyme as control.The termination of 5mL methanol is added instead after carrying out 12h at 37 DEG C in reaction
It answers.
(3) it AFB1 assay: using the concentration of high performance liquid chromatography detection AFB1, is determined according to the variation of AFB1 concentration
Microbial enzyme has bioconversion activity to AFB1.
The chromatographic condition of high performance liquid chromatography detection AFB1 are as follows: chromatographic column: Agilent C18 chromatographic column, 4.6mm ×
150mm×5μm；Mobile phase: methanol-water (45:55)；Flow velocity: 1mL/min；Pump pressure: 75bar；Sample volume: 20 μ L；Fluorescence detection
Device Detection wavelength: λ ex=360nm, λ em=440nm；Acquisition time: 20min.
95% AFB1 is converted after measured.
(4) identification of product AFQ1 and epi-AFQ1: analyze what micro bioenzyme catalysis AFB1 was generated by UPLC-TOF-MS
The molecular weight of two kinds of products, Fig. 1 are that AFB1 blank control group and AFB1 add the total of microbial enzyme processing group in the positive-ion mode
Ion flow graph (A is AFB1 blank control group, and B is that AFB1 adds microbial enzyme processing group)；Fig. 2 is AFB1 in the positive-ion mode
Add the peak H m/z=313, two products add the peak H m/z=329；Nuclear magnetic resonance (NMR) result (Fig. 3) show the microbial enzyme with
AFB1 effect, adds a molecular oxygen on No. 3 position C of AFB1 molecule, generates a pair of of epimer (AFQ1 and epi-AFQ1).Table
1 is the 1H-NMR spectroscopic data of two kinds of converted products.Circular dichroism (CD) further determines that product 1 is epi-AFQ1, product 2
It is AFQ1 the and epi-AFQ1 circular dichroism figure of reference under Fig. 4 for AFQ1 (on Fig. 4), quoted from Buchi et al.,
1975.Synthesis of aflatoxin Q1.J.Org.Chem.。
The 1H-NMR spectroscopic data of 1 two catabolites of table
(5) AFQ1 and epi-AFQ1 is isolated and purified: 40mg AFB1 and 100mg recombinant C otA albumen is added to 500mL
In the sodium phosphate buffer (pH 7.0) of 100mM, reaction 48h is stood under the conditions of 37 DEG C.500mL acetic acid is added after reaction
Ethyl ester, mixing of turning upside down, stands in separatory funnel, collects upper organic phase；500mL acetic acid is added in lower layer's water phase
Ethyl ester repeats to extract organic phase merging that is primary, extracting twice.The organic phase of collection is evaporated in batches with Rotary Evaporators, is added
Residue in the molten cucurbit of 5mL acetone weight, and be transferred in 10mL vial, it is dried with nitrogen, addition 1.5mL acetone weight is molten, adopts
It is isolated and purified with HPLC.
HPLC condition: ultraviolet wavelength 210nm；Mobile phase: methanol: water=45:55；Flow velocity 2mL/min；Column model:
YMC-Pack ODS-A(5μm,120A)250X 4.6mm I.D.；Sample volume: 50 μ L.
As shown in figure 5, A is the HPLC figure of 2 mixture of the product 1 finally prepared and product, B is the product 1epi- of purifying
The HPLC of AFQ1 schemes, content 2.7mg；Scheme the HPLC that C is product 2AFQ1 to scheme, content 2.5mg.
