Preparation of sebacic acid-modified magnetic nanoparticles and their immobilized β-glucosidase

Abstract

The present invention relates to the field of enzyme engineering technology, and specifically to the preparation of sebacic acid-modified magnetic nanoparticles and immobilized β-glucosidase thereof. The surface of iron oxide magnetic nanoparticles is modified with sebacic acid, and the enzyme is adsorbed and immobilized. The immobilized β-glucosidase obtained by the method is superior to the free enzyme in terms of pH stability, thermal stability, mechanical stability, storage resistance, etc., has enhanced tolerance to organic solvents, and can be repeatedly used for the decomposition of natural materials such as rice husk powder and corn cob powder. The immobilization strategy based on sebacic acid-modified iron oxide magnetic nanoparticles provides a more efficient method for immobilizing enzymes.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to the preparation of sebaceous acid-modified magnetic nanoparticles and immobilized beta-glucosidase thereof. Background Art

[0002] Enzymes are a type of protein or nucleic acid produced in organisms that can catalyze reactions. They have higher catalytic activity than chemical catalysts, mild reaction conditions, strong substrate specificity, and are in line with the development trend of green chemistry. In recent years, enzymes have been widely used in many fields such as agriculture, food, and light industry. However, there are also some problems in the application process. For example, many enzymes are easily degraded after separation and purification, and have poor stability. In addition, after the catalytic reaction, the remaining active enzymes cannot be reused. Enzyme immobilization technology provides a solution to overcome these shortcomings by recycling and repeatedly using immobilized enzymes (Zhang Jingxuan et al. Bioprocessing, 2022, 20(1): 9-19). β-Glucosidase is a common biodegradable enzyme in nature. Its main function is to hydrolyze glucosidic bonds and produce glucose. In recent years, scientists have used Eudragit L-100 (E) as a carrier to immobilize and modify β-glucosidase (CB). The thermal stability of the immobilized enzyme has been improved to a certain extent, and the immobilized enzyme still retains 41% of the initial enzyme activity after being reused 5 times (Yan Jieshan et al. Chemical Technology, 2018, 26 (3): 5-10). By preparing chitosan cross-linked SBA-15 mesoporous silica (SBA-15) immobilized β-glucosidase (β-Glc), the efficient conversion of icariin to rare baohuoside I was promoted (Liu Congyan et al. Chinese Herbal Medicine 2021. 52 (3): 685-691). The β-glucosidase from Trichoderma reesei has high enzyme activity and thermal stability, and is suitable for the degradation of various types of cellulose (Xing Shengli et al. Journal of Light Industry, 2022, 37 (1): 20-25). However, so far, the catalytic ability, stability and reusability of immobilized Trichoderma reesei β-glucosidase still need to be improved. The reason is mainly because the surface modification effect of the immobilized carrier is not good, which causes the interaction between the carrier and the enzyme to be unfavorable for the performance of the enzymatic properties, thereby affecting the catalytic function and stability of the enzyme. Therefore, we have established an effective immobilized enzyme strategy and used to realize the immobilization of β-glucosidase from Trichoderma reesei. In brief, it is to modify the iron oxide magnetic nanoparticles with sebacic acid and immobilize the enzyme. After the surface of the iron oxide magnetic nanoparticles is modified, a core-shell stable structure is formed, and a carboxyl group is introduced on the surface, which can form a stable interaction with the amino group of the enzyme. The present invention uses sebacic acid to modify the iron oxide magnetic nanoparticles to realize the immobilization of β-glucosidase, which not only enhances the tolerance of the enzyme to certain organic solvents, but also significantly enhances enzyme activity, pH stability, thermal stability, storage resistance, mechanical stability, etc., and increases the prospects for the industrial application of the enzyme. In addition, the β-glucosidase immobilized enzyme constructed by the present invention also performs well in decomposing agricultural and sideline product polysaccharides such as corn cob powder and rice husk powder, and further shows good application potential. Summary of the invention

