Immobilized lipase and method for preparing the same

Abstract

This invention belongs to the field of lipase immobilization technology, specifically relating to an immobilized lipase and its preparation method. The preparation method includes two steps: (I) preparing activated amino-type silica resin: mixing amino-type silica resin, nonionic surfactant 1308, and deionized water at a mass ratio of 1000-1100:2000-2200:5-8, stirring at 1-5℃ for 10-15 min, adding cyanuric chloride, and adding 30-35wt% sodium hydroxide solution. After the reaction, centrifuge; (II) preparing the immobilized lipase: mixing the activated resin, surfactant, deionized water, and lipase solution, adding an alkaline solution at 40-45℃ and pH=5.0-5.5, centrifuging, washing, and drying after the reaction. The obtained immobilized lipase has a strong covalent bond, and after repeated use 6 times, the enzyme activity is ≥50%. It exhibits excellent acid and alkali tolerance and storage stability, making it suitable for industrial catalysis in multiple industries.

Description

Technical Field

[0001] This invention belongs to the field of lipase immobilization technology, specifically relating to an immobilized lipase and its preparation method. Background Technology

[0002] Lipases [EC3.1.3.1], as a class of highly efficient and specific hydrolases, occupy a core position in many fields such as detergents, leather making, aquatic feed, biochemicals, oil processing, pharmaceutical synthesis, environmental protection, and the food industry. They can catalyze various organic reactions such as triglyceride hydrolysis, transesterification, and stereoisomer resolution, making them one of the key enzyme preparations in the field of industrial catalysis. However, the widespread application of lipases has long been constrained by two major issues: first, the preparation cost of free lipases as catalysts is high, and they are difficult to recover after a single use, resulting in high industrial production costs; second, free lipases have poor structural stability and are easily deactivated by environmental factors such as temperature, pH, and organic solvents during practical applications, and their enzyme activity is rapidly lost during storage, severely limiting their industrial application.

[0003] To address these issues, enzyme immobilization technology has emerged. Enzyme immobilization involves binding free enzymes to the surface or interior of a specific carrier material using physical or chemical means. This preserves the enzyme's catalytic activity while enabling its recovery and reuse, thereby reducing application costs and improving operational stability. Currently, immobilized enzyme preparation methods are mainly classified into four categories: adsorption, covalent bonding, encapsulation, and cross-linking. Adsorption is simple to operate and low in cost, but the binding force between the enzyme and the carrier is weak due to physical adsorption, making it prone to detachment during stirring, washing, and other operations, resulting in poor stability for repeated use. Encapsulation can better preserve the enzyme's natural conformation, but the network structure formed by the carrier can easily generate mass transfer resistance, affecting catalytic efficiency, and the encapsulation material is easily damaged under extreme environments. Cross-linking uses cross-linking agents to directly cross-link enzyme molecules to form polymers, resulting in strong stability, but it can easily lead to the shielding of enzyme active sites and a decrease in catalytic activity. Covalent bonding uses chemical bonds to firmly bind enzyme molecules to the carrier, offering advantages such as good operational stability and low enzyme loss rate, and has become the most widely used immobilization method in industry.

[0004] In existing technologies, commonly used bifunctional cross-linking agents for covalent bonding methods include glutaraldehyde and polyethylene glycol diglycidyl ether. For example, the paper "Immobilized Lipase by Polyethylene Glycol Diglycidyl Ether Cross-linking Amino Support LX-1000EA" discloses the use of polyethylene glycol diglycidyl ether and glutaraldehyde as bifunctional cross-linking agents. The amino-type silicone resin is first activated, and then covalently cross-linked with the amino groups in the lipase molecule to prepare immobilized lipase. However, this technical solution has significant drawbacks: on the one hand, glutaraldehyde, as a cross-linking agent, has high reactivity, easily leading to excessive cross-linking of enzyme molecules, disrupting the spatial conformation of the lipase, and reducing catalytic activity; on the other hand, the chemical bonds formed by polyethylene glycol diglycidyl ether cross-linking are not stable enough and easily break during repeated use, leading to enzyme molecule detachment. Experimental data shows that the immobilized lipase prepared using the above-mentioned existing technology experiences a decrease in enzyme activity of more than 60% after six repeated uses, which is insufficient to meet the requirements for long-term stable use of enzyme preparations in industrial production. Furthermore, existing immobilized lipases exhibit poor acid-base tolerance and are easily inactivated in reaction systems that deviate from neutrality, further limiting their application scenarios. Therefore, developing a simple preparation process for immobilized lipases with high reusability and strong acid-base tolerance has become an urgent technical problem to be solved in this field.

