阅读说明：本技术 一种具有光热增强酶活性的磁性氧化石墨烯基固定化乳糖酶及其制备方法 (Magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity and preparation method thereof ) 是由 郇伟伟 郭建忠 李洁 李迎龙 李高羽 于 2020-07-13 设计创作，主要内容包括：本发明提供了一种具有光热增强酶活性的磁性氧化石墨烯基固定化乳糖酶及其制备方法,属于生物化学领域。本发明首先制备磁性纳米颗粒-氧化石墨烯,并用芘丁酸和聚乙烯亚胺对其进行改性,所得材料负载能力高,稳定性好,能够实现近红外光照射下酶活性的光热增强,然后通过戊二醛活化该材料与乳糖酶共价偶联,得到了固定化乳糖酶。本发明突破了以往不能分离和回收可溶性乳糖,限制实践中的连续和长期应用。本发明所得固定化乳糖酶在近红外光照射下酶活性约为可溶性乳糖酶活性的152％,而且酶具有很高的稳定性和可重复利用性,有助于微球反应器的连续运行,使固定化酶技术在未来的工业领域具有广阔的应用前景。(The invention provides a magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity and a preparation method thereof, belonging to the field of biochemistry. According to the invention, firstly, magnetic nanoparticles, namely graphene oxide, are prepared, pyrenebutyric acid and polyethyleneimine are used for modifying the magnetic nanoparticles, the obtained material has high loading capacity and good stability, the photo-thermal enhancement of enzyme activity under near infrared light irradiation can be realized, and then the immobilized lactase is obtained by activating the material by glutaraldehyde and covalently coupling the material with lactase. The invention breaks through the previous problem that the soluble lactose can not be separated and recovered, and limits the continuous and long-term application in practice. The immobilized lactase obtained by the invention has the enzyme activity about 152% of that of soluble lactase under the irradiation of near infrared light, and the enzyme has very high stability and reusability, thereby being beneficial to the continuous operation of a microsphere reactor and leading the immobilized enzyme technology to have wide application prospect in the future industrial field.)

1. A magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity is characterized in that the immobilized lactase is bonded with lactase by adopting amino on a glutaraldehyde activated functional carrier;

the functional carrier is a modified magnetic graphene oxide nano composite material, the material is obtained by synthesizing a magnetic nanoparticle-graphene oxide nano composite material by taking graphene oxide as a raw material through solvothermal reaction, and modifying the magnetic nanoparticle-graphene oxide nano composite material by pyrenebutyric acid and polyethyleneimine.

2. The magnetic graphene oxide-based immobilized lactase with photothermal enhancing enzyme activity according to claim 1, wherein the immobilized lactase has an optimal reaction pH of 5.0, an optimal reaction temperature of 38-41 ℃, a reaction kinetic constant Km of 8.52mmol/L, and a Vmax of 38.2 mmol/min.

3. The magnetic graphene oxide-based immobilized lactase with photothermal enhancement enzyme activity according to claim 1, wherein the loading ratio of the modified magnetic graphene oxide nanocomposite immobilized lactase is 278.8 mg/g.

4. The magnetic graphene oxide-based immobilized lactase with photothermal enhancement enzyme activity according to claim 1, wherein the preparation of the modified magnetic graphene oxide nanocomposite and the process of immobilizing the enzyme make the formed structure rougher, and ensure the stability and reusability of the enzyme.

5. The magnetic graphene oxide-based immobilized lactase with photothermal enhancement enzyme activity according to claim 1, wherein the immobilized lactase is dispersed with nano-magnetic particles, and can exhibit good reusability during continuous operation of magnetic separation and hydrolysis reaction.