[0003] 1. The present invention aims to provide an efficient enzyme immobilization technology, and an immobilized β-glucosidase with industrial application prospects obtained by applying the technology. Iron oxide magnetic nanoparticles are modified by sebacic acid so that the surface of the iron oxide magnetic nanoparticles carries carboxyl groups, and the enzyme is immobilized by combining with the amino group of the enzyme. After a round of catalytic reaction, the immobilized enzyme is separated by magnetic field force, so that it can be reused. The immobilized β-glucosidase enzyme obtained by this method has significantly enhanced tolerance to certain organic solvents than the free enzyme. The thermal stability, pH stability, mechanical stability and storage resistance of the immobilized enzyme are also significantly higher than those of the free enzyme. More importantly, due to the modification of sebacic acid, the interaction between the enzyme and the immobilized carrier is strong, the immobilized enzyme particles are stable, and repeated catalysis of 10 reactions can maintain more than 60% of the original activity. In addition, the immobilized β-glucosidase enzyme can be repeatedly used to hydrolyze the glucosidic bonds in agricultural and sideline products such as corn cob powder and rice husk powder, and has strong tolerance to impurities in these agricultural and sideline products. To achieve the above purpose, the reaction process is as follows:

[0004] 1) First, the recombinant plasmid expressing the recombinase gene is transformed into Escherichia coli BL21 (DE3) cells and cultured in a medium containing appropriate antibiotics. When the bacteria grow to the logarithmic growth phase, an appropriate concentration of IPTG is added to induce the expression of the target protein. The bacteria are collected by centrifugation and lysed, and the protein is purified by chromatography columns and other methods for immobilization; 2) Trivalent and divalent iron ions are mixed in a certain proportion and co-precipitated in the presence of ammonia water to obtain magnetic iron oxide nanoparticles, which are then mixed with sebaceous acid at high temperature and surface-modified with sebaceous acid (i.e., DMNPs are obtained) so that the surface of the nanoparticles is coated with a layer of carboxyl groups; 3) The above-mentioned purified enzyme is fully mixed with DMNPs and fully fixed in a constant temperature oscillator. After fixation, an external magnetic field is applied for separation and 5 washings, and the washing solution uses a phosphate buffer solution (pH=7.0) to finally obtain immobilized DMNPs.

[0005] Compared with the existing immobilized enzyme method and the obtained immobilized β-glucosidase, the present invention has significant advantages, which are specifically manifested as follows:

[0006] 1) The iron oxide magnetic nanoparticles designed by the present invention have a large surface area and can be separated from the reaction system under the action of an external magnetic field. After being modified with sebaceous acid, the surface of the nanoparticles contains carboxyl groups, which can form a stable interaction with the amino groups on the enzyme molecules. This enzyme immobilization method has good preparation cost and stability, and the separation process of the immobilized enzyme and the reaction solution is simple and convenient.

[0007] 2) The immobilized β-glucosidase enzyme designed by the present invention shows strong tolerance to certain organic solvents, even high-concentration organic solvents: the immobilized β-glucosidase enzyme maintains up to 35% activity in 50% alcohol solution, while the free β-glucosidase enzyme only has less than 10% activity left. Meanwhile, the free β-glucosidase loses its activity substantially in low-concentration butanol solution, while the immobilized β-glucosidase enzyme maintains a relative activity of more than 20%. Since the chemical industry often needs to carry out catalytic reaction in the presence of organic solvents, the enzyme immobilization method involved in the present invention and the immobilized β-glucosidase obtained will have unique application potential.

[0008] 3) The immobilized β-glucosidase designed and constructed by the present invention can be repeatedly used to hydrolyze the glucosidic bonds in agricultural and sideline products such as corn cob powder and rice husk powder, which is specifically manifested as follows: the activity of the enzyme is reduced to 0 after 11 cycles of using the immobilized β-glucosidase to decompose rice husk powder; the activity of the enzyme is reduced to 0 after 6 cycles of using the immobilized β-glucosidase to decompose corn cob powder. It is well known that natural substrates of agricultural and sideline products often contain many impurities, which have a strong inhibitory effect on enzymes, and the immobilized β-glucosidase constructed by the present invention is insensitive to these inhibitors, indicating that the enzyme will have a wide range of application prospects in the degradation of natural substrates of agricultural and sideline products.