[0005] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a method for preparing immobilized lipase, the method comprising the following steps:

[0007] (I) Preparation of activated amino-type silicone resin

[0008] (I-1) Mix amino-type silicone resin, surfactant and deionized water, and stir until homogeneous to obtain a mixture;

[0009] (I-2) Add a bifunctional crosslinking agent to the mixture and add an alkaline solution dropwise to react. After the reaction is completed, centrifuge to obtain the activated amino-type silicone resin.

[0010] (II) Preparation of immobilized lipase

[0011] The activated amino-type silica resin, surfactant, deionized water and lipase solution were mixed and stirred evenly. Then, an alkaline solution was added dropwise to carry out the reaction. The mixture was then centrifuged, washed and dried in sequence to obtain the immobilized lipase.

[0012] Preferably, in step (I-1):

[0013] The primary amine functional group capacity of the amino-type silica resin is 0.75-0.80 mmol / g. It should be noted that the amino-type silica resin is an enzyme immobilization carrier, including but not limited to: Haipu New Materials HP-HA200, HP-EA200, Lanxiao Technology Seplite LX1000HA, Seplite LX1000EA, etc.

[0014] And / or, the surfactants mainly serve a wetting function, including but not limited to: Lutensol TO8 (1308), Lutensol XL80, fatty alcohol polyoxyethylene ether AEO8, etc.

[0015] Preferably, in step (I-1), the mass ratio of the amino-type silicone resin, surfactant, and deionized water is 1000-1100:2000-2200:5-8.

[0016] Preferably, in step (I-1), the stirring temperature is 1-5℃ and the stirring time is 10-15min.

[0017] Preferably, in step (I-2):

[0018] The bifunctional crosslinking agent is cyanuryl chloride;

[0019] And / or, the alkaline solution is a sodium hydroxide solution with a concentration of 30-35 wt%.

[0020] Preferably, in step (I-2), the temperature is controlled at ≤15℃ during the addition of alkaline solution, the pH of the system is 5.0-6.0, the total addition time is 3-5h, the temperature is controlled at 13-15℃ after the addition is completed, and the reaction continues for 2-3h.

[0021] Preferably, in step (II), the activity of the lipase solution is 1×10⁻⁶. 5 -2×10 5 U / mL. This includes, but is not limited to: Novozymes Greasex Ultra G and Greasex 200MG in liquid form.

[0022] Preferably, in step (II), the temperature is controlled at 40-45℃, the pH of the system is 5.0-5.5, and the total dropping time is 3-4h during the process of adding alkaline solution.

[0023] Based on the same technical concept, another aspect of the present invention is to provide an immobilized lipase obtained by the above preparation method.

[0024] To facilitate understanding of the present invention, the reaction process of the present invention is illustrated below:

[0025] The reaction process in step (I) is as follows:Figure 1 As shown, the reaction process in step (II) is as follows: Figure 2 As shown.

[0026] The beneficial effects of this invention are as follows:

[0027] The immobilized lipase and its preparation method provided by this invention address the technical pain points of existing immobilized lipases, such as poor stability during repeated use, insufficient acid and alkali tolerance, and rapid loss of enzyme activity during storage, by optimizing the carrier activation process, screening efficient bifunctional cross-linking agents, and precisely controlling reaction conditions. This significantly improves the overall performance of the product, and the preparation process is mild and controllable, suitable for large-scale industrial production, and has important practical value and promotion prospects.

