阅读说明：本技术 基于淀粉基纳米材料固定化生物酶微反应器及其应用 (Immobilized biological enzyme microreactor based on starch-based nano material and application thereof ) 是由 蒋育澄 何濛 胡满成 李淑妮 翟全国 于 2021-09-29 设计创作，主要内容包括：本发明公开了一种基于淀粉基纳米材料固定化生物酶微反应器及其应用,该生物酶微反应器是在淀粉水凝胶的制备过程中修饰氧化石墨烯,将修饰氧化石墨烯后的淀粉水凝胶作为载体,通过静电作用和氢键作用固定生物酶得到。本发明生物酶微反应器以淀粉基纳米材料为载体,生产成本低廉,具有工业化应用前景,且该载体具有致密的网络结构、高比表面积,同时富含羟基等亲水基团,既保留了游离酶高的催化活性,同时提高了酶的负载量,具有良好的催化活性、热稳定性及重复使用性,在DMF、甲醇、乙腈等有机溶剂中耐受性好,并且固定化辣根过氧化物酶微反应器用于降解废水中双酚A和2,4-二氯苯酚等有机物,降解速度快、降解率高。(The invention discloses a starch-based nano material-based immobilized bio-enzyme microreactor and application thereof. The bio-enzyme microreactor takes starch-based nano materials as a carrier, has low production cost and industrial application prospect, has a compact network structure and a high specific surface area, is rich in hydrophilic groups such as hydroxyl groups and the like, retains the high catalytic activity of free enzyme, improves the loading capacity of the enzyme, has good catalytic activity, thermal stability and reusability, has good tolerance in organic solvents such as DMF, methanol, acetonitrile and the like, is used for degrading organic matters such as bisphenol A, 2, 4-dichlorophenol and the like in wastewater, and has high degradation speed and high degradation rate.)

1. A immobilized biological enzyme microreactor based on starch-based nanomaterials is characterized in that: adding graphene oxide in the preparation process of the starch hydrogel to obtain graphene oxide modified starch hydrogel, and fixing biological enzyme by taking the hydrogel as a carrier through electrostatic action and hydrogen bond action to obtain the biological enzyme microreactor;

the biological enzyme is any one of horseradish peroxidase, catalase, hemoglobinoprotease, chloroperoxidase and cytochrome c.

2. The graphene oxide modified starch hydrogel immobilized bio-enzyme microreactor of claim 1, wherein the graphene oxide modified starch hydrogel is prepared by the following method: uniformly dispersing graphene oxide in deionized water by ultrasonic waves to obtain a graphene oxide suspension; uniformly stirring starch and dispersing the starch in deionized water to obtain a starch suspension; and adding the graphene oxide suspension into the starch suspension, fully mixing, heating the obtained mixed solution to 80-90 ℃, continuously stirring for 3-5 h, cooling to room temperature, and freeze-drying to obtain the graphene oxide modified starch hydrogel.

3. The graphene oxide-modified starch hydrogel immobilized bio-enzyme microreactor of claim 2, wherein: the concentration of the starch suspension is 0.08-0.12 g/mL, and the concentration of the graphene oxide suspension is 0.5-1 mg/mL; the final concentration of the graphene oxide in the obtained mixed solution is 0.2-0.4 mg/mL.

4. The graphene oxide-modified starch hydrogel immobilized bio-enzyme microreactor of claim 1, wherein: the graphene oxide modified starch hydrogel is used for fixing biological enzymes through electrostatic action and hydrogen bond action in PBS (phosphate buffer solution) with the pH value of 3-5.

5. The use of the microreactor based on starch-based nanomaterial immobilized biological enzyme of claim 1 for degrading bisphenol a in wastewater, wherein the biological enzyme is horseradish peroxidase.

6. The use of the microreactor based on starch-based nanomaterial immobilized bio-enzyme of claim 1 for degrading 2, 4-dichlorophenol in wastewater, wherein the bio-enzyme is horseradish peroxidase.

