CN114350716A - Method for promoting bisphenol A conversion by laccase induced copolymerization - Google Patents
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Abstract
The invention belongs to the interdiscipline of microbiology, enzyme catalysis kinetics, water pollution control and the like, and relates to a method for promoting bisphenol A conversion by laccase induced copolymerization. According to the method, coriolus versicolor (Trametes versicolor) laccase is used as a biocatalyst, bisphenol A and humic acid are used as coexisting substrates, and the trapping and sequestration effects of long-chain bisphenol A autopolymers on laccase molecules are eliminated through a free radical cross-bonding mechanism started by coriolus versicolor laccase, so that the enzyme catalysis activity, stability and durability are maintained, and the simple, rapid and efficient removal and conversion of the bisphenol A are realized. The laccase from coriolus versicolor induces the supermolecule copolymerization particles with complex and stable chemical structures generated by the bisphenol A and the humic acid, and the biological toxic effect of the bisphenol A monomer on the cherry radish is thoroughly avoided. The method has the advantages of controllable catalytic process, high removal efficiency, greenness, low carbon, energy conservation, environmental protection and the like, and has great application potential in the aspects of water pollution treatment, global carbon sequestration and the like.
Description
Technical Field
The invention belongs to the cross-fusion field of applied microbiology, enzyme catalysis kinetics, water pollution control science and the like, and relates to a method for promoting bisphenol A conversion by laccase induced copolymerization.
Background
Bisphenol A (BPA) is an important organic plasticizer in the industrial manufacturing field and is widely applied to the production of plastic products containing high polymer materials such as polycarbonate, epoxy resin and the like. BPA in a water ecosystem is mainly derived from urban sewage and industrial wastewater discharge, BPA exposed to the environment shows a typical endocrine disrupting effect on organisms and can be absorbed and accumulated by human bodies through paths such as food network transmission, respiration and skin surface contact, and further the risk of crowd health is caused. Currently, high doses of BPA are detected in natural water, tap water, and even human urine, serum, breast milk, and placenta. BPA in human exposure causes diseases closely related to reproduction and development, such as ovarian cancer, breast cancer, prostate cancer and the like. In addition, excessive intake of BPA can also cause cognitive impairment and memory deficits in children. Therefore, how to simply, rapidly and efficiently eliminate BPA pollution in water and avoid the ecological environment risk? Has become one of the key scientific problems which needs to be solved urgently by the learners.
Extracellular enzyme-induced oxidative polymerization is an important pathway for the reduction and conversion of BPA in water bodies. Laccase (EC 1.10.3.2) as an extracellular copper-containing polyphenol oxidoreductase commonly existing in white rot fungi can catalyze the one-electron oxidation of various phenolic and non-phenolic substrates. Among them, Coriolus versicolor (Trametes versicolor) laccase has been attracting much attention from researchers because of its strong oxidizing property, cost effectiveness and high selectivity. Compared with the traditional method for advanced oxidation and biodegradation of BPA, the oxidative polymerization reaction induced by the fungal laccase has the characteristics of simple operation process, high catalytic efficiency, low energy loss, greenness, no pollution and the like. For example, the single electron oxidation of BPA initiated by Coriolus versicolor laccase can form unstable phenol radical reactive intermediates, and meanwhile, the unstable phenol radical reactive intermediates are reduced into water along with dissolved oxygen in water, and hydrogen peroxide is not needed in the process. The resulting phenolic free radical intermediates then randomly and spontaneously combine to form stable macromolecular C-C and/or C-O-C covalent autopolymers, outside the enzymatically catalytically active sites. With increasing oxidation time and degree of polymerization, BPA autopolymers produced during the enzymatic reaction gradually increase in molecular weight and gradually stabilize in chemical structure, but gradually decrease in solubility in water and precipitate. Thus, these macromolecular polymerization products can be removed by simple centrifugation or filtration processes. The generation of high molecular BPA covalent autopolymer can effectively reduce the estrogenic activity and the biological toxicity of parent compounds. It is emphasized that, as the polymerization time and degree of BPA induced by Coriolus versicolor laccase increase, the formation of long-chain BPA autopolymers gradually increases, which leads to a significant decrease in the catalytic activity of the laccase and even to inactivation, thus seriously hindering the subsequent enzymatic reaction rate and removal effect of BPA monomers.