Microbial enzyme used in the method for the present invention is made by microbial fermentation, and the microorganism can be conversion
There is the recombinant bacterium of the CotA laccase plasmid of aflatoxin degradation to be also possible to the microorganism of itself secretion laccase CotA albumen, wraps
Include one of bacillus, lactic acid bacteria and Bifidobacterium, preferably bacillus；The enzyme is bacterial laccase, fungi paint
Enzyme, arabinofuranosidase, cellulase, chitinase, cutinase, deoxyribonuclease, ribalgilase, esterase, Portugal
Polysaccharide lyase, glucose oxidase, glycuronidase, hydrolase, invertase, isomerase, lipolytic enzyme, lyases, oxidizing ferment,
Blue multicopper oxidase, oxidoreducing enzyme, transelminase, peroxidase, catalase, polyphenol oxidase, Carboxylesterase,
One of amino polyol amine oxidase, carboxypeptidase and glucosidase are a variety of；Preferably bacterial laccase.
The invention has the advantages that the method for the present invention for preparing AFQ1 and epi-AFQ1 standard items it is short when good,
It is at low cost, be easily achieved large-scale production and preparation.Ginseng is provided to the detoxication mechanisms of AFB1 simultaneously for further investigation animal's liver
According to object, to further investigate the toxicity of AFQ1 and its providing raw material in the intracorporal catabolism of machine.
Detailed description of the invention
Fig. 1 show AFB1 blank control group and AFB1 under UPLC-TOF-MS positive ion mode and microbial enzyme is added to handle
The total ion current figure of group (A is AFB1 blank control group, and B is that AFB1 adds microbial enzyme processing group)；
Fig. 2, which show the MS of AFB1 and two catabolite under UPLC-TOF-MS positive ion mode, to scheme；
Fig. 3 show 1D-NMR and the 2D-NMR figure of AFQ1 and epi-AFQ1；
Fig. 4 show AFQ1 and epi-AFQ1 circular dichroism (CD) spectrogram (A) product AFQ1 produced by the present invention and
The CD figure of the AFQ1 and epi-AFQ1 of CD figure (B) Buechi et al. (1975) report of epi-AFQ1；
Fig. 5 show HPLC figure (A) product 1 and 2 mixture of product that two kinds of products are prepared with micro bioenzyme catalysis AFB1
HPLC figure (B) purifying product 1 HPLC figure (C) purifying product 2 HPLC figure.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means.
Embodiment 1 is a kind of to prepare aflatoxin Q 1 (AFQ1) and its poor using CotA laccase oxidation catalysis AFB1 standard items
To the method for isomers (epi-AFQ1)
(1) preparation of microbial enzyme: the conversion of -80 DEG C of preservations is taken to have the recombination bacillus coli of PET-31b-CotA plasmid
Rosseta (DE3) is inoculated in 5mL LB liquid medium and activates overnight, and being transferred to 500mL liquid amount in 1:100 ratio is
In the triangular flask of 300mL, 180r/min, culture is 0.6 to OD600 under the conditions of 37 DEG C, adds final concentration of 0.1mM IPTG and lures
Lead destination protein expression.Fermentation liquid is collected, 4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant；With the phosphate buffer weight of pH7.0
Outstanding thallus, 4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant, repeated washing thallus is three times.Then somatic cells are resuspended in 1mg/
In the binding buffer of mL, ultrasonication, 4 DEG C, 12000rpm is centrifuged 10min, collects supernatant and filters.Use nickel ion
Affinity column (Ni2+- NTA) purification of recombinant proteins, microbial enzyme is made, which being capable of bioconversion AFB1.
(2) aflatoxin B1: being dissolved into the mother liquor that 20 μ g/mL are made into dimethyl sulfoxide by the bioconversion of AFB1,
It is tested by following 5mL reaction system: 4.3mL sodium phosphate buffer (0.1M, pH8.0), 0.2mL CotA albumen (100 μ
G), 0.5mL aflatoxin B1 solution.Not add the system of microbial enzyme as control.Reaction adds after carrying out 12h at 37 DEG C
Enter 5mL methanol and terminates reaction.
(3) it AFB1 assay: using the concentration of high performance liquid chromatography detection AFB1, is determined according to the variation of AFB1 concentration
Microbial enzyme has bioconversion activity to AFB1.
