CN110408662B

CN110408662B - Method for preparing AFQ1 and epi-AFQ1 by oxidizing and catalyzing AFB1 with microbial enzyme

Info

Publication number: CN110408662B
Application number: CN201910700119.2A
Authority: CN
Inventors: 赵丽红; 郭永鹏; 秦晓娟; 郑雅文; 马景林; 张琼琼
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2021-10-29
Anticipated expiration: 2039-07-31
Also published as: CN110408662A

Abstract

The invention provides a method for preparing aflatoxin Q1(AFQ1) and an epimer (epi-AFQ1) thereof by oxidizing and catalyzing aflatoxin B1(AFB1) by using microbial enzyme. The preparation method of the AFQ1 and epi-AFQ1 mycotoxin standard substance provided by the invention is simple in process, low in cost and easy for realizing large-scale preparation and production.

Description

Method for preparing AFQ1 and epi-AFQ1 by oxidizing and catalyzing AFB1 with microbial enzyme

Technical Field

The invention belongs to the technical field of biochemical engineering, and mainly relates to a method for preparing aflatoxin Q1(AFQ1) and an epimer (epi-AFQ1) thereof by using microbial enzyme and application thereof.

Background

Aflatoxin Q1(AFQ1) is one of the metabolites of the animal liver in metabolizing aflatoxin B1(AFB 1). The microsome P450 enzyme system (CYP1A2, CYP2A6, CYP2B7, CYP3A3, CYP3A4) of animal liver has a certain metabolism effect on AFBl, and the main pathways are hydroxylation, demethylation and epoxidation. AFBl is epoxidised by the P450 enzyme system to generate AFBO, which induces gene mutations when added to DNA. Studies have shown that different P450 s activate AFBl with different activity for AFBO production, and at low substrate concentrations, CYP1a2 is primarily responsible for the epoxidation of AFBl to AFBO. At high substrate concentrations, CYP3a4, in addition to CYP1a2, also exerts a certain epoxidation effect, oxidizing AFBl to AFBO. However, studies in human liver have shown that the major function of CYP3A4 is to detoxify it by oxidizing AFBl to form AFQl (Gallagher et al, 1994). At present, only this stage is studied, and there is no report on how the animal body metabolizes AFQ1 further in vivo. Toxicity studies on AFQ1 are also very limited. Indeed, these studies are necessary to understand the toxicity of AFQ1 to the body, what is the further metabolic processes and metabolic end products of AFQ1 in animals. Of course, if these studies were to be carried out, AFQ1 standard was used.

The current research and report related to the preparation and production of AFQ1 standard is very limited, and a complete and detailed production method cannot be found. The reasons for this are the following two main points: 1) the AFQ1 is only an intermediate metabolite of AFB1 metabolized by animal liver, the yield is low, and a pure product is difficult to obtain; 2) no other biological pathway has been found to produce and chemically synthesize AFQ 1. The production of the AFQ1 standard is limited for the reasons mentioned above, resulting in its extreme cost and also limiting the progress of the research work.

Disclosure of Invention

The invention aims to provide a method for producing aflatoxin Q1(AFQ1) and an epimer (epi-AFQ1) thereof, which has short production period and low cost, aiming at the defects of the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing aflatoxin Q1(AFQ1) and an epimer thereof (epi-AFQ1) by oxidizing and catalyzing AFB1 by using microbial enzyme comprises the following steps:

(1) preparation of microbial enzyme: recombinant E.coli transformed with PET-31b-CotA plasmid, stored at-80 ℃ was inoculated into 5mL of liquid LB medium for overnight activation (DE3) at a ratio of 1: transferring 100 proportion into 500mL triangular flask with liquid loading capacity of 300mL, culturing at 37 ℃ at 180r/min until OD600 is 0.6, and adding IPTG with final concentration of 0.1mM to induce target protein expression. Collecting fermentation liquor, centrifuging at 4 deg.C and 12000rpm for 30min, and removing supernatant; the cells were resuspended in phosphate buffer pH7.0, centrifuged at 12000rpm for 30min at 4 ℃ and the supernatant discarded, and the cells were washed three times. Then, the bacterial cells were resuspended in a binding buffer of 1mg/mL, disrupted by sonication, centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant was collected and filtered. Using a nickel ion affinity chromatography column (Ni)²⁺-NTA)Purifying the recombinant protein to produce a microbial enzyme capable of oxidizing AFB 1.

