Immobilized enzyme and method for producing the same

Abstract

The application discloses an immobilized enzyme and a preparation method thereof, wherein the immobilized enzyme comprises a substrate formed by bamboo charcoal powder, a linker deposited on the surface of the substrate, and an enzyme combined with the linker. The immobilized enzyme uses the bamboo charcoal powder which is widely sourced, environmentally friendly, low in price and high in porosity as a carrier substrate, and the linker is deposited on the surface of the substrate, the linker can improve the chemical stability and biocompatibility of the immobilized enzyme carrier material, and the lipase is adsorbed and fixed through the mediation of the linker. The immobilized enzyme is good in stability, high in catalytic activity, low in separation cost and reusable.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to immobilized enzymes and methods for their preparation. Background Technology

[0002] Enzymes are highly efficient, green, and widely used biocatalysts. Because their immobilized forms are superior to their free forms in many properties, enzyme immobilization technology has emerged and continues to develop. Research on immobilization technology in my country began in the 1970s. Currently, immobilized enzymes are widely used in food, medicine, energy, and environmental remediation, and immobilized enzyme technology has become one of the key and hot topics in enzyme engineering research.

[0003] The performance of immobilized enzymes is largely influenced by the immobilization carrier and the immobilization method. Developing carrier materials and immobilization techniques that minimize the impact on enzyme activity and reduce the risk of leakage has always been a research goal for immobilized enzymes. With the continuous interdisciplinary development of biotechnology and materials science, chemistry, and other disciplines, new carrier modification methods and novel materials are constantly emerging, enriching the sources of carriers for immobilization technology research and leading to a number of immobilization strategy studies centered around novel carriers. To date, materials including metal-organic frameworks, magnetic nanoparticles, and silica have been successfully used for enzyme immobilization. Each immobilized enzyme carrier material has its own advantages, and corresponding research and development have made significant progress, yielding many important results. Currently, research on novel immobilization technologies mainly focuses on further improving enzyme activity, enhancing the environmental tolerance and operational stability of immobilized enzymes. However, research on novel immobilization technologies is prone to the drawbacks of cumbersome carrier preparation processes and high production costs.

[0004] Therefore, constructing a simple, efficient, green, economical, and high-performance synthetic methodology for immobilized enzymes has significant scientific research and practical application value. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, one object of this invention is to provide an immobilized enzyme that uses inexpensive natural bamboo charcoal, which has a porous structure and high porosity, as a matrix material. Lipase is adsorbed and immobilized via linkers, overcoming the shortcomings of free enzymes such as poor stability, high separation costs, and lack of reusability. Furthermore, its preparation method is simple, and the reaction conditions are mild. Compared to direct adsorption of lipase by bamboo charcoal, the bamboo charcoal carrier with deposited linkers not only has a high enzyme loading capacity, but the resulting immobilized enzyme also exhibits higher affinity for the substrate and higher enzyme catalytic efficiency.

[0006] It should be noted that this invention was completed based on the following work of the inventors:

[0007] The carrier is a crucial component in enzyme immobilization; therefore, novel carriers such as metal-organic frameworks and magnetic nanomaterials have attracted significant attention in the field of enzyme immobilization technology in recent years. However, research on novel immobilization technologies is prone to drawbacks such as cumbersome preparation processes and high production costs. The inventors have developed a method using inexpensive, highly porous, and environmentally friendly bamboo charcoal as the carrier matrix. By utilizing dopamine as a reducing agent to deposit gold nanoparticles on the bamboo charcoal surface, this method not only improves the chemical stability and biocompatibility of the immobilized enzyme carrier material but also enhances the enzyme's affinity for the substrate and its catalytic efficiency. The preparation method is simple to operate and the reaction conditions are mild.

[0008] Therefore, according to one aspect of the present invention, an immobilized enzyme is provided. According to an embodiment of the present invention, the immobilized enzyme comprises: a matrix formed of bamboo charcoal powder; a linker deposited on the surface of the matrix; and an enzyme bound to the linker.

[0009] The immobilized enzyme according to embodiments of the present invention uses bamboo charcoal powder, which is widely available, inexpensive, has high porosity, and is environmentally friendly, as a carrier matrix. Linkers are deposited on the matrix surface, which improve the chemical stability and biocompatibility of the immobilized enzyme carrier material. Lipase is immobilized through linker-mediated adsorption, resulting in low separation cost and reusability. Compared to free enzymes, immobilized lipases exhibit enhanced tolerance to catalytic environments, significantly improved thermal stability, and reusability. Simultaneously, the presence of linkers promotes the affinity of the immobilized enzyme for the substrate and enhances its catalytic efficiency.