The chromatographic condition of high performance liquid chromatography detection AFB1 are as follows: chromatographic column: Agilent C18 chromatographic column, 4.6mm ×
150mm×5μm；Mobile phase: methanol-water (45:55)；Flow velocity: 1mL/min；Pump pressure: 75bar；Sample volume: 20 μ L；Fluorescence detection
Device Detection wavelength: λ ex=360nm, λ em=440nm；Acquisition time: 20min.
96% AFB1 is converted after measured.
(4) identification of product AFQ1 and epi-AFQ1: micro bioenzyme catalysis AFB1 is analyzed by UPLC-TOF-MS and generates two
The molecular weight of kind product, in the positive-ion mode, AFB1 adds the peak H m/z=313, and two products add the peak H m/z=329；Nuclear-magnetism is total
Shake (NMR) the result shows that, CotA laccase and AFB1 are acted on, and a molecular oxygen is added on No. 3 position C of AFB1 molecule, and it is a pair of poor to generate
To isomers (AFQ1 and epi-AFQ1).Circular dichroism (CD) further determines that product 1 is epi-AFQ1, and product 2 is AFQ1.
(5) preparation and purification of AFQ1 and epi-AFQ1: 40mg AFB1 and 100mg recombinant C otA albumen is added to
In the sodium phosphate buffer (pH 7.0) of 500mL 100mM, reaction 48h is stood under the conditions of 37 DEG C.It is added after reaction
500mL ethyl acetate, mixing of turning upside down, stands in separatory funnel, collects upper organic phase；It is added in lower layer's water phase
500mL ethyl acetate repeats to extract organic phase merging that is primary, extracting twice.In batches with Rotary Evaporators by the organic phase of collection
It is evaporated, the residue in the molten cucurbit of 5mL acetone weight is added, and be transferred in 10mL vial, is dried with nitrogen, 1.5mL third is added
Ketone weight is molten, is isolated and purified using HPLC.
HPLC condition: ultraviolet wavelength 210nm；Mobile phase: methanol: water=45:55；Flow velocity 2mL/min；Column model:
YMC-Pack ODS-A(5μm,120A)250X 4.6mm I.D.；Sample volume: 50 μ L.
Final preparation epi-AFQ1 is 2.7mg；AFQ1 is 2.5mg.
Embodiment 2 it is a kind of using CotA laccase oxidation catalysis AFB1 crude product prepare aflatoxin Q 1 (AFQ1) and its difference to
The method of isomers (epi-AFQ1)
(1) preparation of microbial enzyme: the conversion of -80 DEG C of preservations is taken to have the recombination bacillus coli of PET-31b-CotA plasmid
Rosseta (DE3) is inoculated in 5mL LB liquid medium and activates overnight, and being transferred to 500mL liquid amount in 1:100 ratio is
In the triangular flask of 300mL, 180r/min, culture is 0.6 to OD600 under the conditions of 37 DEG C, adds final concentration of 0.1mM IPTG and lures
Lead destination protein expression.Fermentation liquid is collected, 4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant；With the phosphate buffer weight of pH7.0
Outstanding thallus, 4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant, repeated washing thallus is three times.Then somatic cells are resuspended in 1mg/
In the binding buffer of mL, ultrasonication, 4 DEG C, 12000rpm is centrifuged 10min, collects supernatant and filters.Use nickel ion
Affinity column (Ni2+- NTA) purification of recombinant proteins, microbial enzyme is made, which being capable of bioconversion AFB1.
(2) ferment preparation AFB1 crude product: 20kg rice culture inoculation aflatoxin superior strain, right after culture 14d
It is crushed, and is extracted, then be extracted with ethyl acetate with 80% ethanol solution, is concentrated to get acetic acid ethyl ester extract
(200g), then carry out silica gel column chromatography with chloroform/acetone system elutions, then carries out petroleum ether/acetone system elutions, obtains Huang
The crude product of aspertoxin B1 obtains aflatoxin B1 (50mg) after chloroform/methanol recrystallizes.