(2) Biotransformation of AFB 1: AFB1 was dissolved in dimethyl sulfoxide to prepare a 20. mu.g/mL stock solution, and the test was performed in a 5mL reaction system as follows: 4.3mL sodium phosphate buffer (0.1M, pH8.0), 0.2mL CotA protein (100. mu.g), 0.5mL AFB1 solution. The system without the microbial enzyme was used as a control. The reaction was carried out at 37 ℃ for 12h and then quenched by the addition of 5mL of methanol.

(3) AFB1 content determination: detecting the concentration of AFB1 by high performance liquid chromatography, and determining that the microbial enzyme has biotransformation activity to AFB1 according to the change of the concentration of AFB 1.

The chromatographic conditions for detecting AFB1 by high performance liquid chromatography are as follows: a chromatographic column: agilent C18 chromatography column, 4.6mm × 150mm × 5 μm; mobile phase: methanol-water (45: 55); flow rate: 1 mL/min; pumping pressure: 75 bar; sample introduction amount: 20 mu L of the solution; fluorescence detector detection wavelength: λ ex-360 nm, λ em-440 nm; collecting time: and 20 min.

It was determined that 95% of AFB1 was converted.

(4) Identification of products AFQ1 and epi-AFQ 1: analyzing the molecular weights of two products generated by catalyzing AFB1 by microbial enzyme through UPLC-TOF-MS, wherein a graph 1 is a total ion flow diagram of an AFB1 blank control group and an AFB1 and microbial enzyme treatment group in a positive ion mode (A is an AFB1 blank control group, and B is an AFB1 and microbial enzyme treatment group); fig. 2 shows that in positive ion mode, AFB1 plus H peak m/z is 313, and two products plus H peak m/z are 329; the Nuclear Magnetic Resonance (NMR) results (FIG. 3) show that the microbial enzyme reacts with AFB1 to add one molecule of oxygen to the 3-position C of AFB1 molecule, resulting in a pair of epimers (AFQ1 and epi-AFQ 1). Table 1 shows the 1H-NMR spectrum data of the two conversion products. Circular dichroism spectroscopy (CD) further confirmed that product 1 was epi-AFQ1, product 2 was AFQ1 (on FIG. 4), and reference AFQ1 and epi-AFQ1 circular dichroism spectrograms are shown below FIG. 4, from Buchi et al, 1975.Synthesis of aflatoxin Q1.J.Org.Chem.

TABLE 1H-NMR spectroscopic data of two degradation products

(5) Separation and purification of AFQ1 and epi-AFQ 1: 40mg of AFB1 and 100mg of recombinant CotA protein were added to 500mL of 100mM sodium phosphate buffer (pH 7.0) and allowed to stand still for reaction at 37 ℃ for 48 hours. After the reaction is finished, adding 500mL of ethyl acetate, reversing the upper part and the lower part, uniformly mixing, standing in a separating funnel, and collecting an upper organic phase; the lower aqueous phase was extracted once more with 500mL of ethyl acetate, and the organic phases from the two extractions were combined. The collected organic phases were evaporated down batchwise using a rotary evaporator, the residue in the distillation flask was redissolved with 5mL of acetone and transferred to a 10mL glass flask, blown dry with nitrogen, redissolved with 1.5mL of acetone and purified by HPLC.

HPLC conditions, ultraviolet wavelength 210 nm; mobile phase: methanol: water 45: 55; the flow rate is 2 mL/min; the type of the chromatographic column: YMC-Pack ODS-A (5 μm,120A)250X 4.6mm I.D.; sample introduction amount: 50 μ L.

As shown in FIG. 5, A is the HPLC chart of the mixture of the final prepared product 1 and product 2, and B is the HPLC chart of the purified product 1epi-AFQ1, the content of which is 2.7 mg; panel C is an HPLC plot of product 2AFQ1, at 2.5 mg.

The microbial enzyme used in the method is prepared by microbial fermentation, the microorganism can be a recombinant bacterium transformed with a CotA laccase plasmid for degrading aflatoxin or a microorganism which secretes laccase CotA protein per se, and the microorganism comprises bacillus, lactic acid bacteria and bifidobacterium, and is preferably bacillus; the enzyme is one or more of bacterial laccase, fungal laccase, arabinofuranosidase, cellulase, chitinase, cutinase, deoxyribonuclease, ribonuclease, esterase, glucan lyase, glucose oxidase, glucuronidase, hydrolase, invertase, isomerase, lipolytic enzyme, lyase, oxidase, multicopper oxidase, oxidoreductase, pectate lyase, peroxidase, catalase, polyphenol oxidase, carboxyl esterase, aminopolyol amine oxidase, carboxypeptidase and glucosidase; preferably a bacterial laccase.