6. The preparation method of the magnetic graphene oxide-based immobilized lactase with photothermal enhancement enzyme activity according to any one of claims 1-5, wherein the preparation method comprises the following steps:

(1) carrying out ultrasonic treatment on graphene oxide, dispersing the graphene oxide in ethylene glycol of which the mass is 900-1100 times that of the graphene oxide, mixing to obtain a mixed solution, sequentially adding ferric chloride hexahydrate of which the mass is 0.48-0.55%, sodium acetate of which the mass is 1.98-2.43% and sodium citrate of which the mass is 0.085-0.11% of the mixed solution, mixing to obtain a mixture, treating the mixture by ultrasonic waves, transferring the mixture to an autoclave to react at 190-220 ℃, cooling, collecting a product, washing the product with ethanol and deionized water, and drying to obtain mGO, wherein the mass ratio of the materials is 4-6: dispersing mGO and pyrenebutyric acid in mGO acetone solution with the mass of 460-520 times, performing ultrasonic treatment on the mixed solution at 25-35 ℃, stirring, performing magnetic separation on a mg-pba product, washing with absolute ethyl alcohol, and drying to obtain mGP, wherein the mass ratio of the acetone solution to the pyrenebutyric acid is 10: 3-4: 700-900, dispersing mGP and polyethyleneimine in an ethanol solution, performing a crosslinking reaction at 28-32 ℃ to obtain mGP-PEI, washing with water, and drying to obtain the modified magnetic graphene oxide nanocomposite;

(2) according to the mass ratio of 1: 560-650 dispersing dried mGP-PEI in acetic acid buffer solution, shaking at 28-33 ℃, washing with acetic acid buffer solution, mixing activated mGP-PEI with acetic acid buffer solution containing lactase of 1.0mg/mL, placing the suspension at 28-32 ℃ for 4-6 h, and washing with acetic acid buffer solution by magnetic separation to obtain the magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity.

7. The method for preparing a magnetic graphene oxide-based immobilized lactase with photothermal enhancement enzyme activity according to claim 6, wherein the product is collected by using a magnet after autoclaving in step (1).

8. The method for preparing magnetic graphene oxide-based immobilized lactase with photothermal enhancement enzyme activity according to claim 6, wherein the ethanol solution in step (1) contains 20 mmol/L1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 8mmol/L N-hydroxysuccinimide.

9. The method for preparing magnetic graphene oxide-based immobilized lactase with photothermal enhancement enzyme activity according to claim 6, wherein the acetic acid buffer solution in step (2) has a concentration of 0.1mol/L, a pH of 5.0, and contains 2.5% by mass of glutaraldehyde.

10. The method for preparing a magnetic graphene oxide-based immobilized lactase with photothermal enhancement enzyme activity according to claim 6, wherein the acetic acid buffer solution in step (2) has a concentration of 0.1mol/L and a pH of 5.0.

Technical Field

The invention relates to the field of biochemistry, and particularly relates to magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity and a preparation method thereof.

Background

The enzyme is a highly specific biocatalyst and has wide applications in the fields of industrial processes, environmental fields, disease diagnosis, and the like. Among the various enzymes, lactase (β -galactosidase) is an important enzyme in the dairy industry for catalyzing the conversion of lactose to galactose and glucose. In recent years, hydrolysis reactions have received much attention in the area of improvement of cheese whey, improvement of sweetness and quality of products, production of valuable galactose and lactose-free dairy products, and the like. Lactose intolerance is a common condition in more than half of the world's population and it can lead to a variety of digestive symptoms, including abdominal pain, diarrhea, and gas discharge. The use of lactase as a supplement and hydrolysis of lactose is a viable means of addressing the symptoms. But its stability in a constantly changing thermochemical environment is poor and it is impossible to separate and recover the soluble lactose, limiting its continuous and long-term use in practice. It is highly desirable to develop efficient methods to improve enzyme stability and reusability.

Immobilizing the enzyme on a suitable solid carrier is an effective method for overcoming the defects of low enzyme activity, poor stability, reusability and the like. Several methods of immobilizing lactase have been developed, including adsorption, encapsulation and covalent bonding. Physical adsorption is convenient, but the interaction between the enzyme and the carrier is weak, so that the enzyme is easy to leak. Hydrogel beads were used for encapsulation of lactase, improving enzyme stability, but larger pore sizes resulted in rapid leakage of the enzyme. In addition, it has poor mechanical strength and large size, which limits further applications. By comparison, the less effective size matrix of chemically covalently immobilized lactase provides good bonding stability and recoverability, and several matrices including organic materials (e.g., natural and synthetic polymers) and inorganic materials (e.g., multiwalled carbon nanotubes, magnetic nanocomposites, and silica particles) have been successfully used to immobilize lactase. The improvement of enzyme stability is beneficial to the continuous operation of the microsphere reactor, so that the immobilized enzyme technology has wide application prospect in the future industrial field.