[0010] In addition, the immobilized enzyme according to the above embodiments of the present invention may also have the following additional technical features:

[0011] According to an embodiment of the present invention, the linker is a gold nanoparticle.

[0012] According to an embodiment of the present invention, the linker is deposited on the matrix surface via dopamine deposition.

[0013] According to an embodiment of the present invention, the catalytic pH of the immobilized enzyme is 6-9, preferably 8.

[0014] According to another aspect of the present invention, the present invention provides a method for preparing the aforementioned immobilized enzyme. According to an embodiment of the present invention, the method includes: first mixing bamboo charcoal powder with a first buffer solution to obtain a first mixed solution; second mixing the first mixed solution with a reducing agent to obtain a second mixed solution; third mixing the second mixed solution with a metal salt to reduce the metal salt using the reducing agent, causing the metal to deposit on the particle surface of the bamboo charcoal powder, obtaining a carrier intermediate; and dispersing the carrier intermediate in a second buffer solution, and then fourth mixing it with an enzyme solution to allow the enzyme in the enzyme solution to bind to the linker, thereby obtaining the immobilized enzyme.

[0015] The method for preparing the aforementioned immobilized enzyme according to embodiments of the present invention uses low-cost, highly porous bamboo charcoal as a carrier matrix. Bamboo charcoal is widely available, has high hardness and specific surface area, exhibits superior biocompatibility compared to other carbon materials, and is highly environmentally friendly, aligning with the principles of green chemistry and sustainable development. Furthermore, by using a reducing agent to reduce metal salts and deposit metals on the surface of the bamboo charcoal, and then using the metal as a linker to connect the enzyme, the chemical stability and biocompatibility of the immobilized enzyme carrier material are improved. Lipase is immobilized through linker-mediated adsorption. The preparation method is simple to operate, with mild reaction conditions; and the enzyme immobilization rate is high, resulting in an immobilized enzyme with higher affinity for the substrate and higher enzyme catalytic efficiency.

[0016] According to an embodiment of the present invention, the pH value of the first buffer solution is 7.5-9.0.

[0017] According to an embodiment of the present invention, the second buffer is a phosphate buffer, preferably, the concentration of the phosphate buffer is 40-60 mM and the pH value is 6.0-9.0.

[0018] According to an embodiment of the present invention, the metal is gold nanoparticles.

[0019] According to an embodiment of the present invention, the metal salt is a gold salt, preferably chloroauric acid.

[0020] According to an embodiment of the present invention, the reducing agent is dopamine.

[0021] According to an embodiment of the present invention, the ratio of the bamboo charcoal powder, the reducing agent, the metal salt and the first buffer solution is 1g:20-40mg:0.3-0.5mmol:20-40mL, preferably 1g:25-35mg:0.35-0.45mmol:25-35mL.

[0022] According to an embodiment of the present invention, the mass ratio of the carrier intermediate to the enzyme in the enzyme solution is 1:0.15-0.4.

[0023] According to an embodiment of the present invention, the second mixing is performed by first magnetic stirring at room temperature for 20-30 minutes.

[0024] According to an embodiment of the present invention, the third mixing is a reaction continued with magnetic stirring at room temperature for 1-2 hours.

[0025] According to an embodiment of the present invention, the fourth mixing is carried out under conditions of 30-65°C with second magnetic stirring for 0.5-4 hours.

[0026] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0028] Figure 1 The image shown is a scanning electron microscope image according to an embodiment of the present invention, wherein A and B are bamboo charcoal, and C is a bamboo charcoal-polydopamine-gold nanoparticle (C-PDA-Au) immobilized enzyme carrier;

[0029] Figure 2 The image shows an X-ray photoelectron spectroscopy (XPS) spectrum according to an embodiment of the present invention, wherein A is bamboo charcoal, B is a bamboo charcoal-polydopamine-gold nanoparticle (C-PDA-Au) immobilized enzyme carrier, and C is an immobilized enzyme;

[0030] Figure 3 A schematic diagram showing the effect of enzyme addition amount on enzyme immobilization and enzyme activity according to an embodiment of the present invention is presented.

[0031] Figure 4 A schematic diagram showing the effect of immobilization time on enzyme loading and enzyme activity according to an embodiment of the present invention is presented.

[0032] Figure 5 A schematic diagram showing the effect of temperature on the catalytic activity of free and immobilized enzymes according to an embodiment of the present invention is displayed.