[0009] 4) Compared with free β-glucosidase, the immobilized β-glucosidase constructed by the present invention also has significant improvements in thermal stability, pH stability, mechanical stability and storage resistance. Although there are many successful examples of previous immobilization methods and the immobilized enzymes produced therefrom, there are no related reports on the comprehensive improvement of enzyme function like the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The present invention discloses the steps of the method for immobilizing enzyme using sebaceous acid-modified iron oxide magnetic nanoparticles and a schematic diagram of using the obtained immobilized β-glucosidase enzyme for catalytic reaction.

[0011] Figure 2 The infrared spectrum IR spectrum and scanning electron microscope SEM spectrum of the sebacic acid modified iron oxide magnetic nanoparticles immobilized β-glucosidase designed by the present invention. a and b are the IR spectrum of the sebacic acid modified iron oxide magnetic nanoparticles before and after immobilization of β-glucosidase, respectively; c and d are the SEM spectrum of the sebacic acid modified iron oxide magnetic nanoparticles before and after immobilization of β-glucosidase, respectively.

[0012] Figure 3The figure is a comparison of the effects of pH and temperature on the free enzyme and the immobilized enzyme in the present invention. a and b respectively show the effects of pH and temperature on the activities of free β-glucosidase and β-glucosidase immobilized by sebaceous acid modified iron oxide magnetic nanoparticles.

[0013] Figure 4 This is a comparative diagram of the effects of different concentrations of organic solvents on free β-glucosidase and β-glucosidase immobilized on sebacic acid-modified iron oxide magnetic nanoparticles involved in the present invention.

[0014] Figure 5 The stability analysis of the free β-glucosidase and the sebaceous acid modified iron oxide magnetic nanoparticle immobilized β-glucosidase involved in the present invention is shown in Fig. a, b, c, and d are comparisons of pH stability, thermal stability, mechanical stability, and storage resistance, respectively.

[0015] Figure 6 is the number of cycles of the sebaceous acid modified iron oxide magnetic nanoparticle immobilized β-glucosidase involved in the present invention. a and b are the number of cycles of decomposing rice husk powder and corn cob powder and their catalytic effects. DETAILED DESCRIPTION

[0016] The present invention aims to provide an efficient enzyme immobilization technology, and an immobilized β-glucosidase with industrial application prospects obtained by applying the technology. Iron oxide magnetic nanoparticles are modified by sebacic acid so that the surface of the iron oxide magnetic nanoparticles carries carboxyl groups, and the enzyme is immobilized by combining with the amino group of the enzyme. The β-glucosidase immobilized enzyme obtained by the method has significantly enhanced tolerance to certain organic solvents than the free enzyme. The thermal stability, pH stability, mechanical stability and storage resistance of the immobilized enzyme are also significantly higher than those of the free enzyme. In addition, the immobilized enzyme particles have strong stability and can maintain more than 60% of the original activity after repeated catalysis of 10 reactions. In addition, the immobilized β-glucosidase can be repeatedly used to hydrolyze the glucosidic bonds in agricultural and sideline products such as corn cob powder and rice husk powder, and has strong tolerance to impurities in these agricultural and sideline products.

[0017] Example 1: Construction of sebaceous acid modified iron oxide magnetic nanoparticles of the present invention

[0018] Dissolve 2.8g of ferrous chloride in 10ml of water and remove impurities with a microporous filter; then draw 7.5mL of ferrous chloride solution and mix with 0.2mol / L ferric chloride solution in a beaker. Raise the temperature to 70℃, quickly add 18mL of 25% NH3·H2O to the solution and stir rapidly for 1min. Then add 3.34g of sebacic acid to the above reaction system and continue to stir rapidly at 70℃ for 1h. After the reaction, the black precipitate obtained is sebacic acid-modified iron oxide magnetic nanoparticles. Separate the above precipitate with an external magnetic field and wash it with alcohol and water three times respectively. Then mix the precipitate with 200mL of water, treat it with ultrasound for 2h, and wash it with water three times. Finally, separate the sebacic acid-modified iron oxide magnetic nanoparticles with an external magnetic field and dry them at room temperature.