[0028] 1. Significantly improved covalent bonding stability: Cyanuryl chloride is selected as a bifunctional crosslinking agent. The active groups in its molecular structure can form stable covalent bonds with the primary amine groups on the surface of amino-type silicone resin and the amino groups in lipase molecules. Compared with glutaraldehyde and polyethylene glycol diglycidyl ether in the prior art, the crosslinking reaction is more specific and the binding is stronger, which effectively avoids the loss of enzyme molecules during repeated use and lays the foundation for improving the stability of repeated use.

[0029] 2. Outstanding advantages in reusability: By optimizing the activation conditions (such as temperature, pH value, and material ratio) and immobilization reaction parameters of amino-type silicone resin, the immobilized lipase prepared can be reused 6 times and the relative enzyme activity can still be maintained above 50%. The relative enzyme activity of the best embodiment can reach 65%, which far exceeds the level of the prior art (27%-36%), and significantly reduces the enzyme preparation consumption cost in industrial applications.

[0030] 3. Optimized storage stability: By precisely controlling the temperature and pH range of each step in the preparation process (such as pH=5.0-6.0 for activation reaction and pH=5.0-5.5 for immobilization reaction), the damage of the reaction environment to the active sites of lipase can be reduced, thus solving the problem of poor storage stability of existing products.

[0031] 4. The preparation process is mild and controllable and suitable for industrialization: The preparation steps of this invention are clear, and the parameters such as the ratio of each material, reaction temperature, and time are well defined. No special and expensive equipment is required. Operations such as stirring, centrifugation, washing, and drying are all conventional industrial processes. Moreover, the reaction conditions are mild (such as activation reaction temperature ≤15℃ and immobilization reaction temperature 40-45℃), avoiding the impact of extreme conditions such as high temperature and high pressure on enzyme activity, and making it easy to achieve large-scale production. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is a reaction process diagram for step (I).

[0034] Figure 2 This is a reaction process diagram for step (II). Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] Example 1

[0037] This embodiment provides a method for preparing immobilized lipase, the method comprising the following steps (detailed):

[0038] (1) Equipment selection: 5L stainless steel reactor (equipped with constant temperature water bath device, pH online monitoring instrument and speed regulating stirrer), high-speed centrifuge (maximum speed 10000r / min), vacuum drying oven are selected. Selecting equipment of appropriate specifications can ensure that the materials are fully mixed and the reaction conditions are accurately controlled to meet the needs of large-scale production.

[0039] (2) Preparation of the mixture: 1000g of dried amino-type silicone resin, 2000g of deionized water, and 5g of nonionic surfactant 1308 were added sequentially to a 5L reactor. The stirrer speed was set to 300r / min, and the constant temperature water bath was turned on to control the temperature of the reaction system at 5℃. The mixture was stirred continuously for 10min to obtain a uniformly dispersed mixture. The stirring speed of 300r / min can ensure sufficient contact of materials and avoid uneven local concentration. Controlling the temperature within the range of 1-5℃ can prevent the subsequent material cyanuric chloride from decomposing due to excessive temperature, while maintaining the dispersion stability of the amino-type silicone resin and providing a uniform reaction environment for the subsequent crosslinking reaction.

[0040] (3) Add crosslinking agent and premix: Slowly add 69g of cyanuric chloride to the above mixture, maintain a stirring speed of 300r / min and a system temperature of 5℃, and continue stirring for 10min to ensure that the cyanuric chloride is completely dissolved and fully contacts the surface of the amino-type silicone resin. As a bifunctional crosslinking agent, cyanuric chloride needs to be premixed evenly with the amino-type silicone resin to ensure that its active groups can fully contact the primary amine groups on the resin surface, laying the foundation for the full progress of the subsequent crosslinking reaction; low-temperature stirring can avoid the hydrolysis and inactivation of cyanuric chloride.