[0033] Figure 6 A schematic diagram showing the effect of pH on the catalytic activity of free and immobilized enzymes according to an embodiment of the present invention is provided.

[0034] Figure 7 A schematic diagram showing the results of a thermostability study of a free enzyme and an immobilized enzyme according to an embodiment of the present invention is displayed.

[0035] Figure 8 A schematic diagram showing the repeatability test results of an immobilized enzyme according to an embodiment of the present invention is displayed. Detailed Implementation

[0036] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0037] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0038] According to one aspect of the present invention, an immobilized enzyme is provided. According to an embodiment of the present invention, the immobilized enzyme comprises: a matrix formed of bamboo charcoal powder; a linker deposited on the surface of the matrix; and an enzyme bound to the linker.

[0039] The immobilized enzyme according to embodiments of the present invention uses bamboo charcoal powder, which is widely available, inexpensive, environmentally friendly, and has high porosity, as a carrier matrix. Linkers are deposited on the matrix surface, which improve the chemical stability and biocompatibility of the immobilized enzyme carrier material. Lipase is immobilized through linker-mediated adsorption, resulting in low separation cost and reusability. Compared to free enzymes, immobilized lipases exhibit enhanced tolerance to catalytic environments, significantly improved thermal stability, and reusability. Simultaneously, the presence of linkers promotes the affinity of the immobilized enzyme for the substrate and enhances its catalytic efficiency.

[0040] According to embodiments of the present invention, China has abundant bamboo forest resources and bamboo has a fast growth cycle. The bamboo charcoal produced by its pyrolysis can be used as a porous medium material. It has a porous structure and high porosity, large hardness and specific surface area, better biocompatibility than other carbon materials, and is highly environmentally friendly. It conforms to the concept of green chemistry and sustainable development and is an excellent carrier matrix.

[0041] According to embodiments of the present invention, the immobilized enzyme exhibits high activity, enzyme loading capacity, and enzyme immobilization rate. In some embodiments, the enzyme activity, enzyme loading capacity, and enzyme immobilization rate can reach 513.5 U / g, 234.78 μg / mg, and 93.91%, respectively. Furthermore, in some embodiments, after the immobilized enzyme is reused 5 times, the remaining relative enzyme activity is still 71.09%. Compared to free enzymes, immobilized lipases exhibit enhanced tolerance to catalytic environments and high thermal stability and reusability.

[0042] It should be noted that the immobilized enzymes in the embodiments of the present invention can be used for the immobilization of various enzymes, and the type of enzyme is not limited. For example, it can be lipase, etc.

[0043] According to an embodiment of the present invention, the linker is a gold nanoparticle. This effectively protects the enzyme and overcomes the shortcomings of free enzymes, such as poor stability, high separation cost, and lack of reusability. Furthermore, the modification of the carrier with Au nanoparticles, which have good biocompatibility, further increases the affinity of the immobilized enzyme for the substrate, improving the enzyme's catalytic efficiency, and its kinetic constant K... m Smaller than free enzyme, K cat Greater than free enzyme.

[0044] According to an embodiment of the present invention, the linker is deposited on the matrix surface via dopamine deposition. Thus, dopamine and its oxidized polymers possess reducing properties due to abundant phenol / quinone conversion, enabling them to reduce metal salts, resulting in metal deposition on the matrix surface to form the linker.

[0045] According to embodiments of the present invention, the catalytic pH of the immobilized enzyme is 7-9, preferably 8. Therefore, the immobilized enzyme exhibits strong catalytic activity, with even better catalytic activity at pH 8.

[0046] According to another aspect of the present invention, the present invention provides a method for preparing the aforementioned immobilized enzyme. According to an embodiment of the present invention, the method includes:

[0047] S100 First Hybrid

[0048] According to an embodiment of the present invention, bamboo charcoal powder is first mixed with a first buffer solution to obtain a first mixed solution. This first mixing facilitates the thorough dispersion of the bamboo charcoal powder in the first buffer solution.

[0049] According to an embodiment of the present invention, the pH value of the first buffer solution is 7.5-9.0. Thus, under alkaline conditions, dopamine can undergo self-polymerization to generate polydopamine. Polydopamine has certain adhesive and reducing properties, promoting the reduction of gold ions to gold nanoparticles and their in-situ deposition on the surface of bamboo charcoal.

[0050] S200 Second Hybrid

[0051] According to an embodiment of the present invention, the first mixed solution is mixed with a reducing agent in a second mixing process to obtain a second mixed solution. This second mixing facilitates the full dispersion of the reducing agent in the first mixed solution.