[0019] Example 2: Expression and purification of the β-glucosidase involved in the present invention, and preparation and characterization of β-glucosidase immobilized on sebaceous acid modified iron oxide magnetic nanoparticles

[0020] The recombinant plasmid pETM11-BglC containing the Trichoderma reesei β-glucosidase gene (NCBI Reference Sequence: XM_006966752.1) was transformed into Enterobacter BL21 (DE3) and spread on an LB plate containing kana resistance. The colony was inoculated into TB liquid culture medium containing kana, and cultured overnight at 37°C with shaking until OD600 = 0.4-0.6, and then IPTG was added at a final concentration of 0.1 mmol / L. After inducing expression at 90 rpm and 25°C for 12 hours, the bacteria were collected by centrifugation. The bacteria were resuspended in a phosphate buffer (50 mmol / L) at pH = 7.0, and the bacteria were broken by ultrasound, and then the crude enzyme solution was collected by high-speed centrifugation, and then protein purification was performed using Ni-NTA. 20 mg of the sebaceous acid modified iron oxide magnetic nanoparticles prepared above were added to 100 μL of phosphate buffer (pH = 7.0) and stirred evenly, and then 200 μL of the purified free enzyme solution was added, and the mixture was placed in a 25°C constant temperature oscillator and shaken at 80 rpm for 4 hours to complete the immobilization. The immobilized product was separated by exogenous magnetic force, and washed 5 times with phosphate buffer (pH = 7.0) to elute the free enzyme, and finally the sebaceous acid modified iron oxide magnetic nanoparticles immobilized β-glucosidase were obtained.

[0021] 1) p-NPG method to determine β-glucosidase activity

[0022] 100 μL of pure enzyme solution or sebacic acid-modified iron oxide magnetic nanoparticles immobilized enzyme suspension was mixed with 815 μL of phosphate buffer (pH = 7.0), and then 25 μL of 100 mmol / L p-NPG solution was added, and the reaction was carried out at 40°C for 30 min. After the reaction was completed, 60 μL of 2 mmol / L sodium carbonate solution was added to the reaction system to terminate the reaction. The supernatant was then centrifuged and the OD400 value was measured.

[0023] 2) Surface characteristics of free β-glucosidase and enzyme immobilized on sebaceous acid-modified iron oxide magnetic nanoparticles

[0024] The sample was subjected to infrared spectroscopy analysis. The prepared sample was mixed with potassium bromide and pressed into tablets, and then subjected to infrared spectroscopy at 4000-500 cm -1 Infrared scanning. Figure 2 a and 2b show that the peak shape of the absorption peak of β-glucosidase before and after immobilization changes greatly, which may be due to the fact that the enzyme blocks the groups on the surface of the nanoparticle carrier after immobilization, causing a significant change in the absorption peak, indicating that the enzyme has been successfully immobilized on sebaceous acid-modified iron oxide magnetic nanoparticles. The SEM analysis of the sample uses an acceleration voltage of 0.05-30kV and a magnification of 20-50k, and the morphology is observed after gold spraying. Figure 2 c and 2d show that the β-glucosidase has nanoscale particle size before and after immobilization, and the particle size increases after immobilization, further indicating that the immobilization is successful.

[0025] Example 3: Study on the enzymatic properties of β-glucosidase immobilized on sebacic acid-modified iron oxide magnetic nanoparticles prepared by the method of the present invention

[0026] 1) Determination of the optimal pH and temperature for immobilization of β-glucosidase on sebaceous acid-modified iron oxide magnetic nanoparticles

[0027] Take 100 μL of free β-glucosidase or sebacic acid modified iron oxide magnetic nanoparticles immobilized enzyme suspension, adjust the pH to 3.0-10.0, and measure the relative enzyme activity. Take the average of three replicates for each reaction, and the percentage ratio of the sample activity to the maximum activity is the relative activity. The results show that there is no difference in the optimal pH of free β-glucosidase and immobilized enzyme, both are pH = 7.0. However, under alkaline conditions (pH>9.0), sebacic acid modified iron oxide magnetic nanoparticles immobilized enzyme showed higher activity (such as Figure 3 This indicates that enzymes immobilized on sebaceous acid-modified iron oxide magnetic nanoparticles are more suitable for enzymatic reactions under alkaline conditions.

