CN110279974B

CN110279974B - Chemical-biological mixing method for degrading 2,4, 6-trinitrotoluene by using flavoenzyme

Info

Publication number: CN110279974B
Application number: CN201910651016.1A
Authority: CN
Inventors: 周阳; 魏桐; 杨治林
Original assignee: Institute of Chemical Material of CAEP
Current assignee: Institute of Chemical Material of CAEP
Priority date: 2019-07-18
Filing date: 2019-07-18
Publication date: 2021-06-29
Anticipated expiration: 2039-07-18
Also published as: CN110279974A

Abstract

The invention discloses a chemical-biological mixing method for degrading 2,4, 6-trinitrotoluene by using flavoenzyme, which comprises the following steps: (1) carrying out chemical modification on TNT to obtain a product OH-TNT or H-TNT so as to achieve the purpose of enhancing the negative charge of the TNT nitryl; (2) carrying out enzyme catalysis degradation on the obtained product and processing the degraded product. The method has the advantages of single product, no nitro reduction product, benzene ring dearomatization, hopeful realization of TNT ring opening degradation and the like.

Description

Chemical-biological mixing method for degrading 2,4, 6-trinitrotoluene by using flavoenzyme

Technical Field

The invention relates to the technical field of degradation of nitroaromatic compounds, in particular to a chemical-biological mixing method for degrading 2,4, 6-trinitrotoluene by using flavoenzyme.

Background

The nitroaromatic compounds are compounds simultaneously having aromatic rings and nitro functional groups, and are widely applied to military and civil fields of dyes, insecticides, explosives, pesticides and the like. In the production and use processes of chemical products, a large amount of nitroaromatics are released into environmental water and soil, and serious threats are caused to microorganisms, plants, animals and surrounding ecological environments through biological enrichment and food chain transmission.

TNT is used as nitro aromatic hydrocarbon explosive which is widely used, and has the advantages of low impact sensitivity, good initiation performance, low cost and the like. But TNT is the same asIt is also a toxic pollutant when the concentration of TNT in the air is 0.85 mg-cm^-3This results in an increase in mean corpuscular volume in the blood and a significant decrease in mean corpuscular hemoglobin content and mean corpuscular hemoglobin concentration. Long-term epidemiological monitoring shows that chronic TNT exposure can cause pancytopenia in toxic cataract, toxic hepatitis, jaundice, aplastic anemia, nerve injury and other toxic effects.

However, because the molecules of TNT are stable and symmetrical, the TNT is difficult to degrade by common biodegradation methods of aromatic compounds, namely, benzene ring is subjected to ring-opening degradation by benzene ring hydroxylation oxygenase or benzene ring cleavage oxygenase. The TNT enzymatic degradation method reported at present mainly comprises two ways, one is a nitro reduction path, namely, TNT is degraded by reducing nitro on a TNT benzene ring into amino, and the other is a Mesenheimer reduction path, namely, TNT is degraded by hydrogenating on a TNT aromatic ring to generate a Mesenheimer compound. However, the nitro reduction path has the problems of toxic intermediate products, incomplete degradation and the like; the Messenleimer reduction pathway is considered to be a promising pathway for TNT ring-opening degradation by means of protonation, hydrolysis and the like. Only three pure enzymes capable of degrading TNT through the pathway of meisenheimer are found in the present experiment, which are: pentaerythritol tetranitroreductase (PENTR), xenobiote reductase (XENBR), N-ethylmaleimide reductase (NEMR). Unfortunately, the three enzymes have been found to degrade TNT through the meisenheimer pathway with the formation of various highly toxic nitro reduction products when TNT is used as a substrate. The generation of a large amount of highly toxic nitro reduction products is not beneficial to the growth of protein expression strains (such as enterobacter cloacae, pseudomonas fluorescens and the like) which depend on enzyme catalytic degradation, and even has a toxic effect.

Disclosure of Invention

In order to overcome the technical defects, the invention provides a chemical-biological mixing method for degrading 2,4, 6-trinitrotoluene (TNT) by using flavoenzyme. The method has the advantages of single product, no nitro reduction product, benzene ring dearomatization, hopeful realization of TNT ring opening degradation and the like.

