CN113772829B

CN113772829B - Immobilized biological enzyme microreactor based on starch-based nano material and application thereof

Info

Publication number: CN113772829B
Application number: CN202111147596.4A
Authority: CN
Inventors: 蒋育澄; 何濛; 胡满成; 李淑妮; 翟全国
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2023-06-16
Anticipated expiration: 2041-09-29
Also published as: CN113772829A

Abstract

The invention discloses a starch-based nanomaterial immobilized biological enzyme microreactor and application thereof, wherein the biological enzyme microreactor is obtained by modifying graphene oxide in the preparation process of starch hydrogel, taking the starch hydrogel after the modification of the graphene oxide as a carrier, and immobilizing biological enzyme through electrostatic action and hydrogen bonding action. The biological enzyme micro-reactor takes the starch-based nano material as a carrier, has low production cost and industrial application prospect, has a compact network structure and high specific surface area, is rich in hydrophilic groups such as hydroxyl groups, retains high catalytic activity of free enzyme, improves enzyme loading capacity, has good catalytic activity, thermal stability and reusability, has good tolerance in organic solvents such as DMF, methanol and acetonitrile, and is used for degrading organic matters such as bisphenol A, 2, 4-dichlorophenol and the like in wastewater, and high in degradation speed and degradation rate.

Description

Immobilized biological enzyme microreactor based on starch-based nano material and application thereof

Technical Field

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

Background

Biological enzymes are organic matters with catalytic action produced by living cells, most of the biological enzymes are proteins, and the biological enzymes are nontoxic and environment-friendly biological catalysts. The biological enzyme has high catalytic efficiency and high specificity, and one enzyme can only catalyze one or a class of chemical reactions, so that the reaction strip is mild. However, the free enzyme is easy to deactivate in high temperature, strong acid, strong alkali, organic solvent and other environments, and the tolerance of the enzyme to temperature, acid, alkali and organic solvent can be effectively improved by adopting an immobilized enzyme mode. The immobilized enzyme has wide application prospect in sewage treatment due to the characteristics of green property, recoverability and the like.

Horseradish peroxidase (HRP) is a glycoprotein complex enzyme containing ferriporphyrin prosthetic groups, and is the most widely studied peroxidase at present. Horseradish peroxidase is capable of catalyzing and oxidizing a variety of compounds, particularly substances containing large pi conjugated systems, such as phenols, aromatics, anilines, indoles, and the like, in the presence of hydrogen peroxide. The specific activity is high, the stability is high, the molecular weight is small, the pure enzyme is easy to prepare, the pure enzyme is widely distributed in the plant kingdom, and the content of horseradish is high.

The starch hydrogel contains a large number of hydroxyl groups, has strong crosslinking capability, and has good biocompatibility and biological dynamic response, so that the starch hydrogel can be used as an immobilized enzyme carrier. However, the disadvantages of poor mechanical properties, large brittleness, low stretchability and the like of natural starch hydrogels generally limit the wide application of the starch hydrogels in the catalysis of biological enzymes. Thus, improving the relevant properties of starch hydrogels to make them play a greater role in bio-enzyme catalysis remains a challenging task.

Disclosure of Invention

The invention aims to provide an immobilized biological enzyme micro-reactor 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 micro-reactor.

Aiming at the purposes, the biological enzyme microreactor adopted by the invention is obtained by adding graphene oxide in the preparation process of starch hydrogel to obtain the starch hydrogel modified by graphene oxide, taking the hydrogel as a carrier, and fixing biological enzyme through electrostatic action and hydrogen bonding action; wherein the biological enzyme is any one of horseradish peroxidase, catalase, haemoglobin, 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 graphene oxide suspension; uniformly stirring and dispersing starch in deionized water to obtain starch suspension; and adding 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 graphene oxide in the obtained mixed solution is 0.2-0.4 mg/mL.

The graphene oxide modified starch hydrogel is preferably immobilized with biological enzymes through electrostatic action and hydrogen bonding in PBS buffer solution with pH of 3-5.