[0028] Take 100 μL of free enzyme and or sebacic acid modified iron oxide magnetic nanoparticles immobilized enzyme, and measure the enzyme activity in the temperature range of 20℃ to 100℃. Take the average of three replicates for each reaction, and the ratio of the sample activity to the maximum activity is the relative activity. The results show that the optimal temperatures for free β-glucosidase or sebacic acid modified iron oxide magnetic nanoparticles immobilized enzyme are 60℃ and 40℃, respectively (e.g. Figure 3 b), indicating that sebaceous acid-modified iron oxide magnetic nanoparticles immobilized β-glucosidase is more suitable for catalytic reactions at low temperatures.

[0029] 2) Effects of organic solvents on the activity of free β-glucosidase and immobilized enzymes on sebaceous acid-modified iron oxide magnetic nanoparticles

[0030] Take 100 μL of free β-glucosidase or sebacic acid modified iron oxide magnetic nanoparticles immobilized enzyme, mix it with 100 μL of 10%-50% alcohol, acetone, or butanol solution, and measure the residual enzyme activity. Take the average value of three repetitions for each reaction, and the percentage ratio of the sample activity to the maximum activity is the relative activity. The results show that the activities of free β-glucosidase and sebacic acid modified iron oxide magnetic nanoparticles immobilized enzyme are inhibited by various organic solvents, and the inhibitory effect increases with the increase of organic solvent concentration. However, under the same conditions, the activity of sebacic acid modified iron oxide magnetic nanoparticles immobilized enzyme is significantly better than that of free β-glucosidase (such as Figure 4 As shown in Figure 2 ). Organic solvents as protein denaturants have hydroxyl and carbonyl groups, which can form stable hydrogen bonds with related groups in proteins and destroy the hydrogen bonds of proteins themselves. The carboxyl groups of sebacic acid-modified iron oxide magnetic nanoparticles may weaken the destructive effect of organic solvents on proteins by interacting with organic solvent-related groups, thereby making the immobilized enzyme have higher residual activity than the free enzyme.

[0031] 3) Determination of various stabilities of β-glucosidase immobilized on sebaceous acid modified iron oxide magnetic nanoparticles prepared by the method of the present invention

[0032] Determination of pH stability: Take 100 μL of free β-glucosidase or sebacic acid-modified iron oxide magnetic nanoparticles immobilized enzyme suspension, adjust the pH to 3.0-10.0, react at 30°C for 30 minutes, and then measure the enzyme activity. Take the average of three repetitions for each reaction, and the ratio of the sample activity to the maximum activity is the relative activity. The results show that at different pH values, the sebacic acid-modified iron oxide magnetic nanoparticles immobilized enzyme exhibits higher pH stability than free β-glucosidase, and this advantage is more significant under alkaline and acidic conditions (such as Figure 5-a). Therefore, β-glucosidase immobilized on sebaceous acid-modified iron oxide magnetic nanoparticles is more suitable for catalytic reactions under extreme environmental conditions than its free enzyme.

[0033] Thermal stability determination: Take 100 μL of free β-glucosidase or sebacic acid-modified iron oxide magnetic nanoparticles immobilized enzyme, incubate at a temperature range of 30°C to 90°C for 30 minutes, and then determine the thermal stability. Take the average of three replicates for each reaction, and the percentage ratio of the sample activity to the maximum activity is the relative activity. The results show that when the temperature is greater than 70°C, the free β-glucosidase is completely inactivated, while the sebacic acid-modified iron oxide magnetic nanoparticles immobilized enzyme still has residual activity after incubation at 90°C for 3 minutes (such as Figure 5 -b). Therefore, the immobilized β-glucosidase on the fatty acid-modified iron oxide magnetic nanoparticles is more resistant to high temperature, indicating that although the optimum temperature is lowered, the thermal stability of the enzyme immobilized on the fatty acid-modified iron oxide magnetic nanoparticles is improved.

[0034] Determination of mechanical stability: Take 100 μL of free β-glucosidase or sebacic acid modified iron oxide magnetic nanoparticles immobilized enzyme suspension, vortex for 0-60 minutes, and then measure the enzyme activity. Take the average of three repetitions for each reaction, and the ratio of the sample activity to the maximum activity is the relative activity. The results show that after vortexing for 1 hour, the residual activity of sebacic acid modified iron oxide magnetic nanoparticles immobilized β-glucosidase is about 40%, which is significantly higher than the value of free β-glucosidase (such as Figure 5 -c), this result indicates that the immobilization process increases the mechanical stability of the enzyme, which has positive significance for the subsequent production and transportation of immobilized enzymes.