In recent years, various plasma nano composite materials are prepared as functional carriers, and the immobilization and the activity enhancement of enzymes are realized under the condition of light radiation. Different from the traditional bulk heating method, the plasma nano structure has good photo-thermal conversion capability and can realize rapid local heating. The enzyme activity can be controlled by adjusting the illumination time and the illumination power. Singmananei et al reported photothermal enhancement of immobilized enzyme activity mediated by gold nanorods. Fan et al use Ti₃C₂T_xThe nanosheet is used as a carrier and a photo-thermal material, so that the catalytic activity of the lipase is improved.

Graphene Oxide (GO) is a typical two-dimensional nanosheet material, having a high specific surface area, a large number of active groups, and excellent photo-thermal properties. Due to these unique physical and chemical properties, graphene oxide and graphene oxide-based nanocomposites have been demonstrated to be a high performance platform for protein immobilization, drug delivery, and photothermal conversion. It is envisioned that graphene oxide is a promising candidate material for achieving both immobilization of lactase and photothermal enhancement of enzymatic activity.

Disclosure of Invention

The invention aims to provide a magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity and a preparation method thereof, which solve the problems that the activity of the lactase (beta-galactosidase) is easy to be influenced, the stability is poor, the reusability of soluble lactase is poor, and the continuous and long-term use of the lactase is hindered.

In order to achieve the purpose, the invention adopts the following technical scheme:

a magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity is disclosed, wherein the immobilized lactase is bonded with lactase by adopting amino on a glutaraldehyde activated functional carrier;

the functional carrier is a modified magnetic graphene oxide nano composite material, the material is obtained by synthesizing a magnetic nanoparticle-graphene oxide nano composite material by taking graphene oxide as a raw material through solvothermal reaction, and modifying the magnetic nanoparticle-graphene oxide nano composite material by pyrenebutyric acid and polyethyleneimine.

Furthermore, the optimum reaction pH value of the immobilized lactase is 5.0, the optimum reaction temperature is 38-41 ℃, the reaction kinetic constant Km is 8.52mmol/L, and Vmax is 38.2 mmol/min.

Furthermore, the load proportion of the immobilized lactase of the modified magnetic graphene oxide nanocomposite reaches 278.8 mg/g.

Furthermore, the preparation of the modified magnetic graphene oxide nanocomposite and the process of fixing the enzyme make the structure rougher, and ensure the stability and the reusability of the enzyme.

Furthermore, the immobilized lactase is dispersed with nano magnetic particles, and can show good reusability in the continuous operation process of magnetic separation and hydrolysis reaction.

A preparation method of magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity comprises the following steps:

(1) carrying out ultrasonic treatment on graphene oxide, dispersing the graphene oxide in ethylene glycol of which the mass is 900-1100 times that of the graphene oxide, mixing to obtain a mixed solution, sequentially adding ferric chloride hexahydrate of which the mass is 0.48-0.55%, sodium acetate of which the mass is 1.98-2.43% and sodium citrate of which the mass is 0.085-0.11% of the mixed solution, mixing to obtain a mixture, treating the mixture by ultrasonic waves, transferring the mixture to an autoclave to react at 190-220 ℃, cooling, collecting a product, washing the product with ethanol and deionized water, drying to obtain mGO, and then carrying out ultrasonic treatment on the mixture according to a mass ratio of 4-6: dispersing mGO and pyrenebutyric acid in mGO acetone solution with the mass of 460-520 times, performing ultrasonic treatment on the mixed solution at 25-35 ℃, stirring, performing magnetic separation on a mg-pba product, washing with absolute ethyl alcohol, and drying to obtain mGP, wherein the mass ratio of the acetone solution to the pyrenebutyric acid is 10: 3-4: 700-900, dispersing mGP and polyethyleneimine in an ethanol solution, performing a crosslinking reaction at 28-32 ℃ to obtain mGP-PEI, washing with water, and drying to obtain the modified magnetic graphene oxide nanocomposite;

(2) according to the mass ratio of 1: 560-650 dispersing dried mGP-PEI in acetic acid buffer solution, shaking at 28-33 ℃, washing with acetic acid buffer solution, mixing activated mGP-PEI with acetic acid buffer solution containing lactase of 1.0mg/mL, standing the suspension at 28-32 ℃ for 4-6 h, and washing with acetic acid buffer solution by magnetic separation to obtain the magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity.

