阅读说明：本技术 一种铜基金属有机框架材料固定化漆酶及其制备方法和应用 (Copper-based metal organic framework material immobilized laccase and preparation method and application thereof ) 是由 程建华 袁宇航 杜克斯 周心慧 于 2020-12-25 设计创作，主要内容包括：本发明涉及环保领域,具体公开了一种铜基金属有机框架材料固定化漆酶及其制备方法和应用,将醋酸铜和漆酶溶解于水中,制得A液；将对氨基苯甲酸溶液溶于乙酸缓冲液中,制得B液,然后将上述两种溶液混合,并搅拌4小时以上；离心收集固体,用水洗涤,冷冻干燥,即制得铜基金属有机框架材料固定化漆酶。本发明首次采用铜基MOF(Cu-PABA)固定化漆酶,使用共沉淀的方法一步合成,更加快捷绿色,使漆酶的稳定性大大提高,在降解双酚A之后可以进行8次以上的回收。本发明固定化漆酶之后可以保留41.7％的活性。本发明产品具有很好的双酚A去除能力,在ABTS存在的情况下,12h双酚A去除率可以达到80％左右。(The invention relates to the field of environmental protection, and particularly discloses a copper-based metal organic framework material immobilized laccase as well as a preparation method and application thereof, wherein copper acetate and laccase are dissolved in water to prepare solution A; dissolving a p-aminobenzoic acid solution in an acetic acid buffer solution to prepare a solution B, mixing the two solutions, and stirring for more than 4 hours; and (4) centrifuging, collecting the solid, washing with water, and freeze-drying to obtain the copper-based metal organic framework material immobilized laccase. According to the invention, copper-based MOF (Cu-PABA) immobilized laccase is adopted for the first time, and the method of coprecipitation is used for one-step synthesis, so that the method is quicker and more green, the stability of the laccase is greatly improved, and the recovery can be carried out for more than 8 times after the bisphenol A is degraded. The immobilized laccase of the invention can retain 41.7% of the activity after the immobilization. The product of the invention has good bisphenol A removal capability, and the removal rate of bisphenol A in 12 hours can reach about 80% under the condition of ABTS.)

1. A preparation method of copper-based metal organic framework immobilized laccase is characterized in that copper acetate and laccase are dissolved in water to prepare solution A; dissolving a p-aminobenzoic acid solution in an acetic acid buffer solution to prepare a solution B, mixing the two solutions, and stirring for more than 4 hours; and (4) centrifuging, collecting the solid, washing with water, and freeze-drying to obtain the copper-based metal organic framework material immobilized laccase.

2. The preparation method according to claim 1, wherein the concentration of copper acetate in the solution A is 10-100 mM, and the concentration of laccase is 0.1-3.0 mg/mL; the concentration of p-aminobenzoic acid in the solution B is 5-25 mM.

3. The preparation method of claim 2, wherein the concentration of copper acetate in the solution A is 50 +/-25 mM, and the concentration of laccase is 0.5 +/-0.3 mg/mL; the concentration of p-aminobenzoic acid in the solution B is 12.5 +/-6.5 mM.

4. The method according to claim 1, 2 or 3, wherein the solution A and the solution B are mixed in equal volumes.

5. The method as claimed in claim 4, wherein the stirring speed is 200-500 rpm.

6. The process according to claim 5, wherein the stirring time is 4 to 12 hours.

7. The method according to claim 6, wherein the stirring is carried out for 8 hours and the washing is carried out 3 times.

8. The method of claim 7, wherein the freeze-drying is carried out at-40 ℃ for 12-24 hours.

9. The copper-based metal organic framework material immobilized laccase prepared by the method of any one of claims 1 to 8.

10. The use of the copper-based metal-organic framework material immobilized laccase for degrading bisphenol A as claimed in claim 9, wherein ABTS is further added into the degradation system.

Technical Field

The invention relates to the field of environmental protection, in particular to a preparation method of immobilized enzyme and application thereof in the field of removing bisphenol A.