[0041] (4) Adding alkaline solution and controlling reaction conditions: Turn on the sodium hydroxide solution adding device (constant pressure dropping funnel) and slowly add 90g of 30wt% sodium hydroxide solution to the reaction system. During the addition process, strictly control the system temperature to not exceed 15℃ through a constant temperature water bath. At the same time, adjust the dropping rate in real time through a pH online monitoring instrument to maintain the pH value of the system at 5.0. The total adding time is controlled to 3h. After the addition is completed, keep the system temperature at 15℃ and continue stirring for 2h. The 30wt% sodium hydroxide solution is used to adjust the acidity and alkalinity of the system to provide a suitable environment for the cross-linking reaction of cyanuric chloride and amino groups. Controlling the temperature to ≤15℃ can avoid rapid hydrolysis of cyanuric chloride. Maintaining the pH value in the range of 5.0-6.0 can maximize the efficiency of the cross-linking reaction. The 3h adding time and the subsequent 2h heat preservation reaction can ensure that the cross-linking reaction is fully carried out and form an activated amino-type silicone resin with abundant active sites.

[0042] (5) Centrifugation to obtain the activated carrier: After the reaction is complete, turn off the stirrer and the constant temperature water bath, transfer the reaction system to a high-speed centrifuge, set the centrifugation speed to 8000 r / min and the centrifugation time to 10 min. After centrifugation, discard the supernatant and collect the bottom precipitate, which is the activated amino-type silica gel resin. High-speed centrifugation at 8000 r / min can quickly achieve solid-liquid separation, efficiently remove unreacted free cyanuric chloride, excess surfactants and other impurities, obtain a pure activated carrier, and avoid impurities affecting the subsequent binding reaction with lipase.

[0043] (6) Mixing and immobilizing reaction materials: Take another 5L reactor, add the activated amino-type silica gel resin obtained in step 5, 5g of nonionic surfactant 1308, 2000g of deionized water at 25℃, and 320mL of active material with an activity of 1×10 5 The lipase solution was prepared at U / mL. The stirrer speed was set to 300 rpm, and the mixture was stirred for 10 minutes to ensure thorough mixing. Deionized water at 25°C can maintain the initial activity of the lipase and prevent changes in the spatial conformation of the enzyme due to excessively high or low temperatures. Nonionic surfactant 1308 can reduce the interfacial tension of the system, promote sufficient contact between the activated amino-type silica resin and the lipase molecules, and improve the binding efficiency.

[0044] (7) Immobilization reaction by adding alkaline solution: 60g of 30wt% sodium hydroxide solution was added dropwise to the above mixture through a constant pressure dropping funnel. The temperature of the system was controlled at 40℃ by turning on the constant temperature water bath. The dropping rate was adjusted in real time by the pH online monitoring instrument to maintain the pH value of the system at 5.0. The total dropping time was controlled at 3h. 40℃ is the optimal temperature for covalent bonding between lipase and the activation carrier, which can ensure the reaction rate and avoid enzyme inactivation due to high temperature. The pH value is kept stable in the range of 5.0-5.5 to ensure that the amino groups in the lipase molecule and the active groups on the surface of the activation carrier undergo efficient covalent cross-linking reaction to form stable chemical bonds.

[0045] (8) Centrifugation and washing to remove free enzyme: After the reaction is complete, transfer the system to a high-speed centrifuge and centrifuge at 8000 r / min for 10 min, collect the precipitate; add 500 mL of deionized water to the precipitate, stir and wash for 5 min, then centrifuge again at 8000 r / min for 10 min, repeat this washing-centrifugation operation 3 times. Multiple centrifugation and washing can completely remove free lipase that is not bound to the carrier, avoid the free enzyme from interfering with the catalytic effect in subsequent applications, and at the same time remove excess sodium hydroxide and surfactant in the system, ensuring the purity of the immobilized lipase.

[0046] (9) Low-temperature vacuum drying: The washed precipitate is transferred to a vacuum drying oven, the drying temperature is set to 30℃ and the vacuum degree to -0.09MPa, and the drying time is 12h. After drying, it is taken out, sealed and stored in a desiccator to obtain the target immobilized lipase. Low-temperature vacuum drying can quickly remove moisture from the material without inactivating the lipase, preventing moisture from causing enzyme activity loss; sealed storage can prevent the product from absorbing moisture, extend the storage period, and at the same time ensure the structural stability of the immobilized lipase.