Humic Acid (HA) is a natural, polydisperse supramolecular polymer ubiquitous in the aquatic environment and having a concentration of up to 30 mg.L-1The above. The polymer is composed of hydrophobic aromatic skeleton, and contains various phenolic hydroxyl (-OH), carboxyl (-COOH), and amino (-NH)2) And quinone (C ═ O) and the like. During the biogeochemical cycle, the phenol-OH of HA HAs an electron donor effect, while C ═ O serves as an electron acceptor, accelerating electron transfer. It is generally accepted that HA interferes with the removal and conversion of many phenolic contaminants through competitive inhibition effects in chemical oxidation, microbial metabolism, and enzyme-induced free radical polymerization reactions. It is likely that the formation of contaminant-unstable reactive phenol intermediates is impaired by HA via a feedback mechanism of single electron transfer.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a method which is green, low-carbon, energy-saving and environment-friendly and is used for simply, efficiently and quickly treating bisphenol A in a water environment.
The technical scheme of the invention is as follows: a method for promoting the conversion of bisphenol A (BPA) in a water body by laccase-induced copolymerization is characterized in that Coriolus versicolor laccase is used as a biocatalyst, and bisphenol A (BPA) and Humic Acid (HA) are used as coexisting substrates to catalyze and generate a supermolecule BPA-HA copolymer.
Further, the concentration of the humic acid is not less than 10 mg.L-1Preference is given toIn addition, the concentration of humic acid is not less than 50 mg.L-1。
Further, the catalysis is carried out in an acidic system, preferably at a pH of 5.
Further, the catalytic time is more than 3h, preferably the catalytic time is more than 9 h.
In the invention, the trapping and the sealing of the laccase by the long-chain BPA autopolymer can be eliminated through a free radical cross-binding mechanism initiated by the laccase, so that the catalytic activity, the stability and the durability of the enzyme are maintained, and the BPA in the water body is simply and quickly removed.
The supermolecule BPA-HA copolymer formed by the method HAs a complex and stable chemical structure, and experiments prove that the biological toxic action of BPA monomers on cherry radishes can be completely avoided.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes coriolus versicolor (T.versicolor) laccase to induce the oxidation and copolymerization of BPA and HA, and the BPA conversion half-life period in the water body is only 0.48 h. Compared with the existing advanced oxidation and biodegradation methods, the method HAs obvious difference, when BPA and HA are used as the catalytic substrates of Coriolus versicolor laccase together, BPA and HA are firstly oxidized into active phenol free radical intermediates, and then the random combination of the phenol free radical intermediates is used for generating the supermolecule BPA-HA copolymer with complex and stable chemical structure. The formation of the BPA-HA copolymer eliminates the inhibiting effect of a long-chain BPA autopolymer on the active site of coriolus versicolor laccase, thereby ensuring the stability, high efficiency and durability of enzymatic reaction and achieving the simple, high-efficiency, rapid oxidation and removal of BPA.
The method for promoting BPA conversion by induced copolymerization of coriolus versicolor laccase can generate a large amount of high-tightly-combined supermolecule BPA-HA copolymerized particle precipitation products; as the enzymatic oxidation time and degree of polymerization increase, the molecular weight of the BPA-HA copolymer gradually increases and the chemical structure tends to stabilize. The method only consumes dissolved oxygen in water to generate only byproduct water without adding other chemical substances, and has the advantages of simple operation process, high conversion efficiency, greenness, low carbon, energy conservation, environmental protection and the like. The generated supermolecule BPA-HA copolymer thoroughly eliminates the biological toxic effect of BPA monomers on cherry radish, remarkably reduces the microbial mineralization of organic carbon in the environment and increases the storage and content of carbon in the world.
Drawings
FIG. 1 Effect of HA concentration on the conversion of BPA induced by Coriolus versicolor laccase;
FIG. 2 Effect of HA addition sequence on BPA conversion and laccase activity;
FIG. 3 Effect of HA on BPA autopolymer production and distribution;
FIG. 4 physical-chemical properties of supramolecular BPA-HA copolymers;
FIG. 5 phytotoxic effect of supramolecular BPA-HA copolymers.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.