(3) microbial enzyme is reacted with AFB1 crude product: AFB1 crude product is dissolved into dimethyl sulfoxide be made into it is certain density
Then mother liquor adds 120mg recombinant C otA laccase and reacts 12h at 37 DEG C, methanol is added after reaction and terminates.Not add
The system of microbial enzyme is as control.
Using the concentration of high performance liquid chromatography detection AFB1, determine 97% or more AFB1 by bioconversion.
(4) identification of product AFQ1 and epi-AFQ1: micro bioenzyme catalysis AFB1 is analyzed by UPLC-TOF-MS and generates two
The molecular weight of kind product, in the positive-ion mode, AFB1 adds the peak H m/z=313, and two products add the peak H m/z=329；Nuclear-magnetism is total
Shake (NMR) the result shows that, CotA laccase and AFB1 are acted on, and a molecular oxygen is added on No. 3 position C of AFB1 molecule, and it is a pair of poor to generate
To isomers (AFQ1 and epi-AFQ1).
(5) preparation and purification of AFQ1 and epi-AFQ1: 500mL ethyl acetate is added in step (3) after reaction, up and down
It is mixed by inversion, is stood in separatory funnel, collect upper organic phase；The repetition of 500mL ethyl acetate is added in lower layer's water phase
Extraction is primary, and the organic phase extracted twice merges.The organic phase of collection is evaporated in batches with Rotary Evaporators, 5mL acetone is added
Residue in the molten cucurbit of weight, and be transferred in 10mL vial, it is dried with nitrogen, addition 1.5mL acetone weight is molten, using HPLC points
From purifying.
HPLC condition: ultraviolet wavelength 210nm；Mobile phase: methanol: water=45:55；Flow velocity 2mL/min；Column model:
YMC-Pack ODS-A(5μm,120A)250X 4.6mm I.D.；Sample volume: 50 μ L.
Final preparation epi-AFQ1 is 3.5mg；AFQ1 is 3.2mg.
Embodiment 3 is a kind of to prepare aflatoxin Q 1 (AFQ1) and its poor using blue multicopper oxidase oxidation catalysis AFB1 crude product
To the method for isomers (epi-AFQ1)
(1) preparation of microbial enzyme: the conversion of -80 DEG C of preservations is taken to have the recombination large intestine of PET-31b- blue multicopper oxidase plasmid
Bacillus Rosseta (DE3) is inoculated in 5mL LB liquid medium and activates overnight, is transferred to 500mL liquid amount in 1:100 ratio
For in the triangular flask of 300mL, 180r/min, culture is 0.6 to OD600 under the conditions of 37 DEG C, adds final concentration of 0.1mM IPTG
Induce destination protein expression.Fermentation liquid is collected, 4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant；With the phosphate buffer of pH7.0
Thallus is resuspended, 4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant, repeated washing thallus is three times.Then somatic cells are resuspended in
In the binding buffer of 1mg/mL, ultrasonication, 4 DEG C, 12000rpm is centrifuged 10min, collects supernatant and filters.Use nickel
Ion affinity chromatography column (Ni2+- NTA) purification of recombinant proteins, microbial enzyme is made, which being capable of bioconversion AFB1.
(2) ferment preparation AFB1 crude product: 20kg rice culture inoculation aflatoxin superior strain, right after culture 14d
It is crushed, and is extracted, then be extracted with ethyl acetate with 80% ethanol solution, is concentrated to get acetic acid ethyl ester extract
(200g), then carry out silica gel column chromatography with chloroform/acetone system elutions, then carries out petroleum ether/acetone system elutions, obtains Huang
The crude product of aspertoxin B1 obtains aflatoxin B1 (52mg) after chloroform/methanol recrystallizes.