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

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

1. according to the invention, the starch-based nano material with high biocompatibility is used as a main carrier, firstly, starch is heated in water to gelatinize the starch, then retrogradation is carried out to form the starch hydrogel with a three-dimensional network structure, and graphene oxide is modified in the forming process of the starch hydrogel, so that the structure of the starch hydrogel can be improved by the introduced graphene oxide, the defect that biological enzymes leak due to overlarge pore diameters of the original structure of the starch hydrogel is overcome, the network structure of the hydrogel is more compact, stacking agglomeration of the graphene oxide can be prevented by introducing starch, the specific surface area is effectively increased, the effective area of biological enzyme load is increased, and the immobilization effect of biological enzymes is improved. Secondly, the hydrogel has rich 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 meanwhile, the loading capacity of the biological enzyme and the reusability of the immobilized biological enzyme of the hydrogel are improved.

2. The biological enzyme micro-reactor can maintain the catalytic activity of biological enzyme and ensure the high dispersibility of 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 operation stability of biological enzyme molecular structure, avoids the defect of biological enzyme molecular leakage in the process of repeated use, and improves the repeated use times, and the biological enzyme reactor has good tolerance in DMF, methanol, acetonitrile and other organic solvents, thereby providing a good method for storing and recycling the free biological enzyme.

3. The biological enzyme micro-reactor adopts the starch-based nano material as the carrier, so that the production cost is low, the industrial application prospect is realized, and the cage-shaped structure of the starch-based nano material is provided with the interconnected macropores, which is beneficial to non-cross diffusion and mass transfer of solutes, so that the immobilized horseradish peroxidase reactor can achieve 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 the catalytic activity of HRP@Starch and HRP@GO@Starch.

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

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

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 showing the effect of DMF on the catalytic activity of HRP@Starch and HRP@GO@Starch.

FIG. 6 is a graph showing the effect of degradation of bisphenol A at various concentrations by HRP@starch and HRP@GO@starch.

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

Detailed Description

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

Example 1

Uniformly dispersing 10mg of Graphene Oxide (GO) in 10mL of deionized water by ultrasonic waves to obtain GO suspension; 1g of Starch (Starch) is stirred and uniformly dispersed in 10mL of deionized water to obtain Starch suspension; adding 2mL of GO suspension into 8mL of starch suspension, fully mixing to ensure that the final concentration of GO in the mixed solution is 0.2mg/mL, heating the mixed solution to 90 ℃ and continuously stirring for 3h at a rotating speed of 300rpm, 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 denoted as GO@starch.

Weigh 5mg GO @Starch, 1400. Mu.L of PBS buffer solution with pH=5 and 100. Mu.L of 0.25 mmol.L were added ^- ¹ And (3) oscillating the HRP solution for 10 hours at the constant temperature of 29 ℃, standing for 6 hours, removing supernatant, washing with PBS buffer solution for 2-3 times, and removing non-immobilized HRP to obtain the immobilized HRP microreactor based on the starch-based nanomaterial, wherein the immobilized HRP microreactor is recorded as HRP@GO@starch.

Performance testing was performed on the above prepared hrp@go@starch, and at the same time, a comparative test was performed on hrp@starch (freeze-dried Starch hydrogel was prepared without adding GO suspension in the above method, and then HRP was immobilized), and the specific test was as follows:

1. catalytic Activity experiments

Catalytic oxidation of ABTS with HRP to produce stable blue-green ABTS ^·+ The free radicals were used as model reactions to determine HRP catalytic activity. The method comprises the following specific steps: the PBS buffer solution with pH=5.0 was taken and 30. Mu.L of 2mmol.L was taken ^-1 ABTS solution, free HRP or starch@HRP or GO@starch@HRP equivalent to the same amount of free enzyme, and finally 20 mu L of 0.1 mol.L of ^-1 H ₂ O ₂ The total volume of the solution was kept at 1.5mL. Shaking, shaking in a shaker for 15min, centrifuging, sucking out supernatant, and measuring absorbance at 415nm with ultraviolet spectrophotometer. The conversion of ABTS is calculated from the following equation:

wherein A: absorbance values actually measured by the ultraviolet spectrophotometer; epsilon _415nm ：ABTS ^·+ Molar absorption coefficient at 415 nm; b: the width (cm) of the cuvette used in the measurement; c (C) _ABTS : ABTS substrate concentration before reaction (mol.L) ^-1 ). The results showed that HRP@Starch and HRP@GO@Starch retained higher catalytic activities, about 90.26% and 93.58%, based on the catalytic activity of free HRP as 100%.