In order to achieve the technical effects, the invention provides the following technical scheme:

a chemical-biological hybrid method for degrading 2,4, 6-trinitrotoluene with flavoenzymes, comprising the steps of: (1) carrying out chemical modification on TNT to obtain a product OH-TNT or H-TNT so as to achieve the purpose of enhancing the negative charge of the TNT nitryl; (2) carrying out enzyme catalysis degradation on the obtained product and processing the degraded product.

The further technical scheme is that the preparation method of the H-TNT in the step (1) specifically comprises the steps of dissolving TNT or a mixture containing TNT and sodium borohydride in deionized water, shaking by hand to fully mix the system, then heating in a water bath to maintain the temperature of the system at 50 ℃ for hydrolysis reaction to obtain a chemically modified product H-TNT, and adding an acidic buffer solution after hydrolysis is completed to maintain the pH of the system at 7 and the temperature at 37 ℃.

The further technical scheme is that the preparation method of the OH-TNT in the step (1) specifically comprises the steps of dissolving TNT or a mixture containing TNT in a phosphoric acid buffer solution, keeping the pH of the solution at 11.7, shaking by hand to fully mix the system, then heating in a water bath to maintain the temperature of the system at 50 ℃ for hydrolysis reaction to obtain a chemically modified product OH-TNT, and adding an acidic buffer solution after the hydrolysis is completed to maintain the pH of the system at 7 and the temperature at 37 ℃.

The further technical scheme is that the preparation method of the phosphoric acid buffer solution comprises the steps of quantitatively mixing potassium dihydrogen phosphate and dipotassium hydrogen phosphate, then adding NaOH to enable the pH of the buffer solution to be 11.7 at room temperature, adding a proper amount of deionized water to dissolve, and finally obtaining the phosphoric acid buffer solution.

The further technical scheme is that the mass ratio of the monopotassium phosphate to the dipotassium phosphate is 1: 13.

The further technical proposal is that the ratio of the TNT to the deionized water is 50 mM: 20 ml.

The further technical proposal is that the ratio of the TNT to the phosphate buffer solution is 50 mM: 20 ml.

The further technical scheme is that the step (2) is specifically that an escherichia coli strain which can generate flavoenzyme through genetic engineering modification is added into the reaction system obtained in the step (1), and carbenicillin and IPTG are added as nutrients of microorganisms to perform enzyme-catalyzed biodegradation.

The further technical proposal is that the ratio of carbenicillin to IPTG is 200g:0.4 mM.

The further technical scheme is that the flavoenzyme is selected from any one of heterotypic biomass reductase, pentaerythritol tetranitrate reductase, morphine reductase, N-maleimide reductase and old yellow enzyme.

The invention utilizes molecular dynamics software AMBER to simulate the activity centers of five flavoenzymes (heterotypic biomass reductase (XENB), pentaerythritol tetranitrate reductase (PETNR), Morphine Reductase (MR), N-maleimide reductase (NEMR) and Old Yellow Enzyme (OYE)) of H-TNT molecules, and finds that the essential reason of the degradation path difference of H-TNT and TNT is caused by the electronic structure distribution difference of H-TNT and TNT. Specifically, compared with TNT, the negative nitro charge on H-TNT is stronger, so that flavin enzyme activity center residues are easier to form strong interaction such as hydrogen bonds, and the like, and form a pi-pi stacking structure with coenzyme flavin adenine mononucleotide (FMN) of an activity center, so that the energy barrier of a Mesonamer reduction path is reduced, and finally the degradation is realized through the Mesonamer pathway.

Therefore, we propose a strategy for degrading TNT by a chemical-biological hybrid method, namely, the TNT is firstly subjected to appropriate chemical modification by a chemical method, such as generating OH-TNT (anion) by means of alkaline hydrolysis; the H-TNT is generated through the reaction with sodium borohydride so as to achieve the purpose of enhancing the negative charge on the nitro group, and then the obtained chemical modification product is used as a substrate for enzyme catalytic degradation to carry out biodegradation through a Messemer pathway. The method has the advantages of single product, no nitro reduction product (high toxicity), dearomatization of benzene ring, hopeful realization of TNT ring-opening degradation and the like.