Further, the product is collected by a magnet after being treated by autoclave in the step (1).

Further, the ethanol solution in the step (1) contains 20mmol/L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 8mmol/L of N-hydroxysuccinimide.

Further, the concentration of the acetic acid buffer solution in the step (2) is 0.1mol/L, the pH is 5.0, and the acetic acid buffer solution contains 2.5% of glutaraldehyde by mass fraction.

Further, the concentration of the acetic acid buffer solution in the step (2) is 0.1mol/L, and the pH value is 5.0.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

(1) the invention discloses a process for preparing a functional magnetic graphene oxide nanocomposite for lactase immobilization, which comprises the steps of synthesizing a magnetic nanoparticle-graphene oxide nanocomposite (MGO) through solvothermal reaction, then modifying the surfaces of pyrenebutyric acid and polyethyleneimine through respective pi interaction and electrostatic interaction, and after glutaraldehyde activation, covalently immobilizing lactase on amino groups on a functional carrier.

(2) The invention breaks through the problem that the continuous and long-term application of the soluble lactose in practice is limited because the soluble lactose cannot be separated and recovered in the past; the enzymatic activity of mGPP-lactase under the irradiation of near infrared light is about 152% of that of soluble lactase, and the polymeric lactose can be effectively degraded; the stability and the reusability of the enzyme are improved, the continuous operation of the microsphere reactor is facilitated, and the immobilized enzyme technology has wide application prospect in the future industrial field.

(3) The invention provides a method for preparing a magnetic nanoparticle-graphene oxide nanocomposite, the obtained material is used as a functional carrier for covalent immobilization of lactase, the influence of several immobilization parameters on loading capacity, immobilization efficiency and enzyme activity is studied in detail, and mGPP has various components and a unique structure, so that mGPP has high loading capacity and good stability under the conditions of a wide pH solution and temperature. In addition, the application also tests the influence of the photo-thermal property of mGPP on the activity of immobilized lactase, and the result shows that mGPP can generate heat under the irradiation of near infrared light to cause the local surface temperature to rise, so that the activity of the immobilized lactase is enhanced compared with that of free lactase, and the mGPP-lactase shows good reusability in the continuous operation process of magnetic separation and hydrolysis reaction.

Drawings

FIG. 1 is a structural characterization of the prepared material by Transmission Electron Microscopy (TEM) with (a) GO, (b) mGO, (c) mGPP, (d) mGPP-lactase.

FIG. 2 is a spectrum obtained by studying the chemical composition of the prepared material using Fourier transform Infrared Spectroscopy (FTIR), wherein (a) FTIR spectrum (b) Zeta potential (i) GO, (ii) mGO, (iii) mGP, (iv) mGPP, (v) mGPP-lactase.

Fig. 3 is a spectrum of crystal structures of GO, mGO and mGPP-lactase determined by X-ray diffraction (XRD) analysis, wherein (a) XRD spectrum: (i) GO, (ii) mGO, and (iii) mbgpp-Lactase, (b) magnetization curve (i) mGO, and (ii) mbgpp-Lactase; the inlays in (b) are digital images of mGPP-Lactase dispersed in an aqueous solution (left) and after magnetic separation (right).

FIG. 4 is a bar graph of the loading capacity and immobilization efficiency of lactase on three different supports, mGO, mGE and mGPP.

FIG. 5 is a graph showing the effect of pH and temperature on soluble and immobilized lactase activity.

FIG. 6 is a bar graph of the change in enzyme activity of mGPP-lactase at different times of near infrared light irradiation, with 100% initial activity.

FIG. 7 is a bar graph of the reusability of mGPP-lactase under near infrared light.