[0052] According to an embodiment of the present invention, the reducing agent is dopamine. Dopamine and its oxidized polymers have reducing properties due to abundant phenol / quinone conversion and can reduce metal salts, with the resulting metal deposited on the matrix surface.

[0053] According to an embodiment of the present invention, the second mixing involves magnetic stirring at room temperature for 20-30 minutes. Under these conditions, dopamine, after oxidative polymerization for a period of time, can be reduced to prepare spherical gold nanoparticles without the addition of an external reducing agent.

[0054] S300 reduction reaction

[0055] According to an embodiment of the present invention, the second mixed solution is mixed with a metal salt in a third mixing process to reduce the metal salt using the reducing agent, causing the metal to deposit on the surface of the bamboo charcoal powder particles, thus obtaining a carrier intermediate. Therefore, by reducing the metal salt using a reducing agent under the third mixing conditions, the reduced metal is deposited on the surface of the bamboo charcoal powder in the form of nanoparticles, forming linkers for subsequent enzyme ligation.

[0056] According to an embodiment of the present invention, the metal is gold nanoparticles. Thus, gold nanoparticles modify the bamboo charcoal powder carrier to protect the enzyme, overcoming the shortcomings of free enzymes such as poor stability, high separation cost, and inability to be reused; simultaneously, the presence of gold nanoparticles promotes the affinity of the immobilized enzyme for the substrate and its catalytic efficiency.

[0057] According to an embodiment of the present invention, the metal salt is a gold salt, preferably chloroauric acid. Thus, dopamine and its oxidized polymer can reduce the gold ions in chloroauric acid to gold nanoparticles, causing the metal to deposit on the surface of the bamboo charcoal powder carrier, forming linkers that bind to enzymes.

[0058] According to an embodiment of the present invention, the ratio of the bamboo charcoal powder, the reducing agent, the metal salt, and the first buffer solution is 1g:20-40mg:0.3-0.5mmol:20-40mL, preferably 1g:25-35mg:0.35-0.45mmol:25-35mL. Thus, at this ratio, an appropriate amount of metal salt is fully reduced to nano-gold, which is deposited on the surface of the bamboo charcoal powder to form linkers. Simultaneously, the nano-gold particles, possessing unique properties such as high biocompatibility, adsorption capacity, and conductivity, further enhance the affinity of enzyme molecules for the substrate and the catalytic efficiency.

[0059] According to an embodiment of the present invention, the third mixing involves continuing the magnetic stirring reaction at room temperature for 1-2 hours. This facilitates thorough mixing and contact between the metal salt, reducing agent, and bamboo charcoal powder, promoting the reduction of the metal salt to metal and its uniform precipitation on the surface of the bamboo charcoal powder.

[0060] S400 enzyme immobilization

[0061] According to an embodiment of the present invention, the carrier intermediate is dispersed in a second buffer solution and then mixed with an enzyme solution in a fourth mixing process to allow the enzyme in the enzyme solution to bind to the linker, thereby obtaining the immobilized enzyme. Thus, through the fourth mixing, the enzyme comes into contact with the linker, thereby binding the enzyme in the enzyme solution to the linker and achieving enzyme immobilization. Compared to the direct adsorption of lipase by bamboo charcoal, the bamboo charcoal carrier with deposited linkers not only has a high enzyme loading capacity, but also produces an immobilized enzyme with higher affinity for the substrate and higher enzyme catalytic efficiency.

[0062] According to an embodiment of the present invention, the second buffer is a phosphate buffer, preferably with a concentration of 40-60 mM and a pH of 6.0-9.0. Enzymes exhibit different ionization states at different pH values, and when immobilized in a preferred enzyme activity state, the resulting immobilized enzyme will maintain this preferred state.

[0063] According to an embodiment of the present invention, the fourth mixing is carried out at 30-65°C with second magnetic stirring for 0.5-4 hours. This facilitates sufficient contact between the appropriate amount of enzyme and the carrier intermediate, and uniform binding to the surface of the carrier intermediate, resulting in immobilized enzymes with better activity.

[0064] According to an embodiment of the present invention, the mass ratio of the carrier intermediate to the enzyme in the enzyme solution is 1:0.15-0.4. This facilitates the full binding of an appropriate amount of enzyme to the surface of the carrier intermediate, resulting in a more effective immobilized enzyme.