Technical Field

The invention belongs to the technical field of enzyme immobilization, and particularly relates to an immobilized biological enzyme microreactor taking graphene oxide modified starch hydrogel as a carrier and application thereof.

Background

The biological enzyme is an organic matter which is generated by living cells and has a catalytic action, most of the biological enzyme is protein, and the biological enzyme is a non-toxic and environment-friendly biocatalyst. The biological enzyme has high catalytic efficiency and high specificity, one enzyme can only catalyze one or one type of chemical reaction, and the reaction strip is mild. However, free enzyme is easy to inactivate in high temperature, strong acid, strong base, organic solvent and other environments, and the enzyme tolerance to temperature, acid, base and organic solvent can be effectively improved by adopting an immobilized enzyme mode. And the immobilized enzyme has wide application prospect in the aspect of sewage treatment due to the characteristics of green, recoverability and the like.

Horse Radish Peroxidase (HRP) is a glycoprotein complex enzyme containing iron porphyrin prosthetic group, and is the most widely studied peroxidase at present. In the presence of hydrogen peroxide, horseradish peroxidase can catalyze the oxidation of various compounds, especially substances containing large pi conjugated systems, such as phenols, aromatics, anilines, indoles and other compounds. It has high and stable specific activity, small molecular weight, easy preparation of pure enzyme, wide distribution in plant kingdom and high content of horseradish.

The starch hydrogel contains a large amount of hydroxyl groups, has strong crosslinking capacity, and simultaneously has good biocompatibility and biodynamic response, so the starch hydrogel can be used as an immobilized enzyme carrier. However, the natural starch hydrogel generally has the defects of poor mechanical properties, high brittleness, low stretchability and the like, and the wide application of the starch hydrogel in the aspect of biological enzyme catalysis is limited. Therefore, it remains a challenging task to improve the relevant properties of starch hydrogels to play a greater role in bio-enzyme catalysis.

Disclosure of Invention

The invention aims to provide an immobilized biological enzyme microreactor which has high catalytic activity, good thermal stability, reusability and organic solvent tolerance, simple preparation, low production cost and industrial application prospect, and provides new application for the biological enzyme microreactor.

Aiming at the purposes, the adopted biological enzyme microreactor is obtained by adding graphene oxide in the preparation process of starch hydrogel to obtain graphene oxide modified starch hydrogel, and fixing biological enzyme by taking the hydrogel as a carrier through electrostatic action and hydrogen bond action; wherein the biological enzyme is any one of horseradish peroxidase, catalase, hemoglobinoprotease, chloroperoxidase and cytochrome c.

The preparation method of the graphene oxide modified starch hydrogel comprises the following steps: uniformly dispersing graphene oxide in deionized water by ultrasonic waves to obtain a graphene oxide suspension; uniformly stirring starch and dispersing the starch in deionized water to obtain a starch suspension; and adding the graphene oxide suspension into the starch suspension, fully mixing, heating the obtained mixed solution to 80-90 ℃, continuously stirring for 3-5 h, cooling to room temperature, and freeze-drying to obtain the graphene oxide modified starch hydrogel.

The concentration of the starch suspension is 0.08-0.12 g/mL, and the concentration of the graphene oxide suspension is 0.5-1 mg/mL; the final concentration of the graphene oxide in the obtained mixed solution is 0.2-0.4 mg/mL.

The graphene oxide modified starch hydrogel is preferably used for fixing biological enzymes through electrostatic action and hydrogen bond action in PBS (phosphate buffer solution) with the pH value of 3-5.

The starch-based nanomaterial-based immobilized bio-enzyme microreactor can be used for degrading bisphenol A or 2, 4-dichlorophenol in wastewater, wherein the bio-enzyme is horseradish peroxidase.