Background

Bisphenol a is a typical endocrine disrupter, widely produced in baby bottles, toys and medical equipment, and BPA is detected in succession in soil, landfill leachate, air and food, in addition to surface and ground water. Common treatment methods are physical, chemical and biological. Physical methods generally include adsorption methods and membrane separation methods, which are relatively fast but not thorough, and are difficult to recycle, so that the application of the methods is limited. Chemical methods mainly include photocatalysis, electrochemistry, Fenton method and the like, generally, more energy sources are needed to be consumed, and the generated intermediate product is likely to have higher toxicity, thereby causing secondary pollution to the environment. The biological enzyme method has entered the field of view of the public due to its green and efficient characteristics.

In general, enzyme preparations are readily water soluble, are hardly recyclable after treatment of contaminants, and have poor free enzyme stability (temperature, chemistry, storage), further limiting their use. After the concept of immobilized enzyme is proposed, the problem is solved to a great extent. Hydrogel, porous carbon materials, polymer microspheres, mesoporous magnetic materials, mesoporous silica and the like are used for fixing enzymes, and metal organic framework materials are also used for fixing enzymes due to the fact that the metal organic framework materials have some excellent characteristics (large specific surface area, tunable functional groups, holes, topological structures and the like).

Compared with an ectopic method for fixing enzyme, the in-situ method directly fixes the enzyme on the MOF in a one-pot synthesis mode, can keep relatively mild environment (water environment, normal temperature and normal pressure), has certain benefit on enzyme activity retention, and is more green and rapid. A copper-based MOF (Cu-PABA) has excellent acid tolerance, and a laccase for degrading bisphenol A can exert the maximum activity under acidic conditions, so that a reliable material is provided for laccase immobilization. However, the existing immobilization method generally has the problem of low laccase retention activity. According to the previous literature research, the retention activity of the mesoporous silica immobilized laccase is 12.1%, the iron-based MOF immobilized laccase causes almost loss of the activity, the retention activity of the ZIF-8 immobilized laccase is 12.3% and 13.0%, and the retention activities of MIL-53 and Mg-MOF-74 immobilized laccase are 9.3% and 14.9%. In addition, the existing immobilization method also has the problems of complex process, higher cost, poor stability of immobilized laccase and the like.

Disclosure of Invention

The invention aims to provide a recyclable environment-friendly functional material for treating bisphenol A. The laccase is fixed on the MOF material by an in-situ method, so that the repeated utilization and the stability of an enzyme preparation are realized, and the cost for treating sewage is reduced.

The purpose of the invention is realized by the following technical scheme:

a method for preparing copper-based metal organic framework material immobilized laccase comprises dissolving copper acetate and laccase in water to obtain solution A; dissolving a p-aminobenzoic acid solution in an acetic acid buffer solution to prepare a solution B, mixing the two solutions, and stirring for more than 4 hours; and (4) centrifuging, collecting the solid, washing with water, and freeze-drying to obtain the copper-based metal organic framework material immobilized laccase.

Preferably, the concentration of copper acetate in the solution A is 10-100 mM, and the concentration of laccase is 0.1-3.0 mg/mL; the concentration of p-aminobenzoic acid in the solution B is 5-25 mM.

Preferably, the concentration of copper acetate in the solution A is 50 +/-25 mM, and the concentration of laccase is 0.5 +/-0.3 mg/mL; the concentration of p-aminobenzoic acid in the solution B is 12.5 +/-6.5 mM.

Preferably, the solution A and the solution B are mixed in equal volume.

Preferably, the stirring speed is 200-500 rpm.

Preferably, the stirring time is 4-12 h.

Preferably, the stirring is carried out for 8 hours, and the washing is carried out for 3 times.

Preferably, the temperature of the freeze drying is-40 ℃ and the time is 12-24 h.

The application of the copper-based metal organic framework material immobilized laccase in degrading bisphenol A is characterized in that ABTS is also added into a degradation system.