The method for preparing the AFQ1 and epi-AFQ1 standard substances has the advantages of short time, low cost and easiness in realization of large-scale production and preparation. Meanwhile, the compound provides a reference substance for deeply researching the detoxification mechanism of animal liver to AFB1, and provides a raw material for deeply researching the toxicity of AFQ1 and the catabolism of the toxicity in the organism.

Drawings

FIG. 1 is a total ion flow graph of AFB1 blank control and AFB1 plus microbial enzyme treated groups in UPLC-TOF-MS positive ion mode (A is AFB1 blank control and B is AFB1 plus microbial enzyme treated group);

FIG. 2 is a MS plot of AFB1 and its two degradation products in the UPLC-TOF-MS positive ion mode;

FIG. 3 shows 1D-NMR and 2D-NMR spectra of AFQ1 and epi-AFQ 1;

FIG. 4 shows the Circular Dichroism (CD) spectra of AFQ1 and epi-AFQ1 (A) the CD spectra of AFQ1 and epi-AFQ1 reported in Buechi et al (1975) of products AFQ1 and epi-AFQ1 produced according to the present invention;

figure 5 shows HPLC plots of two products prepared by catalytic AFB1 with microbial enzymes (a) HPLC plot of product 1 and product 2 mixture (B) HPLC plot of purified product 1 (C) HPLC plot of purified product 2.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 method for preparing aflatoxin Q1(AFQ1) and epimer (epi-AFQ1) thereof by using CotA laccase to oxidize and catalyze AFB1 standard substance

(1) Preparation of microbial enzyme: recombinant E.coli transformed with PET-31b-CotA plasmid, stored at-80 ℃ was inoculated into 5mL of liquid LB medium for overnight activation (DE3) at a ratio of 1: transferring 100 proportion into 500mL triangular flask with liquid loading capacity of 300mL, culturing at 37 ℃ at 180r/min until OD600 is 0.6, and adding IPTG with final concentration of 0.1mM to induce target protein expression. Collecting fermentation liquor, centrifuging at 4 deg.C and 12000rpm for 30min, and removing supernatant; the cells were resuspended in phosphate buffer pH7.0, centrifuged at 12000rpm for 30min at 4 ℃ and the supernatant discarded, and the cells were washed three times. Then, the bacterial cells were resuspended in binding bu at 1mg/mLIn buffer, sonicate, centrifuge at 12000rpm for 10min at 4 ℃, collect supernatant and filter. Using a nickel ion affinity chromatography column (Ni)²⁺NTA) purification of the recombinant protein to produce a microbial enzyme capable of bioconversion of AFB 1.

(2) Biotransformation of AFB 1: aflatoxin B1 was dissolved in dimethyl sulfoxide to make a 20 μ g/mL mother liquor, and the test was performed in a 5mL reaction system as follows: 4.3mL of sodium phosphate buffer (0.1M, pH8.0), 0.2mL of CotA protein (100. mu.g), 0.5mL of aflatoxin B1 solution. The system without the microbial enzyme was used as a control. The reaction was carried out at 37 ℃ for 12h and then quenched by the addition of 5mL of methanol.

96% of the AFB1 was determined to be converted.

(4) Identification of products AFQ1 and epi-AFQ 1: analyzing the molecular weight of the microbial enzyme catalyzing AFB1 to generate two products by UPLC-TOF-MS, wherein in a positive ion mode, AFB1 plus H peak m/z is 313, and two products plus H peak m/z is 329; nuclear Magnetic Resonance (NMR) results show that CotA laccase reacts with AFB1, and one molecule of oxygen is added to position C of AFB1 molecule to generate a pair of epimers (AFQ1 and epi-AFQ 1). Circular Dichroism (CD) further confirmed that product 1 was epi-AFQ1 and product 2 was AFQ 1.