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

1. according to the invention, a starch-based nano material with high biocompatibility is used as a main carrier, firstly, starch is heated in water to be gelatinized and then retrograded to form starch hydrogel with a three-dimensional network structure, and graphene oxide is modified in the formation process of the starch hydrogel, so that the introduced graphene oxide can improve the structure of the starch hydrogel, overcome the defect that the biological enzyme leaks due to overlarge pore diameter of the original structure of the starch hydrogel, enable the network structure of the hydrogel to be more compact, prevent the graphene oxide from stacking and aggregating through the introduction of the starch, effectively improve the specific surface area, increase the effective area of biological enzyme load and improve the immobilization effect of the biological enzyme. Secondly, the obtained hydrogel has abundant hydrophilic groups, so that the interaction between the hydrogel and the biological enzyme can be enhanced, the immobilization efficiency of the biological enzyme is enhanced, and the loading capacity of the biological enzyme and the reusability of the hydrogel immobilized biological enzyme are improved.

2. The biological enzyme microreactor can maintain the catalytic activity of biological enzyme and ensure the high dispersibility of the biological enzyme, overcomes the defects of low stability, short service life, sensitivity to various environmental factors, difficulty in recycling and the like of free biological enzyme, improves the stability and the operation stability of a biological enzyme molecular structure, avoids the defect of biological enzyme molecular leakage in the recycling process, improves the recycling times, and has good tolerance in organic solvents such as DMF, methanol, acetonitrile and the like, thereby providing a good method for the storage and the recovery of the free biological enzyme.

3. The biological enzyme microreactor has low production cost and industrial application prospect due to the adoption of the starch-based nano material as a carrier, and the starch-based nano material has interconnected macropores in a cage-shaped structure, so that the non-cross diffusion and mass transfer of solutes are facilitated, and the immobilized horseradish peroxidase reactor can achieve a better degradation effect in a shorter time in the application of degrading bisphenol A and 2, 4-dichlorophenol in wastewater.

Drawings

FIG. 1 is a graph of the effect of temperature on HRP @ Starch and HRP @ GO @ Starch catalytic activities.

FIG. 2 is a graph of reusability of HRP @ Starch and HRP @ GO @ Starch in buffer solution.

FIG. 3 is a graph of the effect of methanol on HRP @ Starch and HRP @ GO @ Starch catalytic activity.

FIG. 4 is a graph of the effect of acetonitrile on the catalytic activity of HRP @ Starch and HRP @ GO @ Starch.

FIG. 5 is a graph of the effect of DMF on HRP @ Starch and HRP @ GO @ Starch catalytic activity.

FIG. 6 is a graph showing the effect of varying concentrations of bisphenol A degraded by HRP @ Starch and HRP @ GO @ Starch.

FIG. 7 is a graph showing the effect of degradation of 2, 4-dichlorophenol by HRP @ Starch and HRP @ GO @ Starch at various concentrations.

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

Example 1

Uniformly dispersing 10mg of Graphene Oxide (GO) in 10mL of deionized water by ultrasonic to obtain a GO suspension; 1g of Starch (Starch) is uniformly stirred and dispersed in 10mL of deionized water to obtain a Starch suspension; adding 2mL of GO suspension into 8mL of Starch suspension, fully mixing to enable the final concentration of GO in the mixed solution to be 0.2mg/mL, heating the mixed solution to 90 ℃, continuously stirring at the rotating speed of 300rpm for 3h, carrying out ultrasonic treatment in an ultrasonic bath at 60 ℃ for 2h to obtain homogeneous hydrogel, cooling to room temperature, and freeze-drying to obtain graphene oxide modified Starch hydrogel which is recorded as GO @ Starch.