[0047] Example 2

[0048] This embodiment provides a method for preparing immobilized lipase, the method comprising the following steps (briefly described):

[0049] (1) Add 1000g of amino-type silicone resin (primary amine functional group capacity is 0.75mmol / g), 2000g of water and 5g of nonionic surfactant 1308 to the reaction vessel, control the temperature at 5℃, stir for 10min to obtain a mixture.

[0050] (2) Add 103.5g of cyanuric chloride, continue to control the temperature at 5℃, stir for 10min, then add 90g of 30% sodium hydroxide solution, control the temperature at 15℃, pH value at 5, the total time of addition is 3h, after the addition is completed, control the temperature at 15℃, continue the reaction for 2h, and then perform centrifugation to obtain activated amino-type silica resin.

[0051] (3) The activated amino-type silica resin, 5g of nonionic surfactant 1308, 2000g of water (water temperature 25℃) and 320mL of lipase solution (100000U / mL) were added to the reaction vessel. After stirring for 10min, 60g of 30% sodium hydroxide solution was added. The temperature was controlled at 40℃ and the pH value was 5.0. The total drop time was 3h. Then, centrifugation was performed. The washing and centrifugation were repeated multiple times to remove the unfixed free lipase. The immobilized lipase was dried at a low temperature of 30℃ to obtain the immobilized lipase.

[0052] Example 3

[0053] This embodiment provides a method for preparing immobilized lipase, the method comprising the following steps (briefly described):

[0054] (1) Add 1000g of amino-type silicone resin (primary amine functional group capacity is 0.75mmol / g), 2000g of water and 5g of nonionic surfactant 1308 to the reaction vessel, control the temperature at 5℃, stir for 10min to obtain a mixture.

[0055] (2) Add 138g of cyanuric chloride, continue to control the temperature at 5℃, stir for 10min, then add 90g of 30% sodium hydroxide solution, control the temperature at 15℃, pH value at 5, the total time of addition is 3h, after the addition is completed, control the temperature at 15℃, continue the reaction for 2h, and then perform centrifugation to obtain activated amino-type silica resin.

[0056] (3) The activated amino-type silica resin, 5g of nonionic surfactant 1308, 2000g of water (water temperature 25℃) and 320mL of lipase solution (100000U / mL) were added to the reaction vessel. After stirring for 10min, 60g of 30% sodium hydroxide solution was added. The temperature was controlled at 40℃ and the pH value was 5.0. The total drop time was 3h. Then, centrifugation was performed. The washing and centrifugation were repeated multiple times to remove the unfixed free lipase. The immobilized lipase was dried at a low temperature of 30℃ to obtain the immobilized lipase.

[0057] Verification Example

[0058] The immobilized lipases of Examples 1-3, Comparative Examples 1 and 2 were tested, as follows:

[0059] (a) Comparative Example 1 is glutaraldehyde-immobilized lipase, and Comparative Example 2 is polyethylene glycol diglycidyl ether-immobilized lipase.

[0060] (II) Enzyme Activity Assay Method: A modified copper soap spectrophotometric method was used to determine enzyme activity. Lipase activity was defined as the amount of enzyme required to catalyze the hydrolysis of a substrate to produce 1 μmol of fatty acid within 1 minute under the assay conditions (40℃ / pH 7.0). Free enzymes were measured by the number of enzyme activity units per milliliter (U / mL), and immobilized enzymes were measured by the number of enzyme activity units per gram of immobilized enzyme (U / g).

[0061] (III) The relative enzyme activity test results of the examples and comparative examples after being used 6 times are shown in Table 1.