[0065] According to an embodiment of the present invention, the rotation speeds of the first and second magnetic stirrers are 400-1000 rpm. This provides a suitable rotation speed for sufficient contact between the reactants.

[0066] The method for preparing the aforementioned immobilized enzyme according to embodiments of the present invention uses low-cost, highly porous bamboo charcoal as a carrier matrix. Bamboo charcoal has a wide range of sources, large hardness and specific surface area, superior biocompatibility compared to other carbon materials, and is highly environmentally friendly, conforming to the concepts of green chemistry and sustainable development. A reducing agent is used to reduce metal salts and deposit metals on the surface of the bamboo charcoal. The enzyme is then linked using the metal as a linker, improving the chemical stability and biocompatibility of the immobilized enzyme carrier material. Lipase is immobilized through linker-mediated adsorption. The preparation method is simple to operate, with mild reaction conditions; and the enzyme immobilization rate is high. In a preferred embodiment of the present invention, the enzyme loading is 234.78 μg / mg, and the enzyme immobilization rate is as high as 93.91%. Furthermore, the prepared immobilized enzyme has higher affinity for the substrate and higher enzyme catalytic efficiency.

[0067] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0068] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products, such as those purchased from Sigma.

[0069] Example 1: Preparation method of immobilized enzyme

[0070] (1) Preparation of C-PDA-Au immobilized enzyme carrier: Weigh 1g of bamboo charcoal powder and disperse it in 30mL of tris(hydroxymethyl)aminomethane hydrochloride buffer (pH=8.5, 50mM). Add 30mg of dopamine salt and stir magnetically for 30min at room temperature. Then add chloroauric acid solution (0.4mM) and continue to stir magnetically for 1-2h at room temperature. After the reaction is complete, centrifuge and wash repeatedly. The product is the C-PDA-Au immobilized enzyme carrier.

[0071] (2) Enzyme immobilization: The C-PDA-Au immobilized enzyme carrier obtained in step (1) was dispersed in 10 mL of phosphate buffer (pH = 8.0, 50 mM), and enzyme solution (Amano lipase PS, purchased from Sigma, catalog number: 534641) was added. The ratio of enzyme to C-PDA-Au immobilized enzyme carrier was 250 mg / g. The mixture was magnetically stirred for 1 h. After the reaction was completed, the mixture was centrifuged and repeatedly washed. The resulting product was the bamboo charcoal immobilized enzyme.

[0072] Scanning electron microscopy of the prepared C-PDA-Au immobilized enzyme carrier is shown below. Figure 1 As shown, (A) and (B) are scanning electron microscope (SEM) images of bamboo charcoal powder. It can be seen from the images that the surface of the bamboo charcoal carrier is dense and porous, providing a large specific surface area for the adsorption of lipase. (C) is a scanning electron microscope (SEM) image of the C-PDA-Au immobilized enzyme carrier. Compared with the SEM image of bamboo charcoal powder, there are more spherical particles on the surface of bamboo charcoal powder, indicating that biocompatible biomimetic polydopamine and nano-gold have been successfully modified on the bamboo charcoal matrix material.

[0073] The X-ray photoelectron spectroscopy (XPS) of the prepared immobilized enzyme is as follows: Figure 2As shown, (A) is the XPS spectrum of bamboo charcoal, and (B) is the XPS spectrum of the C-PDA-Au immobilized enzyme carrier. Compared with bamboo charcoal, the spectrum of the C-PDA-Au immobilized enzyme carrier has an additional Au4f peak, which further indicates that the nano-gold was successfully modified on the bamboo charcoal matrix material. Compared with the XPS spectra of bamboo charcoal and the carrier, the O1s and N1s peaks of the immobilized enzyme (C) are significantly enhanced. This should be due to the enzyme adsorbing on the surface of bamboo charcoal, which increases the ratio of O1s and N1s, indicating that the enzyme was successfully immobilized on the carrier.

[0074] Example 2: Determination of enzyme immobilization capacity

[0075] In this embodiment, the enzyme loading capacity of the immobilized enzyme prepared in Example 1 was measured, as follows:

[0076] 100 mg of the C-PDA-Au immobilized enzyme carrier prepared in Example 1 was dispersed in 10 mL of phosphate buffer (50 mM, pH = 7.0). 25 mg of enzyme powder was weighed and added to the solution. The mixture was magnetically stirred at room temperature for 1 h. After the reaction was complete, the mixture was centrifuged, and the concentration of residual lipase in the supernatant was determined using the BCA quantitative method. The lipase loading and immobilization rate were calculated according to the following formula:

[0077] Enzyme loading (μg / mg) = (M-CV) / W

[0078] In the formula, M represents the initial amount of enzyme used (μg), C represents the residual enzyme concentration in the reaction solution after immobilization (μg / mL), V represents the volume of the immobilized enzyme reaction solution (mL), and W represents the mass of the carrier (mg).