Drawings

FIG. 1 is a comparison of the electrostatic potentials and partial charges of TNT and H-TNT;

FIG. 2 shows the conformation of the TNT molecule in the OYE active center and its two-pathway reaction energy barrier;

FIG. 3 is the conformation of the H-TNT molecule at the OYE active center and its two pathway reaction energy barriers;

FIG. 4 shows the electrostatic potential distribution of OH-TNT (anion) molecules and the conformation at the PETNR active center.

Detailed Description

Example 1

A chemo-biological hybrid method for degrading 2,4, 6-trinitrotoluene using Old Yellow Enzyme (OYE), comprising the steps of:

(1) chemical modification of a substrate: 50mM TNT or a mixture containing TNT and sodium borohydride were dissolved in 20ml of deionized water and the system was mixed thoroughly by shaking with hand. Then heating in water bath to maintain the system temperature at 50 ℃ for reaction to obtain a chemically modified product (H-TNT);

(2) after the hydrolysis is finished, a proper amount of acid buffer solution is added to keep the pH of the system at 7 and the temperature at about 37 ℃, so that the method is suitable for the next enzyme-catalyzed reaction.

(3) Modifying a substrate for enzymatic degradation: adding an Escherichia coli strain which can generate OYE through genetic engineering modification into the reaction system in the step (2), and adding 200g of carbenicillin (carbenicillin) and 0.4mM of IPTG (isoproyl-beta-D-thiogalactopyranoside) as nutrients of the microorganism to carry out enzymatic biodegradation.

(4) And (3) post-treatment of degradation products: and taking the biodegradable mixture out of the reaction kettle for drying.

Experiments prove that: the OYE cannot degrade TNT through the Mesonamer pathway when TNT is used as a substrate, and can degrade through the Mesonamer pathway when a chemically modified Mesonamer monohydrogen compound (H-TNT) of TNT is used as a substrate. The OYE active center residues are predominantly Tyr196, His191, Asn194, Phe250, Asn294, Phe296 and Tyr 375. The conformation of the TNT molecule in the active center is shown in figure 2 through molecular dynamics simulation, and the TNT and FMN do not form a pi-pi stacking structure. Analysis of the interactions revealed that Phe250 and Phe296 of the OYE active center can form pi-pi stacking with TNT molecules simultaneously. Through reaction energy barrier calculation, the energy barrier of a nitro reduction path is lower than that of a Messemer reduction path, and degradation cannot be carried out through the Messemer pathway. After the substrate is replaced by H-TNT with stronger negative charge on the nitro group, the fact that the H-TNT can form a pi-pi stacking structure with FMN is found, the energy barrier of a nitro reduction path is higher than that of a Messenberger reduction path, and finally the H-TNT can be degraded through the Messenberger path, wherein the reaction energy barrier of the H-TNT in the OYE active center conformation and two paths is shown in figure 3.

Example 2

A chemo-biological hybrid process for the degradation of 2,4, 6-trinitrotoluene using tai-ampere enzyme (PETNR) comprising the steps of:

(1) preparing a buffer solution: quantitatively mixing potassium dihydrogen phosphate and dipotassium hydrogen phosphate, adding NaOH to enable the pH of a buffer solution to be about 11.7 at room temperature, adding a proper amount of deionized water to dissolve, and finally fixing the volume to 20 mL.

(2) Chemical modification of a substrate: 50mM TNT or a mixture containing TNT was dissolved in 20ml of phosphate buffer solution, the pH of the solution was kept at about 11.7, and the system was thoroughly mixed by shaking with hand. Then, water bath heating is carried out, so that the temperature of the system is maintained at 50 ℃ to carry out hydrolysis reaction, and a chemically modified product (OH-TNT (anion)) is obtained.

(3) After the hydrolysis is finished, an equivalent amount of acidic buffer solution is added, so that the pH of the system is kept at 7, and the temperature is kept to be about 37 ℃, and the method is suitable for the next enzyme-catalyzed reaction.