According to the invention, copper-based MOF (Cu-PABA) immobilized laccase is adopted for the first time, and the laccase is synthesized in one step by using a coprecipitation method, so that the method is quicker and more green, the stability (heat, pH and organic reagents) of the laccase is greatly improved, and the recovery can be carried out for more than 8 times after the bisphenol A is degraded. The immobilized laccase of the invention can retain 41.7% of the activity after the immobilization.

Compared with the prior art, the invention has the following beneficial effects:

(1) the product of the invention has good activity retention, and can retain about 40% of relative activity after the laccase is immobilized by an in-situ one-pot method. Due to the synergistic effect of the copper ions of the MOF material and the active center (copper ion cluster) of the laccase, the immobilized laccase has better affinity with guaiacol, K_mThe value decreases.

(2) The product of the invention has good stability, and the pH, temperature and organic reagent stability of the immobilized laccase and the free enzyme are all improved, which shows that the rigid skeleton of Cu-PABA has a certain protection effect on the space structure of the laccase, and the inactivation caused by the change of the conformation of the laccase is reduced.

(3) The product of the invention has good bisphenol A removal capability, the removal rate of bisphenol A in 12 hours can reach about 80% under the condition of ABTS, the introduction of ABTS accords with an electron transfer mechanism, and the electron transfer of a system is enhanced, so that the removal rate of bisphenol A is more efficient.

Drawings

FIG. 1 is an XRD pattern of Cu-PABA and Cu-PABA @ Lac.

FIG. 2 is a TGA curve of Cu-PABA and Cu-PABA @ Lac.

FIG. 3 is a Michaels-Menten plot of free laccase and immobilized laccase.

FIG. 4 is a Lineweaverv-Burk plot of free laccase and immobilized laccase.

FIG. 5 is a graph of the temperature stability of Cu-PABA and Cu-PABA @ Lac.

FIG. 6 is a graph of the pH stability of Cu-PABA and Cu-PABA @ Lac.

FIG. 7 is a graph of organic reagent stability for Cu-PABA and Cu-PABA @ Lac.

FIG. 8 is a graph showing the degradation curve of Cu-PABA @ Lac for 12h of bisphenol A at different dosages of immobilized enzyme.

FIG. 9 is a graph showing the degradation curves of Cu-PABA @ Lac for 12h of bisphenol A at different temperatures.

FIG. 10 is a graph of the degradation curves of Cu-PABA @ Lac for 12h bisphenol A at various initial concentrations of bisphenol A.

FIG. 11 is a graph of BPA removal for different conditions.

FIG. 12 is a graph showing the effect of Cu-PABA @ Lac on the recycling of degraded bisphenol A.

Detailed Description

The technical solution of the present invention is described in detail and completely with reference to the following specific examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention. All other embodiments obtained by a person skilled in the art without making any inventive step are within the scope of protection of the present invention.

Example 1

The synthesis method of Cu-PABA @ Lac comprises the following steps: copper acetate (50mM), laccase (0.5mg/mL, from sigma) was first dissolved in water; PABA (12.5mM) was dissolved in acetate buffer (ABS, pH 7.0,50 mM). The two solutions were then mixed in equal volumes and stirred at room temperature for more than 4 hours. The solid was collected by centrifugation and washed 3 times with deionized water.

Example 2

The synthesis method of Cu-PABA comprises the following steps: first copper acetate (50mM) was dissolved in water; PABA (12.5mM) was dissolved in acetate buffer (ABS, pH 7.0,50 mM). The two solutions were then mixed and stirred at room temperature for 8 hours. The solid was collected by centrifugation and washed 3 times with deionized water.

From the XRD spectrum of FIG. 1, there was no significant difference in the crystal structures of the Cu-PABA and Cu-PABA @ lac biocomposites. The diffraction peak intensity of Cu-PABA @ lac is reduced, and the result shows that laccase is successfully loaded and the structure of Cu-PABA is not greatly influenced by the introduced laccase.