5mg of GO @ Starch was weighed, and 1400. mu.L of PBS buffer solution with pH 5 and 100. mu.L of 0.25 mmol.L^{- 1}And (3) shaking the HRP solution at the constant temperature of 29 ℃ for 10h, standing for 6h, removing the supernatant, washing for 2-3 times by using PBS (phosphate buffer solution), removing the unfixed HRP, and obtaining the Starch-based nano material-based immobilized HRP microreactor which is marked as HRP @ GO @ Starch.

The performance of the HRP @ GO @ Starch prepared above was tested, and a comparative test was performed with HRP @ Starch (freeze-dried Starch hydrogel prepared by the above method without adding GO suspension, and then with immobilized HRP), and the specific test was as follows:

1. experiment of catalytic Activity

Generation of stable blue-green ABTS by HRP catalytic oxidation ABTS^·+Free radicals were used as model reactions to determine HRP catalytic activity. The method comprises the following specific steps: PBS buffer solution (pH 5.0) was taken and 30. mu.L of 2 mmol. multidot.L was added^-1ABTS solution, free HRP or equivalentAn equivalent amount of free enzyme Starch @ HRP or GO @ Starch @ HRP, followed by the final addition of 20. mu.L of 0.1 mol. L^-1H₂O₂Aqueous solution, keeping the total volume of the solution at 1.5 mL. Shaking, shaking in shaking table for 15min, centrifuging, sucking out supernatant, and measuring absorbance at 415nm with ultraviolet spectrophotometer. The conversion of ABTS is calculated by the following formula:

in the formula, A: actually measuring an absorbance value by an ultraviolet spectrophotometer; epsilon_415nm：ABTS^·+Molar absorption coefficient at 415 nm; b: width (cm) of cuvette used in measurement; c_ABTS: ABTS substrate concentration (mol. L) before reaction^-1). The results show that HRP @ Starch and HRP @ GO @ Starch retain relatively high catalytic activity, about 90.26% and 93.58%, based on 100% catalytic activity of free HRP.

2. Thermal stability test

Incubating 10mg of HRP @ Starch, 10mg of HRP @ GO @ Starch and an equal amount of free HRP in water baths at different temperatures (60-90 ℃) for 3 hours, taking out the free HRP, the HRP @ Starch and the HRP @ GO @ Starch after 3 hours, and measuring the catalytic activity of the HRP @ Starch, the HRP @ Starch and the HRP @ GO @ Starch by using a catalytic ABTS conversion reaction after the temperature is reduced to room temperature. The thermostability of free HRP, HRP @ Starch and HRP @ GO @ Starch was characterized by plotting the catalytic activity retained after 3h incubation versus temperature for 100% of the enzyme catalytic activity before incubation at the specified temperature, and the results are shown in FIG. 1.

It can be seen from the figure that the residual activity of both enzyme reactors for catalyzing ABTS conversion decreases with increasing temperature, and at each temperature the retained activity of the enzyme reactor is higher than that of free HRP. Wherein the GO @ Starch @ HRP and the HRP @ Starch can respectively maintain 88.58% and 74.62% of catalytic activity after being placed for 1h at 80 ℃; after standing at 90 ℃ for 3h, GO @ Starch @ HRP and HRP @ Starch still retained 56.48% and 48.36% catalytic activity, respectively, and had good thermal stability at high temperatures compared to free HRP.

3. Reusability test

5mg of HRP @ Starch and HRP @ GO @ Starch were weighed into a 2mL centrifuge tube, and 1.2mL of a pH 5 PBS buffer solution and 30. mu.L of 2 mmol.L^-1ABTS solution, finally H is added₂O₂(0.1mmol·L^-1) The reaction was started, maintaining a total solution volume of 1.5 mL. After a certain period of reaction, centrifugation was carried out, the absorbance value of the supernatant at 415nm was measured, and the above operation was repeated after washing the material. The reusability of HRP @ Starch and HRP @ GO @ Starch was characterized in terms of residual activity with 100% conversion of the first ABTS, after which each conversion was compared to the first, and the results are shown in figure 2.