(3) microbial enzyme is reacted with AFB1 crude product: AFB1 crude product is dissolved into dimethyl sulfoxide be made into it is certain density
Then mother liquor adds 120mg recombination blue multicopper oxidase and reacts 12h at 37 DEG C, methanol is added after reaction and terminates.With not
Add the system of microbial enzyme as control.
Using the concentration of high performance liquid chromatography detection AFB1, determine 96% or more AFB1 by bioconversion.
(4) identification of product AFQ1 and epi-AFQ1: micro bioenzyme catalysis AFB1 is analyzed by UPLC-TOF-MS and generates two
The molecular weight of kind product, in the positive-ion mode, AFB1 adds the peak H m/z=313, and two products add the peak H m/z=329；Nuclear-magnetism is total
Shake (NMR) the result shows that, blue multicopper oxidase and AFB1 are acted on, and a molecular oxygen is added on No. 3 position C of AFB1 molecule, are generated a pair of
Epimer (AFQ1 and epi-AFQ1).
(5) preparation and purification of AFQ1 and epi-AFQ1: 500mL ethyl acetate is added in step (3) after reaction, up and down
It is mixed by inversion, is stood in separatory funnel, collect upper organic phase；The repetition of 500mL ethyl acetate is added in lower layer's water phase
Extraction is primary, and the organic phase extracted twice merges.The organic phase of collection is evaporated in batches with Rotary Evaporators, 5mL acetone is added
Residue in the molten cucurbit of weight, and be transferred in 10mL vial, it is dried with nitrogen, addition 1.5mL acetone weight is molten, using HPLC points
From purifying.
HPLC condition: ultraviolet wavelength 210nm；Mobile phase: methanol: water=45:55；Flow velocity 2mL/min；Column model:
YMC-Pack ODS-A(5μm,120A)250X 4.6mm I.D.；Sample volume: 50 μ L.
Final preparation epi-AFQ1 is 3.3mg；AFQ1 is 3.2mg.
Claims (7)
1. a kind of method for preparing AFQ1 and its epimer epi-AFQ1 using microbial enzyme oxidation catalysis AFB1, feature
It is, comprising the following steps:
(1) preparation of microbial enzyme: the conversion of -80 DEG C of preservations is taken to have the recombination bacillus coli of PET-31b-CotA plasmid
Rosseta (DE3) is inoculated in 5mL LB liquid medium and activates overnight, and being transferred to 500mL liquid amount in 1:100 ratio is
In the triangular flask of 300mL, culture is 0.6 to OD600 under the conditions of 37 DEG C, adds final concentration of 0.1mM IPTG induction destination protein
Expression；Fermentation liquid is collected, 4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant；Thallus is resuspended with the phosphate buffer of pH7.0, repeats
Washing thalline is three times；Then somatic cells are resuspended in the binding buffer of 1mg/mL, ultrasonication, 4 DEG C,
12000rpm is centrifuged 10min, collects supernatant and filters；Using nickel ion affinity chromatograph column purification recombinant protein, microorganism is made
Enzyme, the enzyme being capable of oxidation catalysis AFB1；
(2) AFB1: being dissolved into the mother liquor that 20 μ g/mL are made into dimethyl sulfoxide by the bioconversion of AFB1, is reacted by following 5mL
System is tested: 4.3mL sodium phosphate buffer (0.1M, pH8.0), and 0.2mL CotA albumen (100 μ g), 0.5mLAFB1 is molten
Liquid；Not add the system of microbial enzyme as control；The termination reaction of 5mL methanol is added after carrying out 12h at 37 DEG C in reaction；Using
The concentration of high performance liquid chromatography detection AFB1 determines that microbial enzyme is living with catalyzed conversion to AFB1 according to the variation of AFB1 concentration
Property；
(3) two kinds that micro bioenzyme catalysis AFB1 is generated the identification of product AFQ1 and epi-AFQ1: are analyzed by UPLC-TOF-MS