FIG. 8 is a spectrum of hydrolysis of lactose by soluble or immobilized lactase under different conditions:

(a) mGPP-lactase under near infrared light irradiation; (b) mGPP-lactase without being irradiated by near infrared light; (c) soluble lactase under near-infrared light irradiation; (d) a soluble lactase that is not irradiated by near infrared light.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

A magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity is disclosed, wherein the immobilized lactase is bonded with lactase by adopting amino on a glutaraldehyde activated functional carrier; the functional carrier is a modified magnetic graphene oxide nano composite material, the material is obtained by synthesizing a magnetic nanoparticle-graphene oxide nano composite material by taking graphene oxide as a raw material through solvothermal reaction, and modifying the magnetic nanoparticle-graphene oxide nano composite material by pyrenebutyric acid and polyethyleneimine. The immobilized lactase has the optimal reaction pH value of 5.0, the optimal reaction temperature of 38-41 ℃, the reaction kinetic constant Km of 8.52mmol/L and the Vmax of 38.2 mmol/min. The load proportion of the immobilized lactase of the modified magnetic graphene oxide nanocomposite reaches 278.8 mg/g. The modified magnetic graphene oxide nanocomposite can generate heat under near-infrared light irradiation, so that the local surface temperature is increased, and the activity of immobilized lactase is enhanced compared with that of free lactase. The preparation of the modified magnetic graphene oxide nanocomposite and the process of fixing the enzyme make the structure rougher, and ensure the stability and the reusability of the enzyme. The immobilized lactase shows good reusability in the continuous operation process of magnetic separation and hydrolysis reaction.

A preparation method of magnetic graphene oxide-based immobilized lactase with photo-thermal enhanced enzyme activity comprises the following steps:

(1) carrying out ultrasonic treatment on graphene oxide, dispersing the graphene oxide in ethylene glycol with the mass of 900-1100 times that of the graphene oxide, mixing for 50-70 min to obtain a mixed solution, sequentially adding ferric chloride hexahydrate with the mass of 0.48-0.55% of the mixed solution, sodium acetate with the mass of 1.98-2.43% of the mixed solution and sodium citrate with the mass of 0.085-0.11% of the mixed solution, mixing to obtain a mixture, carrying out ultrasonic treatment on the mixture for 1-1.8 h, transferring the mixture to a Teflon-lined stainless steel autoclave, reacting for 5-7 h at the temperature of 190-220 ℃, cooling to room temperature, collecting an obtained product with a magnet, washing with ethanol and deionized water for three times, drying at the temperature of 50-60 ℃ for 7-10 h to obtain mGO, and then carrying out ultrasonic treatment on the graphene oxide in an amount of 4-6: dispersing mGO and pyrenebutyric acid in an acetone solution with the mass of 460-520 times of mGO, performing ultrasonic treatment on the mixed solution at the temperature of 25-35 ℃ for 25-45 min, continuing stirring for 20-24 h, performing magnetic separation on a mg-pba (mGP for short) product, washing for 3 times with absolute ethyl alcohol, drying at the temperature of 50-60 ℃ for 7-10 h, and mixing the obtained product according to the mass ratio of 10: 3-4: 700-900, dispersing mGP and polyethyleneimine in an ethanol solution with the volume fraction of 25%, wherein the ethanol solution simultaneously contains 20 mmol/L1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 8mmol/L N-hydroxysuccinimide, carrying out a crosslinking reaction at 28-32 ℃ for 7-10 h, finally washing mGP-PEI (abbreviated as mGPP) with deionized water for 3 times, and drying to obtain the modified magnetic graphene oxide nanocomposite;

(2) according to the mass ratio of 1: 560-650 dispersing dried mGPP in acetic acid buffer solution (0.1mol/L, pH5.0) containing 2.5% glutaraldehyde, shaking the mixed solution gently at 28-33 ℃ for 3-5 h, washing the glutaraldehyde-activated mGPP with acetic acid buffer solution (0.1mol/L, pH5.0) three times, then mixing the activated mGPP with acetic acid buffer solution (0.1mol/L, pH5.0) containing lactase (1.0mg/mL), placing the suspension at 28-32 ℃ for 4-6 h, washing with acetic acid buffer solution magnetically for three times (0.1mol/L, pH5.0), and obtaining lactase immobilized carrier (mGPP-lactase).

The wash liquor during the cleaning was collected and the protein concentration was determined by the Bradford protein method. The loading amount of immobilized lactase is determined by dividing the amount of immobilized lactase by the amount of carrier, and the immobilization efficiency is determined by dividing the amount of immobilized lactase by the initial amount of lactase.