The TGA curve of fig. 2 may also demonstrate the success of laccase loading. As can be seen from fig. 2, in the Cu-PABA curve, the mass loss below 160 ℃ (about 10%) is due to water evaporation, and the structure above 260 ℃ is destroyed. The TGA curve of Cu-PABA @ Lac, however, shows a clear difference between 240 ℃ and 260 ℃, which is related to the laccase loss.

Example 3: determination of the Retention Activity of an immobilized enzyme

50mmol/L sodium succinate as buffer (pH 4.5), 0.04mmol guaiacol and laccase/immobilized enzyme in 10mL reaction mixture, reacting at 30 deg.C for 30min, and measuring absorbance at 465 nm. The activity of the immobilized enzyme is calculated by taking the activity of the free enzyme as 100 percent, and the enzyme activity is kept. The product obtained in example 1 had a maximum retained enzyme activity of 41.7% as measured.

Example 4: kinetics of enzymatic reaction

Kinetic parameters were evaluated by varying the concentration of guaiacol in sodium succinate buffer. Figure 3 is a graph of the concentration of different guaiacols plotted against the initial rate of the enzymatic reaction, and figure 4 is a graph of the reciprocal of the figure 3. K_mThe value refers to the affinity between the enzyme and the substrate. Thus the smaller the Km value, the higher the affinity of the substrate for the laccase. V_maxThe values refer to the maximum enzymatic reaction rate.

As can be seen from FIG. 3, the enzymatic reaction rate increased first and then leveled off as the substrate concentration increased. In addition, the reaction rate of immobilized laccase is lower compared to free laccase due to limited mass transfer.

As shown in FIG. 4, K of free laccase_m0.0024mM, while Cu-PABA @ Lac shows K_mA decrease (0.0014mM) indicates an increased affinity of the immobilized enzyme for the substrate. Vmax after immobilization is from 1.80 mM. min^-1Down to 0.76mM min^-1This is due to the Cu-PABA shell preventing the substrate from contacting the laccase active site.

Example 5: stability test of immobilized enzyme

And (3) soaking the Cu-PABA and the Cu-PABA @ Lac for 1h at the temperature of 30-70 ℃, measuring the activities before and after soaking, and calculating the relative activities of the immobilized laccase and the free enzyme at different temperatures by taking the highest activity as 100%.

As shown in FIG. 5, the thermal stability of Cu-PABA @ Lac is higher than that of the free laccase at 30-70 ℃, which is due to the rigid skeleton of Cu-PABA and the interaction between MOF and enzyme (metal ions of MOF, organic ligands and laccase free amino and carboxyl carbonyl) and protects the spatial conformation of laccase from being changed.

Example 6: stability test of immobilized enzyme

And (3) soaking the Cu-PABA and the Cu-PABA @ Lac for 1h under the condition that the pH value is 3.5-9.5 (acetate buffer solution and phosphate buffer solution), measuring the activity before and after soaking, taking the highest activity as 100%, and calculating the relative activity of the immobilized laccase and the free enzyme under different pH values.

As shown in FIG. 6, the Cu-PABA @ Lac shows the same trend with the free laccase along with the increase of the pH value, the relative activity shows the trend of increasing firstly and then decreasing after soaking for 1h, and the pH stability of the immobilized laccase is obviously superior to that of the free laccase. Particularly under acidic conditions, the Cu-PABA @ Lac biological composite material shows better stability. Probably due to the attraction of the amine function of the ligand 4-aminobenzoic acid of the Cu-PABA to H⁺Ions, thereby forming an acidic microenvironment more suitable for the laccase.

Example 7: stability test of immobilized enzyme

Soaking Cu-PABA and Cu-PABA @ Lac in different organic reagents (methanol, ethanol, DMF, acetonitrile) for 1h, measuring the activity before and after soaking, taking the activity in water as 100%, and calculating the relative activity of immobilized laccase and free enzyme in different organic reagent environments.

Also, as shown in FIG. 7, Cu-PABA @ Lac has higher organic agent stability than free laccase. Generally, the organic solvent adsorbs the "essential water" of the laccase, resulting in denaturation of the free laccase. In the presence of the Cu-PABA protective shell, mass transfer of the organic reagent contacted with laccase is hindered, so that higher activity is retained.