Example 1 Effect of HA on Coriolus versicolor laccase induced BPA Oxidation and removal
The effect of HA concentration on BPA conversion induced by Coriolus versicolor (T. versicolor) laccase was investigated. The key test steps are briefly described as follows: (1) a50 mL heat-resistant brown glass bottle was selected as the container for laccase initiated radical polymerization, containing 20mL 10mM citrate-phosphate buffer, 100. mu.M BPA and 0-50 (i.e., 0, 10, 20, 30, 40 and 50) mg.L-1HA; (2) accurately adding 2 U.mL into buffer solution-1Coriolus versicolor laccase (light brown, not less than 0.5U. mg)-1) Starting enzymatic polymerization; (3) performing standing, oxidizing and polymerizing for 3h under the conditions of pH 5.0, 25 ℃ and dark black by using laccase-initiated free radical polymerization; (4) further, after 3 hours of the enzymatic reaction, 30 mg. multidot.L was added to the system-1HA, the effect of HA addition sequence on laccase induced BPA conversion and enzyme activity was further investigated. 0.5mL of the reaction sample was periodically aspirated through a pipette and the highly active phenol radical intermediate generated in the enzymatic reaction was quenched with 0.5mL of chromatographically pure methanol. The concentration of BPA remaining in the quenched solution was determined by Waters high Performance liquid chromatography (HPLC, Milford, MA, USA) analysis. HPLC detection conditions: the mobile phase is chromatographic pure acetonitrile and super-acetonitrilePure water (7:3, v/v) at a flow rate of 1.0 mL/min-1Injection volume 20. mu.L, detection wavelength 278 nm. And measuring the absorbance change of the reaction solution at 468nm by using a cuvette color development method, and calculating the catalytic activity of the coriolus versicolor laccase. All experiments were set up in 3 replicates and mean values were calculated.
The results are shown in fig. 1, the trametes versicolor laccase has a high conversion rate to BPA within 1h, and then shows hysteresis, mainly due to the fact that the catalytic activity of the enzyme is remarkably inhibited. However, in the system with the coexistence of BPA and HA, BPA can be continuously, rapidly and efficiently oxidized and removed by coriolus versicolor laccase. Moreover, with the increase of HA concentration, the conversion rate of BPA is gradually increased, and the catalytic activity of Coriolus versicolor laccase is more durable and stable. The reaction conforms to a pseudo first order kinetic model (R) within 1h20.91-0.99), when the HA concentration is changed from 10 mg.L-1Increased to 50 mg.L-1Time, BPA conversion kinetic constant (k)prcs) From 1.08h-1Raise to 1.43h-1Is not added with HA (k)prcs＝0.61h-1) 1.77-2.34 times of treatment group, and conversion half-life (t)1/2) The minimum time is only 0.48 h. Therefore, HA can significantly improve BPA removal and conversion by maintaining the catalytic activity, stability and durability of Coriolus versicolor laccase. However, the effect of HA addition on BPA removal and laccase activity after 3h of reaction was negligible, suggesting that the blocking effect of BPA on enzyme activity is mainly due to the accumulation of the intermediate products formed by itself (fig. 2). Thus, the simultaneous presence of BPA and HA may facilitate the rapid incorporation of BPA conversion intermediates into supramolecular HA, thereby eliminating the trapping and sequestration of enzymes by hydrophobic intermediates formed from BPA.
Example 2 Effect of HA on BPA autopolymer production and distribution
Using LTQ-Orbitrap high resolution Mass Spectrometry (HRMS, Thermo Fisher Scientific, Bremen, Germany; molecular Mass accuracy)<5.0ppm) combined with the Xcalibur software analysis method, the elemental composition, molecular formula and chemical structure of the BPA conversion product were identified, and HA (concentration 30mg · L) was defined-1) Influence on yield and distribution of BPA autopolymers induced by coriolus versicolor laccase. A total of 3 macromolecular BPA autopolymers were selected, which had m/z values of 453.2071, 679.3054 and905.4067. based on the exact molecular masses and elemental compositions of the 3 compounds, these products were initially identified as BPA dimers (C)30H30O4) Trimer (C)45H44O6) And tetramer (C)60H58O8) (precision of molecular Mass)<2.0 ppm). It is noted that no small molecule BPA degradation products and no supramolecular BPA-HA copolymer production was detected. Mainly because of the complexity and uncertainty of HA structure, it is difficult to reveal the molecular mass and chemical structure of supramolecular BPA-HA copolymers from the molecular level. Therefore, 3 oligomers of BPA were selected and the effect of HA on BPA dimer, trimer, tetramer production and distribution was qualitatively studied (due to the lack of standard samples, the production of 3 BPA oligomers could not be quantitatively analyzed).