[0079] Enzyme immobilization rate (%) = (MT) * 100 / M

[0080] In the formula, M represents the initial amount of enzyme used (μg), and T is the residual enzyme mass in the reaction solution after immobilization (μg).

[0081] The immobilized enzyme prepared according to the conditions of the example had an enzyme loading of 234.78 μg / mg and an enzyme immobilization rate of 93.91%.

[0082] Example 3: Enzyme Activity Assay

[0083] In this embodiment, the activity of the immobilized enzyme prepared in Example 1 was determined using the p-nitrophenol method, as detailed below:

[0084] 1. Enzyme activity assay

[0085] (1) Weigh 30 mg of p-nitrophenol palmitate and disperse it in 10 mL of isopropanol;

[0086] (2) Take 1 mL of the solution obtained in step (1) and dilute it 10 times with a phosphate buffer solution with pH = 8.0;

[0087] (3) Take 2.4 mL of the diluted solution from step (2) into a test tube, preheat at 37°C for 5 min, add 300 μL of enzyme solution, react for 10 min, and immediately add 1 mL of 95% ethanol to mix evenly to terminate the reaction (300 μL of deionized water replaces 300 μL of enzyme solution, and other conditions remain unchanged to obtain a blank).

[0088] (4) After the reaction is complete, dilute it 10 times and measure its absorbance at 410 nm.

[0089] 2. Calculate the hydrolysis activity using the p-nitrophenol standard curve.

[0090] The enzyme activity unit is defined as the amount of enzyme required per minute to catalyze the decomposition of a substrate and release 1 μmol of p-nitrophenol under the specified assay conditions.

[0091] Enzyme activity calculation formula:

[0092] X = (A * V * N * 1000) / T * m

[0093] Where X is the enzyme activity (U / g), A is the concentration calculated from the standard curve of p-nitrophenol (μmol / mL), N is the dilution factor, T is the reaction time (min), V is the total volume of the reaction system (mL), m is the amount of enzyme used (g), and 1000 is the ratio from mg to g.

[0094] The immobilized enzyme prepared according to the conditions of the example had an enzyme activity of 513.5 U / g.

[0095] Example 4: Optimization of enzyme immobilization conditions

[0096] In this embodiment, the experimental conditions during enzyme immobilization were optimized as follows:

[0097] (1) Effect of enzyme addition amount on immobilization

[0098] The amount of enzyme used affects the activity and immobilization capacity of the immobilized enzyme. This example investigated the effect of different enzyme amounts on the generation of immobilized enzymes. The immobilized enzymes were prepared according to the method in Example 1, except that the ratio of enzyme to C-PDA-Au immobilized enzyme carrier was 150 mg / g, 200 mg / g, 250 mg / g, 300 mg / g, 350 mg / g, 400 mg / g, and 450 mg / g, respectively.

[0099] The immobilized enzyme loading and activity were determined according to the methods described in Examples 2 and 3. The results are as follows: Figure 3As shown, there is a positive correlation between enzyme loading capacity and enzyme dosage. The immobilized enzyme exhibits the highest activity when the enzyme dosage increases to 250 mg / g. However, as the lipase dosage further increases to 400 mg / g, the enzyme activity decreases. This phenomenon can be attributed to excessive enzyme loading, leading to steric hindrance and reduced activity due to overcrowding on the carrier surface. Therefore, the optimal enzyme loading capacity for preparing bamboo charcoal-immobilized enzymes is 250 mg / g.

[0100] (2) The effect of immobilization time

[0101] To investigate the immobilization time, this experiment measured the enzyme activity and enzyme loading of bamboo charcoal immobilized enzymes at different immobilization times. The immobilized enzymes were prepared according to the method in Example 1, except that the immobilization time ranged from 0.5 h to 4 h, with a time interval of 0.5 h.