(5) Preparation and purification of AFQ1 and epi-AFQ 1: 40mg of AFB1 and 100mg of recombinant CotA protein were added to 500mL of 100mM sodium phosphate buffer (pH 7.0) and allowed to stand still for reaction at 37 ℃ for 48 hours. After the reaction is finished, adding 500mL of ethyl acetate, reversing the upper part and the lower part, uniformly mixing, standing in a separating funnel, and collecting an upper organic phase; the lower aqueous phase was extracted once more with 500mL of ethyl acetate, and the organic phases from the two extractions were combined. The collected organic phases were evaporated down batchwise using a rotary evaporator, the residue in the distillation flask was redissolved with 5mL of acetone and transferred to a 10mL glass flask, blown dry with nitrogen, redissolved with 1.5mL of acetone and purified by HPLC.

Final preparation epi-AFQ1 was 2.7 mg; AFQ1 was 2.5 mg.

Example 2 method for preparing aflatoxin Q1(AFQ1) and epimer (epi-AFQ1) thereof by using CotA laccase to oxidize and catalyze AFB1 crude product

(1) Preparation of microbial enzyme: recombinant E.coli transformed with PET-31b-CotA plasmid, stored at-80 ℃ was inoculated into 5mL of liquid LB medium for overnight activation (DE3) at a ratio of 1: transferring 100 proportion into 500mL triangular flask with liquid loading capacity of 300mL, culturing at 37 ℃ at 180r/min until OD600 is 0.6, and adding IPTG with final concentration of 0.1mM to induce target protein expression. Collecting fermentation liquor, centrifuging at 4 deg.C and 12000rpm for 30min, and removing supernatant; the cells were resuspended in phosphate buffer pH7.0, centrifuged at 12000rpm for 30min at 4 ℃ and the supernatant discarded, and the cells were washed three times. Then, the bacterial cells were resuspended in a binding buffer of 1mg/mL, disrupted by sonication, centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant was collected and filtered. Using a nickel ion affinity chromatography column (Ni)²⁺NTA) purification of the recombinant protein to produce a microbial enzyme capable of bioconversion of AFB 1.

(2) Preparing a crude AFB1 product by fermentation: inoculating 20kg rice culture with high-yield strain of aflatoxin, culturing for 14 days, pulverizing, extracting with 80% ethanol solution, extracting with ethyl acetate, concentrating to obtain ethyl acetate extract (200g), performing silica gel column chromatography, eluting with chloroform/acetone system, and eluting with petroleum ether/acetone system to obtain crude product of aflatoxin B1, and recrystallizing with chloroform/methanol to obtain aflatoxin B1(50 mg).

(3) Reacting microbial enzyme with AFB1 crude product: dissolving the AFB1 crude product into dimethyl sulfoxide to prepare mother liquor with a certain concentration, then adding 120mg of recombinant CotA laccase to react for 12 hours at 37 ℃, and adding methanol to terminate the reaction after the reaction is finished. The system without the microbial enzyme was used as a control.

The concentration of AFB1 was measured by HPLC to determine that more than 97% of AFB1 was biotransformed.

(4) Identification of products AFQ1 and epi-AFQ 1: analyzing the molecular weight of the microbial enzyme catalyzing AFB1 to generate two products by UPLC-TOF-MS, wherein in a positive ion mode, AFB1 plus H peak m/z is 313, and two products plus H peak m/z is 329; nuclear Magnetic Resonance (NMR) results show that CotA laccase reacts with AFB1, and one molecule of oxygen is added to position C of AFB1 molecule to generate a pair of epimers (AFQ1 and epi-AFQ 1).

(5) Preparation and purification of AFQ1 and epi-AFQ 1: after the reaction in the step (3) is finished, adding 500mL of ethyl acetate, reversing the upper part and the lower part, uniformly mixing, standing in a separating funnel, and collecting an upper organic phase; the lower aqueous phase was extracted once more with 500mL of ethyl acetate, and the organic phases from the two extractions were combined. The collected organic phases were evaporated down batchwise using a rotary evaporator, the residue in the distillation flask was redissolved with 5mL of acetone and transferred to a 10mL glass flask, blown dry with nitrogen, redissolved with 1.5mL of acetone and purified by HPLC.

Final preparation epi-AFQ1 was 3.5 mg; AFQ1 was 3.2 mg.