As shown in FIG. 3, in Coriolus versicolor laccase-induced BPA conversion, the relative content of its monomers gradually decreased due to rapid oxidation and self-polymerization of BPA (similar to the conclusions in FIG. 1); the yield of BPA dimers reached a maximum within 0.5h of the enzymatic polymerization and then gradually decreased as they continued to be oxidatively polymerized into BPA trimers and tetramers. The addition of HA effectively promoted the rapid conversion and elimination of BPA self-aggregates, particularly BPA tetramer product, compared to the non-HA-added treatment group. After 0.5-3h of enzymatic polymerization, the yield of BPA tetramer in the HA-deficient treated group was 1.58-5.97 times that of the HA-added treated group. It can be seen that HA can facilitate the oxidation and removal of BPA monomer by effectively reducing the production of BPA autopolymers. This is because the BPA autopolymer is rapidly incorporated into supramolecular HA, generating supramolecular BPA-HA copolymers, eliminating the trapping and sequestering effect of long-chain BPA autopolymers on laccases. The enhancement mechanism mainly comprises the following key steps: (1) production of reactive phenolic free radical intermediates. Firstly, the coriolus versicolor laccase induces BPA and HA to carry out single electron oxidation at a T1-Cu binding site to form an unstable active phenol free radical intermediate. Meanwhile, electrons from the substrate are transferred to the center of a trinuclear copper cluster consisting of T2/T3-Cu, and the solution oxygen in the water body is reduced into water molecules. (2) BPA autopolymerization. These reactive phenolic intermediates are then free to polymerize beyond the enzymatic oxidation site to form C-C and/or C-O-C dimers, trimers, tetramers, oligomers and polymers of BPA. The formed long-chain BPA self-polymer can quickly trap and store laccase, so that the catalytic activity of the laccase is reduced and even inactivated, and further oxidation and removal of BPA monomers are hindered. (3) BPA autopolymer and HA were copolymerized. The copolymerization mechanism of BPA is similar to that of BPA autopolymerization, and BPA autopolymers have higher hydrophobicity and binding energy, so that the BPA autopolymers are easily covalently bound with an active intermediate of HA to generate supermolecular BPA-HA copolymers. The formation of the copolymer effectively eliminates the inhibitory effect of BPA autopolymers on enzyme activity, significantly facilitates BPA removal and conversion, but this process impairs the BPA autopolymerization pathway.
Example 3 physicochemical Properties of supramolecular BPA-HA copolymers
A continuous large-batch test method is adopted to obtain a large amount of macroscopic supramolecular BPA-HA copolymerization particle products, and the basic physicochemical properties of the supramolecular BPA-HA copolymer are analyzed. The main test operation steps are briefly described as follows: (1) the reactor contained 1L of 10mM citrate-phosphate buffer, 100. mu.M BPA, 30 mg.L-1HA and 2 U.mL-1Laccase from coriolus versicolor; (2) the reaction system is placed in a dark place at 25 ℃ and under the condition of pH 5.0 for 150r min-1Carrying out enzymatic oxidative polymerization for 3h, immediately adding high-concentration hydrochloric acid to acidify the enzymatic reaction liquid to pH 1.0; the process can quench the active phenol free radical intermediate and promote the precipitation and precipitation of the supermolecule BPA-HA copolymer; (3) standing the reaction solution quenched by the free radicals for 24 hours in a dark place to obtain a large amount of visual supramolecular BPA-HA copolymerized particle precipitation products; (4) the collected BPA-HA copolymer is dried, ground and screened, and then the surface morphology, functional groups and chemical structure characteristics are measured.