[0102] Experimental results are as follows Figure 4 As shown, both the activity and immobilized enzyme load of the immobilized enzyme reached their peak values ​​when the immobilization time was 1 hour. Further extending the immobilization time slightly reduced both enzyme activity and immobilized enzyme load. This may be because excessively long immobilization times result in a larger amount of enzyme being immobilized, burying the enzyme's active sites and causing steric hindrance, which leads to a decrease in immobilized enzyme activity. Therefore, the optimal reaction time for immobilizing the enzyme is 1 hour. Under the optimal preparation conditions (enzyme dosage 250 mg / g, immobilization time 1 hour), the prepared immobilized enzyme had an activity of 513.5 U / g, an enzyme load of 234.78 μg / mg, and an immobilization rate of 93.91%.

[0103] Example 5: Enzymatic properties of immobilized enzymes

[0104] In this embodiment, the enzymatic properties of the immobilized enzyme prepared in Example 1 were tested, as follows:

[0105] (1) Preferred catalytic temperature

[0106] Temperature is an important factor affecting the activity of enzyme catalytic reactions. Therefore, in this example, the catalytic activities of free enzymes and immobilized enzymes at different temperature systems were investigated.

[0107] Appropriate amounts of free enzyme and immobilized enzyme were dispersed separately in 50 mM phosphate buffer solution, pH = 8.0. Following the method for determining enzyme activity described in Example 3, the enzyme-catalyzed reaction was carried out for 10 min in water baths at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, and 65℃. Enzyme activity was measured, with the highest enzyme activity taken as 100%. The relative enzyme activities at different temperatures were obtained, and the results are as follows: Figure 5As shown, the preferred reaction temperature for the free enzyme is 40℃, and the preferred reaction temperature for the immobilized enzyme is 10℃ higher than that for the free enzyme. The preferred reaction temperature for the immobilized enzyme is 50℃. The results indicate that the C-PDA-Au porous immobilized enzyme carrier has a certain protective effect on the enzyme.

[0108] (2) Preferred catalytic pH

[0109] The conformation of enzymes is easily altered by pH changes, which can lead to a loss of enzyme activity. Therefore, pH is another important factor affecting enzyme activity.

[0110] This example investigated the catalytic activities of free and immobilized enzymes under different pH systems. Appropriate amounts of free and immobilized enzymes were dispersed in 50 mM phosphate buffer solutions at different pH values ​​(6.0-9.0). Following the method described in Example 3 for determining enzyme activity, the enzyme activity was measured after 10 minutes of enzymatic reaction in a 37°C water bath. The highest enzyme activity was taken as 100%, and the relative enzyme activities under different pH systems were obtained. The results are as follows: Figure 6 As shown, both immobilized and free enzymes reach peak catalytic activity at pH 8.0. Their relative enzyme activity decreases with decreasing or increasing pH. However, the graph shows that the activity decrease of the immobilized enzyme is less than that of the free enzyme, which maintains over 70% activity over a wider pH range (6.0-9.0), exhibiting stronger acid-base tolerance, which is crucial in practical applications.

[0111] (3) Thermal stability of free enzymes and immobilized enzymes

[0112] The prepared immobilized and free enzymes were placed in a 50℃ water bath for different times, and enzyme activities were measured at regular intervals. The highest enzyme activity in each group was taken as 100%, and the activities of other enzymes were used as ratios to 100%. Thermostability curves of the enzymes were plotted. Figure 7 It can be seen that the activities of both free and immobilized enzymes showed a decreasing trend, but the relative activity of the free enzyme decreased at a faster rate and with a more pronounced trend than that of the immobilized enzyme. After incubation at 50℃ for 180 min, the activity of the free enzyme was only 50.2%, while the activity of the immobilized enzyme was 72.4%. After the same temperature and incubation time, the activity of the immobilized enzyme was significantly higher than that of the free enzyme, exhibiting stronger thermal stability.

[0113] (4) Investigation of enzyme kinetic constants

[0114] In this experiment, substrates with concentrations ranging from 0.24 to 0.96 mM were prepared, and the absorbance at 410 nm was measured after different time intervals of catalysis by the free enzyme, the immobilized enzyme (lipase-C-PDA) using C-PDA as a carrier, and the immobilized enzyme (lipase-C-PDA-Au) using C-PDA-Au as a carrier. The Kc values ​​of the free and immobilized enzymes were obtained based on the slope and intercept of the Eadie-Hofstee method. m and V m value.