The structure of product, in the positive-ion mode, AFB1 add the peak H m/z=313, and two products add the peak H m/z=329；Using nuclear-magnetism
Determine that two kinds of substances that molecular weight is 328 are AFQ1 and epi-AFQ1；Further determine that product 1 is finally by circular dichroism
Epi-AFQ1, product 2 are AFQ1；
(4) AFQ1 and epi-AFQ1 is isolated and purified: 40mg AFB1 and 100mg recombinant C otA albumen is added to 500mL
In the sodium phosphate buffer (Ph7.0) of 100mM, reaction 48h is stood under the conditions of 37 DEG C；500mL acetic acid is added after reaction
Ethyl ester, mixing of turning upside down, stands in separatory funnel, collects upper organic phase；500mL acetic acid is added in lower layer's water phase
Ethyl ester repeats to extract organic phase merging that is primary, extracting twice；The organic phase of collection is evaporated in batches with Rotary Evaporators, is added
Residue in the molten cucurbit of 5mL acetone weight, and be transferred in 10mL vial, it is dried with nitrogen, addition 1.5mL acetone weight is molten, adopts
It is isolated and purified with HPLC；HPLC condition: ultraviolet wavelength 210nm, mobile phase: methanol: water=45:55, flow velocity 2mL/min, sample introduction
Measure 50 μ L；Two kinds of products are collected respectively.
2. the method according to claim 1, wherein the AFB1 can be the toxin mark that purity is greater than 95%
Quasi- product are also possible to the AFB1 crude product by being inoculated with aflatoxin superior strain fermenting and producing.
3. the method according to claim 1, wherein the microbial enzyme be as made from microbial fermentation,
The microorganism is the recombinant bacterium for the CotA laccase plasmid that conversion has aflatoxin degradation, this can be secreted by being also possible to itself
One of microorganism of fermentoid, including bacillus, lactic acid bacteria and Bifidobacterium, preferably bacillus.
4. the method according to claim 1, wherein the microbial enzyme be bacterial laccase, fungal laccase, I
Primary furans carbohydrase, cellulase, chitinase, cutinase, deoxyribonuclease, ribalgilase, esterase, glucan cracking
Enzyme, glucose oxidase, glycuronidase, hydrolase, invertase, isomerase, lipolytic enzyme, lyases, oxidizing ferment, the oxidation of more copper
Enzyme, oxidoreducing enzyme, transelminase, peroxidase, catalase, polyphenol oxidase, Carboxylesterase, amino are polynary
One of alcohol amine oxidase, carboxypeptidase and glucosidase are a variety of.
5. claim 1, claim 3 and microbial enzyme as claimed in claim 4 are in preparation AFQ1 and epi-AFQ1 toxin mark
Application in quasi- product.
6. method described in claim 1 is preparing the application in AFQ1 toxin standard items.
7. method described in claim 1 is preparing the application in epi-AFQ1 toxin standard items.
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| CN102250848A (en) * | 2011-07-12 | 2011-11-23 | 湖北工业大学 | Method for purifying recombinant aspergillus flavus uricase expressed by bacillus coli |
| CN105112394A (en) * | 2015-09-11 | 2015-12-02 | 江南大学 | Separation and purification method of lyase capable of degrading aflatoxin B1 |
| CN109820132A (en) * | 2018-12-07 | 2019-05-31 | 中国农业大学 | Application of the bacterial laccase CotA albumen in degradation mycotoxin |
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| CN102250848A (en) * | 2011-07-12 | 2011-11-23 | 湖北工业大学 | Method for purifying recombinant aspergillus flavus uricase expressed by bacillus coli |
| CN105112394A (en) * | 2015-09-11 | 2015-12-02 | 江南大学 | Separation and purification method of lyase capable of degrading aflatoxin B1 |
| CN109820132A (en) * | 2018-12-07 | 2019-05-31 | 中国农业大学 | Application of the bacterial laccase CotA albumen in degradation mycotoxin |
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