The supramolecular BPA-HA copolymerized particle product is black, compact in texture and irregular in surface morphology (a in figure 4). Fourier Infrared Spectroscopy (FTIR, NEXUS870, NICOLET, USA) data show that these copolymerised particle products contain reactive groups such as phenolic-OH, -COOH and aromatic ethers (b in FIG. 4).1H-nuclear magnetic resonance (1H-NMR, Agilent Technologies, USA) spectra indicated that they exhibited significant chemical bands in the high field region (1.0-4.0ppm), indicating that the BPA-HA copolymerIs composed of oligomer and high polymer; the disappearance of the C-H protons in the low field region (6.5-7.2ppm) confirms that the phenol-OH of the BPA and HA monomers is oxidized and forms C-C and/or C-O-C polymeric products (C in FIG. 4) by free radical induced dehydrogenation. Thus, Coriolus versicolor laccase can initiate free radical cross-polymerization of BPA and HA to produce BPA-HA copolymerized particle precipitation products with high aromaticity and humification degree. The resulting BPA-HA copolymer released only 0.3% to 0.5% loosely bound BPA monomer at pH2.0 to 11.0, indicating that the pH and environmental stability of the BPA-HA copolymer was high (d in FIG. 4).
Example 4 phytotoxicity evaluation of supramolecular BPA-HA copolymers
Evaluating the phytotoxic effect of the generated supermolecule BPA-HA copolymer on cherry radish within 9h of BPA and HA oxidative polymerization induced by coriolus versicolor laccase. A250 mL glass reactor contained 100mL of 10mM citrate-phosphate buffer, 0.5mM BPA, 150 mg.L-1HA and 10 U.mL-1And (3) quenching laccase for inducing copolymerization reaction after 3 hours, 6 hours and 9 hours respectively, adding 0.3% agar (w/v), heating and dissolving at high temperature, and preparing a plant solid culture medium. Subsequently, the cherry radish seeds (purchased from agricultural academy of sciences, Anhui province, China fertilizer combinations) were placed in 75% ethanol, surface sterilized for 2-3min, and washed several times with autoclaved deionized water. Seeds, saturated in size, uniform in size, and pregerminated, were selected and distributed evenly on 30mL solid plates containing 0.3% agar (w/v), 15 seeds per plate. A blank was set up by adding only sterilized 10mM citrate-phosphate buffer solid culture medium. Artificial climate box culture conditions: the humidity of the climatic chamber was 65%, and the light/dark cultivation period and temperature were 14/10h and 25/20 ℃ respectively. Culturing seedlings of the cherry radish seeds for 3d, measuring the germination rate (cotyledon and radicle appear) and root length of the seeds, and calculating the germination index.
As shown in fig. 5, the addition of BPA-HA monomer mixture (reaction 0h) significantly suppressed the germination rate, root elongation and germination index of cherry radish seeds compared to the blank control, mainly because high concentrations of BPA had a negative effect on plant seed germination and root development; whereas the toxic effect of supramolecular BPA-HA copolymers on plants gradually diminished or even disappeared with increasing oxidation time and degree of polymerization (a in fig. 5). For example, after the coriolus versicolor laccase initiated the copolymerization of BPA and HA for 3h, the germination indexes of the cherries and radish seeds were 99.9%, 5.1% and 69.8%, respectively, in the blank control, BPA-HA monomer mixture and supramolecular BPA-HA copolymer treatment groups (b in fig. 5), indicating that the BPA-HA monomer mixture had very high phytotoxicity to the cherries and radish seeds, whereas the BPA-HA copolymer showed only slight inhibition of plant growth (toxicity: germination index > 80%, no effect; 60% < germination index < 80%, slight effect; 40% < germination index < 60%, strong effect; germination index < 40%, severe effect). With the increase of the Coriolus versicolor laccase induced copolymerization reaction time from 3h to 9h, the germination index of the cherry radish seeds reaches 89.8%, and the longer the laccase induced copolymerization reaction time is, the larger the molecular weight, the more complex and stable the structure of the BPA-HA copolymer are, and the smaller the influence on the plant growth is.
Claims (7)
1. A method for promoting the conversion of bisphenol A (BPA) in a water body by laccase-induced copolymerization is characterized in that Coriolus versicolor laccase is used as a biocatalyst, BPA and Humic Acid (HA) are used as coexisting substrates, and a supramolecular BPA-HA copolymer is generated by catalysis.
2. The method according to claim 1, wherein the concentration of humic acid is not less than 10 mg-L-1。
3. The method according to claim 2, wherein the concentration of humic acid is not less than 50 mg-L-1。
4. The method of claim 1, wherein the catalyzing is performed in an acidic system.
5. The method of claim 4, wherein the catalytic system has a pH of 5.
6. The method of claim 1, wherein the catalytic time is greater than 3 hours.
7. The method of claim 6, wherein the catalytic time is greater than 9 hours.
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