As can be seen, after 10 times of repeated use, the catalytic activity of HRP @ GO @ Starch can be kept at 62.08%, and the catalytic activity of HRP @ GO @ Starch can be kept at 75.42%, which is obviously higher than that of HRP @ Starch, thus indicating that the reusability of HRP @ GO @ Starch is good.

4. Organic solvent resistance test

(1) Tolerance to methanol

To a volume fraction of 5% to 30% in aqueous methanol, 10mg of HRP @ Starch or HRP @ GO @ Starch or an equal amount of free HRP was added and their retained activity was tested by ABTS conversion after standing for 1h at room temperature. The catalytic activity of HRP @ Starch, HRP @ GO @ Starch and free HRP without added methanol was taken as 100%, and the catalytic activity retained by HRP @ Starch, HRP @ GO @ Starch and free HRP at each volume fraction relative to that without added methanol was used to characterize the tolerance to methanol, and the results are shown in figure 3.

As can be seen, after treatment with 30% by volume aqueous methanol, the retained activity of free HRP was only 30.61%, and the retained catalytic activity of HRP @ Starch was 75.88%, while the retained catalytic activity of HRP @ GO @ Starch under the same conditions was 90.23%, significantly higher than that of free enzyme and higher than that of HRP @ Starch.

(2) Tolerance to acetonitrile

To a volume fraction of 5% to 30% in acetonitrile in water, 10mg of HRP @ Starch or HRP @ GO @ Starch or an equal amount of free HRP was added and their retained activity was tested by ABTS conversion after standing for 1h at room temperature. The catalytic activity of HRP @ Starch, HRP @ GO @ Starch and free HRP without acetonitrile was taken as 100%, and the catalytic activity retained by HRP @ Starch, HRP @ GO @ Starch and free HRP at each volume fraction relative to that without acetonitrile was taken to characterize the tolerance to acetonitrile, and the results are shown in figure 4.

As can be seen, the retained activity of free HRP after treatment with 30% volume fraction acetonitrile in water was only 32.09%, HRP @ Starch retained 80.02% catalytic activity, while HRP @ GO @ Starch retained 89.65% catalytic activity, which is significantly higher than that of free enzyme and higher than that of HRP @ Starch.

(3) Tolerance to N, N-Dimethylformamide (DMF)

10mg of HRP @ Starch or HRP @ GO @ Starch or an equal amount of free HRP was added to a 5% to 30% volume fraction aqueous DMF solution and tested for retained activity by ABTS conversion after standing for 1h at room temperature. The catalytic activity of HRP @ Starch, HRP @ GO @ Starch and free HRP without DMF addition was taken as 100%, and the catalytic activity retained by HRP @ Starch, HRP @ GO @ Starch and free HRP at each volume fraction was compared to the catalytic activity without DMF addition to characterize the tolerance to DMF, and the results are shown in figure 5.

As can be seen, the residual activity of free HRP after treatment with 15% by volume of DMF in water was 20.75%, while the residual activities of HRP @ GO @ Starch and HRP @ Starch were 73.63% and 65.44%, respectively.

5. Electrostatic driving force of HRP and carrier combination

Starch and GO @ Starch were ultrasonically dispersed in PBS buffer at pH 3 and pH 5, respectively, and their Zeta potentials at different pH were measured with a laser particle sizer, as in table 1.

TABLE 1

As can be seen from table 1, Starch and GO @ Starch are negatively charged in both buffers. The isoelectric point pI of HRP is known to be 7.2, and it is preliminarily considered that a PBS buffer solution with a pH of 3-5 can be selected for immobilizing HRP. HRP binds to carrier Starch and GO @ Starch primarily via electrostatic interactions.