2. Thermal stability test

10mg of HRP@Starch and HRP@GO@Starch and an equal amount of free HRP are incubated in water baths at different temperatures (60-90 ℃) for 3 hours, the free HRP, the HRP@Starch and the HRP@GO@Starch are taken out after 3 hours, and after the temperature is reduced to room temperature, the catalytic activity is determined by utilizing a catalytic ABTS conversion reaction. The catalytic activity of the enzyme before incubation at the specified temperature was regarded as 100%, and the thermal stability of free HRP, hrp@starch and hrp@go@starch was characterized by plotting the catalytic activity remaining after incubation for 3h versus the catalytic activity before incubation, the results being shown in fig. 1.

It can be seen that the residual activity of the two enzyme reactors catalyzing ABTS conversion reactions decreases with increasing temperature, and at each temperature the enzyme reactor retains activity higher than the free HRP. Wherein, after the GO@starch@HRP and the HRP@starch are placed for 1 hour at 80 ℃, the catalytic activities of 88.58% and 74.62% can be respectively maintained; after 3h at 90 ℃, the GO@starch@HRP and the HRP@starch still can respectively retain 56.48% and 48.36% of catalytic activity, and have good thermal stability at high temperature compared with free HRP.

3. Reusability test

5mg of HRP@Starch and HRP@GO@Starch were weighed into a 2mL centrifuge tube, respectively, and 1.2mL of PBS buffer solution with pH=5 and 30. Mu.L of 2mmol.L were added ^-1 ABTS solution, finally add H ₂ O ₂ (0.1mmol·L ^-1 ) The reaction was started and the total volume of the solution was kept at 1.5mL. After a period of reaction, the supernatant was centrifuged to measure the absorbance at 415nm, and the material was washed and the above procedure was repeated. The reusability of hrp@starch and hrp@go@starch was characterized by residual activity at 100% conversion of the first ABTS, after which each conversion was compared to the first, and the results are shown in fig. 2.

It can be seen from the graph that after 10 times of repeated use, the HRP@Starch can retain 62.08% of catalytic activity, the HRP@GO@Starch can retain 75.42% of activity, and the activity is obviously higher than that of the HRP@Starch, which indicates that the reusability of the HRP@GO@Starch is good.

4. Organic solvent tolerance test

(1) Resistance to methanol

10mg of HRP@Starch or HRP@GO@Starch or an equivalent amount of free HRP is added to an aqueous methanol solution with a volume fraction of 5% -30%, and their retention activity is tested by ABTS conversion reaction after being left at room temperature for 1 h. The catalytic activities of hrp@starch, hrp@go@starch and free HRP without methanol added were regarded as 100%, and the tolerance to methanol was characterized by the catalytic activities retained by hrp@starch, hrp@go@starch and free HRP at the respective volume fractions relative to the catalytic activity without methanol added, and the results are shown in fig. 3.

As can be seen from the graph, after the methanol aqueous solution with the volume fraction of 30%, the activity of the free HRP is only 30.61%, the catalytic activity of HRP@Starch is 75.88%, and the catalytic activity of HRP@GO@Starch under the same conditions can reach 90.23%, which is obviously higher than that of the free enzyme and higher than that of the HRP@Starch.

(2) Resistance to acetonitrile

10mg of HRP@Starch or HRP@GO@Starch or an equivalent amount of free HRP is added to an aqueous acetonitrile solution with a volume fraction of 5% -30%, and their retention activity is tested by ABTS conversion reaction after being left at room temperature for 1 h. The catalytic activities of hrp@starch, hrp@go@starch and free HRP without acetonitrile added were regarded as 100%, and the tolerance to acetonitrile was characterized by the catalytic activities retained by hrp@starch, hrp@go@starch and free HRP at the respective volume fractions relative to the catalytic activity without acetonitrile added, and the results are shown in fig. 4.

It can be seen from the graph that after the treatment of 30% acetonitrile aqueous solution by volume fraction, the retention activity of free HRP is only 32.09%, HRP@Starch retains 80.02% of catalytic activity, while HRP@GO@Starch can retain 89.65% of 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 equivalent amount of free HRP is added to a 5% -30% by volume of DMF aqueous solution, and their retention activity is tested by ABTS conversion reaction after 1h of standing at room temperature. The catalytic activities of hrp@starch, hrp@go@starch and free HRP without DMF added were regarded as 100%, and the tolerance to DMF was characterized by the catalytic activities retained by hrp@starch, hrp@go@starch and free HRP at the respective volume fractions relative to the catalytic activity without DMF added, the results are shown in fig. 5.

It can be seen that the residual activity of free HRP was 20.75% after treatment with 15% by volume of DMF aqueous solution, while the residual activities of HRP@GO@Starch and HRP@Starch could still reach 73.63% and 65.44%, respectively.