[0115] As shown in Table 1, the K values ​​of the immobilized enzymes lipase-C-PDA and lipase-C-PDA-Au are... m The values ​​were 0.8907 mM and 0.6728 mM, respectively, which are lower than the K of the free enzyme. m The value of 0.9073 mM indicates that the two immobilized enzymes have a greater affinity for the substrate than the free enzyme, and the modification of the carrier with Au nanoparticles, which have good biocompatibility, further increases the affinity of the immobilized enzymes for the substrate. m The value is related to the enzyme concentration; the Vc of immobilized enzymes lipase-C-PDA and lipase-C-PDA-Au is... m The values ​​were 0.015 mM·min -1 and 0.0218 mM·min -1 V compared to free enzyme m The value increases. V m The value can be obtained by comparing the enzyme concentration to the K value. cat K cat Kcat represents the number of substrates converted into products by one enzyme molecule per unit time. It can be used to measure the catalytic efficiency of an enzyme. cat The larger the value, the higher the catalytic efficiency of the enzyme. The Kc of immobilized enzymes lipase-C-PDA and lipase-C-PDA-Au... cat The value is greater than the K of the free enzyme. cat The higher K value may be because, after the enzyme is immobilized on the surface of a hydrophobic support, the cap opens to a greater extent, allowing its active site to more easily contact the substrate compared to free enzymes, thus improving catalytic efficiency. Compared to lipase-C-PDA, lipase-C-PDA-Au has a higher K value. cat The value further increases, indicating that the presence of gold nanoparticles improves the catalytic efficiency of the enzyme.

[0116] Table 1 Kinetic parameters of free enzyme, lipase-C-PDA, and lipase-C-PDA-Au

[0117]

[0118] (5) Reproducibility of immobilized enzymes

[0119] The good reusability of immobilized enzymes is crucial for their application in practical industry. This example demonstrates the reusability of the immobilized enzyme, and the results are as follows: Figure 8 As shown, the experimental results indicate that the residual enzyme activity of the immobilized enzyme lipase-C-PDA-Au gradually decreases with the increase of the number of uses. After being used repeatedly for 5 times, the immobilized enzyme lipase-C-PDA-Au still retains more than 70% of the initial enzyme activity, indicating that the immobilized enzyme lipase-C-PDA-Au has good reusability.

[0120] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0121] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. An immobilized enzyme, characterized in that, include: The matrix is ​​formed from bamboo charcoal powder; Linkers, which are deposited on the surface of the matrix, are gold nanoparticles, and are deposited on the surface of the matrix via dopamine; as well as The enzyme, which binds to the linker, The method for preparing the immobilized enzyme includes: Bamboo charcoal powder is mixed with a first buffer solution to obtain a first mixed solution, wherein the first buffer solution is tris(hydroxymethyl)aminomethane hydrochloride buffer solution and the pH value is 7.5-9.0; The first mixed solution is mixed with a reducing agent to obtain a second mixed solution; The second mixed solution is then mixed with a metal salt in a third step to reduce the metal salt using the reducing agent, causing the metal to deposit on the surface of the bamboo charcoal powder particles, thus obtaining a carrier intermediate. The metal is gold nanoparticles, the metal salt is chloroauric acid, and the reducing agent is dopamine. The carrier intermediate is dispersed in a second buffer solution and then mixed with an enzyme solution in a fourth mixing process to allow the enzyme in the enzyme solution to bind to the linker, thereby obtaining the immobilized enzyme. The second buffer solution is a phosphate buffer with a concentration of 40-60 mM and a pH of 6.0-9.

0.

2. The immobilized enzyme according to claim 1, characterized in that, The immobilized enzyme has a catalytic pH of 6-9.

3. The immobilized enzyme according to claim 2, characterized in that, The catalytic pH of the immobilized enzyme is 8.

4. The immobilized enzyme according to claim 1, characterized in that, The ratio of the bamboo charcoal powder, the reducing agent, the metal salt, and the first buffer solution is 1g: 20-40mg: 0.3-0.5mmol: 20-40mL.

5. The immobilized enzyme according to claim 4, characterized in that, The ratio of the bamboo charcoal powder, the reducing agent, the metal salt, and the first buffer solution is 1g: 25-35mg: 0.35-0.45mmol: 25-35mL.

6. The immobilized enzyme according to claim 1, characterized in that, The mass ratio of the carrier intermediate to the enzyme in the enzyme solution is 1:0.15-0.

4.

7. The immobilized enzyme according to claim 1, characterized in that, The second mixing is performed by first magnetic stirring at room temperature for 20-30 minutes.

8. The immobilized enzyme according to claim 1, characterized in that, The third mixing involves continuing the magnetic stirring reaction at room temperature for 1-2 hours.

9. The immobilized enzyme according to claim 1, characterized in that, The fourth mixing is carried out under conditions of 30-65 °C with second magnetic stirring for 0.5-4 h.

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