Example 3A method for preparing aflatoxin Q1(AFQ1) and its epimer (epi-AFQ1) by using a crude AFB1 product oxidized and catalyzed by a multi-copper oxidase

(1) Preparation of microbial enzyme: recombinant E.coli Rosseta (DE3) transformed with PET-31 b-multicopper oxidase plasmid, stored at-80 ℃ was inoculated in 5mL of liquid LB medium and activated overnight as 1: transferring 100 proportion into 500mL triangular flask with liquid loading capacity of 300mL, culturing at 37 ℃ at 180r/min until OD600 is 0.6, and adding IPTG with final concentration of 0.1mM to induce target protein expression. Collecting fermentation liquor, centrifuging at 4 deg.C and 12000rpm for 30min, and removing supernatant; the cells were resuspended in phosphate buffer pH7.0, centrifuged at 12000rpm for 30min at 4 ℃ and the supernatant discarded, and the cells were washed three times. Then the somatic cells are resuspendedIn a binding buffer of 1mg/mL, sonicated, centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant collected and filtered. Using a nickel ion affinity chromatography column (Ni)²⁺NTA) purification of the recombinant protein to produce a microbial enzyme capable of bioconversion of AFB 1.

(2) Preparing a crude AFB1 product by fermentation: inoculating 20kg rice culture with high-yield strain of aflatoxin, culturing for 14 days, pulverizing, extracting with 80% ethanol solution, extracting with ethyl acetate, concentrating to obtain ethyl acetate extract (200g), performing silica gel column chromatography, eluting with chloroform/acetone system, and eluting with petroleum ether/acetone system to obtain crude product of aflatoxin B1, and recrystallizing with chloroform/methanol to obtain aflatoxin B1(52 mg).

(3) Reacting microbial enzyme with AFB1 crude product: dissolving the AFB1 crude product into dimethyl sulfoxide to prepare mother liquor with a certain concentration, then adding 120mg of recombinant copper-rich oxidase to react at 37 ℃ for 12 hours, and adding methanol to terminate the reaction after the reaction is finished. The system without the microbial enzyme was used as a control.

The concentration of AFB1 was measured by HPLC to determine that more than 96% of AFB1 was biotransformed.

(4) Identification of products AFQ1 and epi-AFQ 1: analyzing the molecular weight of the microbial enzyme catalyzing AFB1 to generate two products by UPLC-TOF-MS, wherein in a positive ion mode, AFB1 plus H peak m/z is 313, and two products plus H peak m/z is 329; nuclear Magnetic Resonance (NMR) results showed that the multicopper oxidase reacted with AFB1 to add one molecule of oxygen to position C3 of AFB1 molecule to form a pair of epimers (AFQ1 and epi-AFQ 1).

Final preparation epi-AFQ1 was 3.3 mg; AFQ1 was 3.2 mg.

Claims

1. A method for preparing AFQ1 and an epimer epi-AFQ1 of the AFQ1 by utilizing multi-copper oxidase to oxidize and catalyze AFB1 is characterized by comprising the following steps:

(1) separation and purification of AFQ1 and epi-AFQ 1: adding 40mg of AFB1 and 100mg of multicopper oxidase into 500mL of 100mM sodium phosphate buffer solution, and standing for reaction at 37 ℃ for 48 hours; after the reaction is finished, adding 500mL of ethyl acetate, reversing the upper part and the lower part, uniformly mixing, standing in a separating funnel, and collecting an upper organic phase; adding 500mL of ethyl acetate into the lower water phase, repeatedly extracting once, and combining the organic phases extracted twice; evaporating the collected organic phase by using a rotary evaporator in batches, adding 5mL of acetone to redissolve the residue in a distillation flask, transferring the residue into a 10mL glass bottle, blowing the residue to dry by using nitrogen, adding 1.5mL of acetone to redissolve, and separating and purifying by using HPLC (high performance liquid chromatography); HPLC conditions comprise ultraviolet wavelength of 210nm, mobile phase of methanol: water 45:55, flow rate 2mL/min, sample volume 50 uL; collecting two products respectively;

(2) identification of AFQ1 and epi-AFQ 1: firstly, analyzing the structures of two products generated by catalyzing AFB1 by using multicopper oxidase through UPLC-TOF-MS, wherein in a positive ion mode, the sum of the AFB1 and the sum of the H peak m/z is 313, and the sum of the H peak m/z of the two products is 329; determining two substances with the molecular weight of 328 as AFQ1 and epi-AFQ1 by using a nuclear magnetic resonance technology; finally, the product 1 is further determined to be epi-AFQ1 by circular dichroism spectrum, and the product 2 is AFQ 1.

2. Use of the multicopper oxidase of claim 1 in the preparation of AFQ1 toxin standards.

3. Use of the multicopper oxidase of claim 1 in the preparation of epi-AFQ1 toxin standard.