[0062] Table 1

[0063]

[0064] As shown in Table 1:

[0065] 1. The relative enzyme activities of Comparative Example 1 (glutaraldehyde immobilization) and Comparative Example 2 (polyethylene glycol diglycidyl ether immobilization) in the prior art are only 27% and 36%, respectively, indicating that the immobilized lipase prepared by traditional cross-linking agents has poor stability for repeated use, and the enzyme molecules are easy to detach or become inactive. In contrast, the relative enzyme activities of Examples 1-3 of the present invention reach 51%, 65%, and 60%, respectively, which are significantly higher than those of the comparative examples. This proves that the technical solution of using cyanuric chloride as a bifunctional cross-linking agent can effectively improve the binding stability of enzyme and carrier, and solve the core pain point of the prior art.

[0066] 2. The amount of cyanuric chloride used in Example 2 (103.5g) was between that in Example 1 (69g) and Example 3 (138g), and its relative enzyme activity (65%) was the highest, indicating that there is an optimal range for the amount of crosslinking agent: (i) When the amount used in Example 1 was insufficient, there were fewer active sites on the activated carrier, and the binding sites between the enzyme and the carrier were limited, resulting in some enzyme molecules easily detaching and thus lower relative enzyme activity; (ii) When the amount used in Example 2 was moderate, the number of active sites formed on the surface of the carrier matched the lipase molecules the most, and the degree of crosslinking was appropriate, which ensured both strong binding and did not block the active sites of the lipase, thus resulting in the best catalytic stability; (iii) When the amount used in Example 3 was excessive, the excessive crosslinking caused changes in the spatial conformation of the lipase molecules, and some active sites were blocked, making it impossible to effectively bind with the substrate, thus the relative enzyme activity was slightly lower than that in Example 2.

[0067] 3. The immobilized lipase of the present invention can still maintain more than 50% of its enzyme activity after being reused 6 times. Compared with the prior art, it can reduce the frequency of enzyme preparation replacement and reduce the raw material cost in industrial production. Among them, Example 2 has the best performance, providing the optimal process parameters for industrial production, and has significant economic value and application prospects.

[0068] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing immobilized lipase, characterized in that, The preparation method includes the following steps: (I) Preparation of activated amino-type silicone resin (I-1) Mix amino-type silicone resin, surfactant and deionized water, and stir until homogeneous to obtain a mixture; (I-2) Add a bifunctional crosslinking agent to the mixture and add an alkaline solution dropwise to react. After the reaction is complete, centrifuge to obtain the activated amino-type silicone resin; wherein: The bifunctional crosslinking agent is cyanuryl chloride; And, the alkaline solution is a sodium hydroxide solution with a concentration of 30-35 wt%; During the addition of alkaline solution, the temperature should be controlled at ≤15℃, the system pH should be 5.0-6.0, the total addition time should be 3-5h, and after the addition is completed, the temperature should be controlled at 13-15℃, and the reaction time should continue for 2-3h. (II) Preparation of immobilized lipase The activated amino-type silica resin, surfactant, deionized water, and lipase solution were mixed and stirred until homogeneous. An alkaline solution was then added dropwise to initiate the reaction. The mixture was then centrifuged, washed, and dried sequentially to obtain the immobilized lipase. Wherein: During the addition of alkaline solution, the temperature was controlled at 40-45℃, the pH of the system was 5.0-5.5, and the total addition time was 3-4 hours.

2. The method for preparing immobilized lipase according to claim 1, characterized in that, In step (I-1): The primary amine functional group capacity of the amino-type silicone resin is 0.75-0.80 mmol / g; And / or, the surfactant is nonionic surfactant 1308.

3. The method for preparing immobilized lipase according to claim 1, characterized in that, In step (I-1), the mass ratio of the amino-type silicone resin, surfactant and deionized water is 1000-1100:2000-2200:5-8.

4. The method for preparing immobilized lipase according to claim 1, characterized in that, In step (I-1), the stirring temperature is 1-5℃ and the stirring time is 10-15min.

5. The method for preparing immobilized lipase according to claim 1, characterized in that, In step (II), the activity of the lipase solution is 1×10⁻⁶. 5 -2×10 5 U / mL.

6. The immobilized lipase obtained by the preparation method according to any one of claims 1-5.

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