[0035] Storage stability test: Take 100 μL of free β-glucosidase or sebacic acid modified iron oxide magnetic nanoparticles immobilized enzyme suspension, place it at 20°C, and take samples every 3 days for residual enzyme activity determination. Take the average of three replicates for each reaction, and the ratio of the sample activity to the maximum activity is the relative activity. The results show that after 34 days of storage at 20°C for β-glucosidase immobilized with sebacic acid modified iron oxide magnetic nanoparticles, the residual activity is about 40%, while the residual activity of free β-glucosidase under the same conditions is only 20% (such as Figure 5 -d). Therefore, β-glucosidase or sebaceous acid modified iron oxide magnetic nanoparticles immobilized enzyme is more conducive to long-term storage than free enzyme.

[0036] Example 4: Study on the recycling effect of sebaceous acid modified iron oxide magnetic nanoparticles prepared by the method of the present invention on the immobilization of β-glucosidase and the degradation effect of natural agricultural and sideline products

[0037] Determination of the recycling effect of immobilized enzyme: After each activity measurement of sebacic acid-modified iron oxide magnetic nanoparticles immobilized β-glucosidase, it was washed three times with phosphate buffer (pH = 7.0), and then the substrate p-NPG dissolved in phosphate buffer (pH = 7.0) was added to continue the enzyme activity measurement, and the above measurement was repeated 10 times. The results showed that sebacic acid-modified iron oxide magnetic nanoparticles immobilized β-glucosidase still maintained a relative activity of > 60% after 10 repeated reactions (such as Figure 6 -a), indicating that the recycling effect of the immobilized enzyme is good.

[0038] Analysis of the degradation effect of natural agricultural and sideline products: 890 μL of pH = 7.0 phosphate buffer and 50 μL of sebacic acid modified iron oxide magnetic nanoparticles immobilized β-glucosidase suspension were added to 20 mg of corn cobs or rice husk powder, and the reaction was carried out at 40°C for 30 minutes, and then the activity was measured. Under the action of an external magnetic field, the sebacic acid modified iron oxide magnetic nanoparticles immobilized β-glucosidase was separated from the reaction mixture and washed three times with sodium phosphate buffer (pH = 7.0). Then it was mixed with new corn cobs or rice husk powder for the next round of reaction until the activity could not be measured. The average value of three repetitions was taken for each reaction, and the percentage ratio of the sample activity to the maximum activity was the relative activity. The results showed that the immobilized enzyme was used to degrade rice husk powder 6 times and the activity was completely lost after degrading corn cob powder 10 times (such as Figure 6 -b). This indicates that sebaceous acid modified iron oxide magnetic nanoparticles immobilized β-glucosidase has important application prospects in the degradation of natural agricultural byproducts.

Claims

1. Use of sebaceous acid modified magnetic nanoparticles in immobilizing β-glucosidase, characterized in that: The method for modifying iron oxide magnetic nanoparticles by sebaceous acid comprises the following steps: dissolving 2.8 g of ferrous chloride in 10 ml of water and removing impurities with a microporous filter; then taking 7.5 mL of the ferrous chloride solution and mixing it with a 0.2 mol / L ferric chloride solution in a beaker, raising the temperature to 70 o С, quickly add 18 mL 25% NH3•H2O to the solution and stir rapidly for 1 min, then add 3.34 g sebaceous acid to the above reaction system and continue to stir at 70 o The reaction was stirred rapidly for 1 h. After the reaction was completed, the obtained black precipitate was the sebaceous acid-modified iron oxide magnetic nanoparticles. The immobilization method of β-glucosidase comprises the following steps: adding 20 mg of the sebaceous acid-modified iron oxide magnetic nanoparticles prepared above to 100 μL of phosphate buffer and stirring evenly, then adding 200 μL of the purified free enzyme solution, and placing the mixture at 25 o The immobilization was completed by shaking at 80 rpm in a constant temperature oscillator for 4 h. The immobilized product was separated by external magnetic force and washed with phosphate buffer for 5 times to elute the free enzyme, and finally the sebaceous acid modified iron oxide magnetic nanoparticles immobilized β-glucosidase were obtained.

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