(4) Modifying a substrate for enzymatic degradation: adding an escherichia coli strain which can generate PETRN through genetic engineering modification into the reaction system in the step (3), and adding 200g of carbenicillin (carbenicillin) and 0.4mM of IPTG (isoproyl-beta-D-thiogalactopyranoside) as nutrients of the microorganism to carry out enzyme-catalyzed biodegradation.

(5) And (3) post-treatment of degradation products: and taking the biodegradable mixture out of the reaction kettle for drying.

Different types of TNT chemical modifiers such as H-TNT (radial), H-TNT (anion), OH-TNT (radial) and the like are obtained by carrying out different chemical modifications on TNT, and the electrostatic potential of modified products is analyzed, so that the following results are found: wherein OH-TNT (anion) and H-TNT have very similar electrostatic potential fractions. Through molecular dynamics simulation, OH-TNT (anion) can form a pi-pi stacking structure with FMN at the PETN active center and can also be degraded by the Messensettimer pathway through the enzyme Perflavin (PETNR), and the electrostatic potential distribution of OH-TNT (anion) molecules and the conformation at the PETNNR active center are shown in FIG. 4.

Although the present invention has been described herein with reference to the illustrated embodiments thereof, which are intended to be preferred embodiments of the present invention, it is to be understood that the invention is not limited thereto, and that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure.

Claims

1. A chemical-biological hybrid process for the enzymatic degradation of 2,4, 6-trinitrotoluene using flavins, comprising the steps of: (1) carrying out chemical modification on TNT to obtain a product OH-TNT so as to achieve the purpose of enhancing the negative charge of TNT nitryl; (2) carrying out enzyme catalytic degradation on the obtained product and treating the degraded product; the preparation method of the OH-TNT in the step (1) specifically comprises the steps of dissolving TNT or a mixture containing TNT in a phosphoric acid buffer solution, keeping the pH of the solution at 11.7, shaking by hand to fully mix the system, then heating in a water bath to maintain the temperature of the system at 50 ℃ for hydrolysis reaction to obtain a chemically modified product OH-TNT, and adding an acidic buffer solution after hydrolysis to maintain the pH of the system at 7 and the temperature at 37 ℃.

2. The chemical-biological mixing method for degrading 2,4, 6-trinitrotoluene with flavoenzymes according to claim 1, wherein the phosphate buffer solution is prepared by quantitatively mixing potassium dihydrogen phosphate and dipotassium hydrogen phosphate, adding NaOH to make the pH of the buffer solution at room temperature to be 11.7, adding an appropriate amount of deionized water to dissolve, and finally obtaining the phosphate buffer solution.

3. The chemo-biological mixing method for degrading 2,4, 6-trinitrotoluene with flavoenzymes according to claim 2, wherein the mass ratio of the potassium dihydrogen phosphate to the dipotassium hydrogen phosphate is 1: 13.

4. The chemo-biological mixing method for degrading 2,4, 6-trinitrotoluene with flavoenzymes according to claim 1, characterized in that the TNT to phosphoric acid buffer solution ratio is 50 mM: 20 ml.

5. The chemo-biological hybrid method for degrading 2,4, 6-trinitrotoluene by flavoenzyme according to claim 1, characterized in that the step (2) is carried out by adding escherichia coli strain capable of producing flavoenzyme through genetic engineering modification into the reaction system obtained in the step (1), and adding carbenicillin and IPTG as nutrients for microorganisms to carry out enzymatic biodegradation.

6. The chemo-biological mixing method for degrading 2,4, 6-trinitrotoluene with flavoenzymes according to claim 5, wherein the ratio of carbenicillin to IPTG is 200g:0.4 mM.

7. The chemo-biological hybrid process for degrading 2,4, 6-trinitrotoluene with flavoenzymes according to claim 5, wherein the flavoenzymes are selected from any one of xenobiotase, pentaerythritol tetranitrate reductase, morphine reductase, N-maleimide reductase and old yellow enzyme.