5. Electrostatic driving force for binding between HRP and carrier

Starch and go@starch were dispersed ultrasonically into PBS buffer solutions at ph=3 and ph=5, respectively, and their Zeta potentials at different pH were measured with a laser particle sizer as shown in table 1.

TABLE 1

As can be seen from Table 1, both Starch and GO@starch are electronegative in both buffers. Given that the isoelectric point pI of HRP is 7.2, it was initially thought that PBS buffer at pH 3-5 could be selected to immobilize HRP. The HRP and the carrier Starch are combined mainly through electrostatic interaction.

Example 2

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

10mg HRP@GO@Starch (simultaneously HRP@Starch is used as a comparison test), artificial wastewater and bisphenol A with different concentrations are added into a 10mL centrifuge tube, and finally 36 mu L of 0.1 mol.L is added ^-1 H ₂ O ₂ The reaction was started with aqueous solution, keeping the total volume of the solution at 3mL. The reaction was carried out at room temperature for 25min under magnetic stirring, and after the completion of the reaction, the mixture was extracted 3 times with ethyl acetate. Finally, the extract is completely evaporated by a rotary evaporator, and then the sample is dissolved by chromatographic pure acetonitrile to obtain a crude sample. The crude sample was filtered through a 0.22 μm organic phase filtration membrane and used for high performance liquid chromatography (HPLC-15C) analysis and determination under the following conditions: acetonitrile-water solution (V) was used in isocratic mode _Acetonitrile :V _{Water and its preparation method} =90:10) is the mobile phase, the flow rate is 0.5ml·min ^-1 The 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: η= (C ₀ -C _t )/C ₀ X 100%, C in _t The concentration of bisphenol A at time t after enzyme addition is shown,C ₀ the bisphenol A concentration of the reaction system without enzyme is shown.

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

Example 3

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

A series of 2, 4-dichlorophenol standard solutions with different concentrations are accurately prepared by taking methanol as a solvent, filtering the solution by using an organic phase filter membrane with the thickness of 0.22 mu m, measuring peak areas corresponding to different substrate concentrations by using a high performance liquid chromatography, drawing the substrate concentrations and the peak areas corresponding to the substrate concentrations, and fitting the peak areas to obtain a standard curve equation. 10mg HRP@GO@Starch (simultaneously HRP@Starch is used as a comparison test), artificial wastewater and 2, 4-dichlorophenol with different concentrations are added into a 10mL centrifuge tube, and 36 mu L of 0.1 mol.L is finally added ^- ¹ H ₂ O ₂ The reaction was started with aqueous solution, keeping the total volume of the solution at 3mL. After 20min of reaction in the dark, the reaction mixture was centrifuged, 1000. Mu.L of the reaction mixture was taken out by a pipette, 1000. Mu.L of ethyl acetate was added to the vessel, and extraction was performed by stirring in the dark, and the operation was repeated 3 times. The extract was rotary evaporated, and after complete evaporation of the solvent, it was dissolved in 1000 μl of methanol and the filtrate was subjected to high performance liquid chromatography. The high performance liquid chromatography measurement conditions are as follows: methanol-water solution (V) is used in the isocratic mode _Methanol :V _{Water and its preparation method} =60:40) is the mobile phase, the flow rate is 1.0ml·min ^-1 The 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 of FIG. 7, when the concentration of 2, 4-dichlorophenol is 1.0 mmol.L ^-1 When the HRP@GO@starch and the HRP@starch are used, the materials can be basically and completely degraded within 20 minutes. Continuously increasing the concentration of the 2, 4-dichlorophenol until the concentration of the 2, 4-dichlorophenol is 3.0 mmol.L ^-1 When the degradation rate of HRP@GO@starch can reach 94.5%,slightly higher than the degradation rate of HRP@starch.

Claims

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

the biological enzyme is horseradish peroxidase;

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 graphene oxide suspension; uniformly stirring and dispersing starch in deionized water to obtain starch suspension; adding 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 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 graphene oxide in the obtained mixed solution is 0.2-0.4 mg/mL;

the graphene oxide modified starch hydrogel is used for fixing biological enzymes through electrostatic action and hydrogen bonding in PBS buffer solution with pH of 3-5.

2. The use of the immobilized bio-enzyme microreactor based on starch-based nanomaterial of claim 1 for degrading bisphenol a in wastewater.

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