Examples 8 to 10: bisphenol A degradation experiment

50mL of the system contains ABTS, immobilized laccase (0.7-5.6 U.mL-1) and bisphenol a (10-100 mg.L-1), and the degradation is carried out for 12h at different temperatures (15-55 ℃). At specific time intervals, samples were taken with a syringe and filtered through a 0.22 micron membrane filter. The sample concentration was then determined colorimetrically (4-aminoantipyrine) at 510 nm.

The 4-aminoantipyrine method comprises the steps of diluting 1mL of sample to 25mL, adding 0.5mL of ammonium chloride buffer solution (pH is 10.7), 1mL of 2% 4-aminoantipyrine solution and 1mL of 8% potassium ferricyanide solution, shaking up, and testing after 10min at a lambda of an ultraviolet spectrophotometer of 510 nm.

With the increase of the initial dosage of the immobilized laccase, the degradation rate of bisphenol A after 12h also increases, and can reach 79.4% at the maximum (figure 8). This is apparently due to the increase in initial dose which enhances a given concentration (20 mg-mL)^-1) The degradation ability of bisphenol A. However, increasing the initial dosage by several times did not result in a corresponding increase in degradation rate, probably due to poor contact of bisphenol-A with laccase due to material accumulation.

The effect of temperature on degradation is shown in figure 9. The removal rate tends to increase and decrease from 15 ℃ to 55 ℃, and reaches a maximum (85.7%) at 35 ℃. The degradation efficiency is not much different at 25 ℃, 35 ℃ and 45 ℃, and the degradation efficiency is lower at 15 ℃ and 55 ℃. This may be associated with a decrease in laccase activity at low and high temperatures. The initial degradation rate of the reaction is slower at 15 ℃ but the final degradation degree is higher compared to 55 ℃. The reason for this is that laccase activity is inhibited and degradation is slow at low temperature, whereas laccase activity is lost with time at high temperature and finally degradation is affected.

FIG. 10 shows the effect of BPA concentration, which can be seen at 20 mg.mL^-1And 30 mg. mL^-1When the degradation rate of the Cu-PABA @ Lac to the bisphenol A is the highest (84.3 percent and 84.6 percent). At low concentration (10 mg. mL)^-1) The reduced degradation of bisphenol-A may be due to a low driving force, resulting in no better contact of bisphenol-A with laccase. 10 mg/mL^-1And 20 mg. mL^-1The degradation equilibrium is reached more quickly no matter the concentration is 10 mg/mL-1, 20 mg/mL-1 or 30 mg/mL^-1Residual concentration of BPA after 12 hours (about 3 mg. mL)^-1) Are substantially the same. These phenomena are consistent with the above conclusions. As the concentration of bisphenol a increased to 50 mg. multidot.mL^-1And 100 mg. mL^-1The degradation rate is significantly reduced because the active site of laccase is blocked due to excessive bisphenol a and the production of intermediates.

As can be seen in fig. 11, the BPA change is not significant in the presence of ABTS alone. The adsorption capacity of pure Cu-PABA is measured, and about 10 percent of pure Cu-PABA is removed in 12 hours. After the laccase was immobilized, the material showed better BPA removal performance, about 26%, indicating that the presence of laccase is beneficial for degrading BPA. When ABTS (final concentration 0.2mM) was added to the system, the BPA degradation rate was close to 84.7% for 12 h.

Example 11: to determine the reusability of the Cu-PABA @ Lac biocomposite, the BPA removal rate was calculated after multiple degradation experiments, with the initial removal rate defined as 100%.

To evaluate the reusability of the prepared Cu-PABA @ Lac, a quantity of biocomposite was mixed with BPA and ABTS, and then collected by centrifugation. As shown in FIG. 12, BPA degradation rate gradually decreased with increasing cycle number, and the degradation rate remained nearly 70% after 8 cycles. This is of great significance for recycling laccase and reducing treatment cost.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.