Example 2

Application of HRP @ GO @ Starch prepared in example 1 in degradation of bisphenol A in wastewater

10mg of HRP @ GO @ Starch (with HRP @ Starch as a comparative test), artificial wastewater, and bisphenol A with different concentrations were added into a 10mL centrifuge tube, and 36. mu.L of 0.1 mol.L was added^-1H₂O₂The reaction was started with aqueous solution, maintaining a total solution volume of 3 mL. The reaction was carried out for 25min at room temperature under magnetic stirring, and after the reaction was completed, it was extracted with ethyl acetate 3 times. And finally, completely evaporating and removing the extract liquor by using a rotary evaporator, and dissolving a sample by using chromatographic pure acetonitrile to obtain a crude sample. Filtering the crude sample by a 0.22 mu m organic phase filter membrane, and analyzing and determining by using a high performance liquid chromatography (HPLC-15C) under the following determination conditions: in isocratic mode with acetonitrile-water solution (V)_Acetonitrile:V_{Water (W)}90:10) as the mobile phase, at a flow rate of 0.5mL · min^-1The detection wavelength is 275nm, the column temperature is 25 ℃, and the sample injection amount is 15 mu L.

The degradation rate (η) is calculated according to the formula: eta ═ C₀-C_t)/C₀X 100%, wherein C_tDenotes the concentration of bisphenol A at time t after addition of the enzyme, C₀The bisphenol A concentration of the reaction system in the absence of the enzyme is shown.

FIG. 6 shows the results when the concentration of bisphenol A is 0.5 mmol.L^-1And meanwhile, the degradation rate of HRP @ GO @ Starch and the degradation rate of HRP @ Starch can reach more than 90% within 20 min. When the substrate concentration continues to increase, the degradation rates of the two immobilized enzymes gradually decrease. When the concentration of bisphenol A is 3.0 mmol.L^-1When the method is used, the degradation rate of the HRP @ GO @ Starch to the bisphenol A in the artificial wastewater is 57.58%, which is higher than that of the HRP @ Starch to the bisphenol A in the artificial wastewater by 46.86%.

Example 3

Application of HRP @ GO @ Starch prepared in example 1 to degradation of 2, 4-dichlorophenol in wastewater

Accurately preparing a series of 2, 4-dichlorophenol standard solutions with different concentrations with methanol as solvent, filtering with 0.22 μm organic phase filter membrane, and measuring peaks corresponding to different substrate concentrations by high performance liquid chromatographyAnd (4) plotting the substrate concentration and the corresponding peak area, and fitting to obtain a standard curve equation. 10mg of HRP @ GO @ Starch (with HRP @ Starch as a comparative test), artificial wastewater, 2, 4-dichlorophenol with different concentrations, 36. mu.L of 0.1 mol.L^{- 1}H₂O₂The reaction was started with aqueous solution, maintaining a total solution volume of 3 mL. After 20min of reaction in the dark, centrifugation was carried out, 1000. mu.L of the reaction solution was taken out into a container by a pipette, 1000. mu.L of ethyl acetate was added, extraction was carried out by stirring in the dark, and the operation was repeated 3 times. And performing rotary evaporation on the extract, completely evaporating the solvent, dissolving with 1000 mu L of methanol, and performing high performance liquid chromatography on the filtered filtrate. The high performance liquid chromatography determination conditions are as follows: methanol-water solution (V) is adopted in an isocratic mode_Methanol:V_{Water (W)}60:40) as the mobile phase, at a flow rate of 1.0mL · min^-1The detection wavelength is 284nm, the column temperature is 25 ℃, and the sample injection amount is 20 mu L.

As can be seen from the results in FIG. 7, when the concentration of 2, 4-dichlorophenol was 1.0 mmol.L^-1HRP @ GO @ Starch and HRP @ Starch were substantially completely degraded within 20 min. Continuously increasing the concentration of the 2, 4-dichlorophenol when the concentration of the 2, 4-dichlorophenol is 3.0 mmol.L^-1And the degradation rate of HRP @ GO @ Starch can reach 94.5%, which is slightly higher than that of HRP @ Starch.