Preparation method and application of sodium alginate in-situ interface modified immobilized aspergillus niger lipase

Abstract

This invention discloses a method for preparing and applying sodium alginate-modified immobilized Aspergillus niger lipase, belonging to the field of enzyme immobilization technology. The invention employs a stepwise pH control strategy for covalent immobilization: first, 1% sodium alginate and free enzyme are dissolved in a phosphate buffer solution at pH 7.0, and after adding pretreated epoxy resin, the reaction is carried out at 25°C and 150 rpm for 9 hours; then, a phosphate buffer solution at pH 9.5 is added to adjust the system to slightly alkaline (pH 8.5-9.0), and the reaction continues for 3 hours. After standing for 12-18 hours, washing until no protein is detected, and vacuum drying for 12 hours, the immobilized lipase is obtained. This invention utilizes the epoxy groups on the epoxy resin surface to simultaneously undergo ring-opening covalent reactions with the amino groups on the lipase surface and the hydroxyl / carboxyl groups of sodium alginate, achieving multi-point anchoring of the enzyme molecule and in-situ grafting of sodium alginate, forming a hydrated protective layer around the enzyme molecule, effectively improving immobilization efficiency, preventing enzyme leakage, and enhancing operational stability.

Description

Technical Field

[0001] This invention relates to the field of enzyme immobilization technology, and in particular to a method for preparing and applying sodium alginate-modified immobilized Aspergillus niger lipase. Background Technology

[0002] Lipase (EC3.1.1.3), as an important industrial enzyme, is widely used in oil hydrolysis, transesterification, biodiesel synthesis, chiral compound resolution, and food processing. However, free lipases suffer from inherent defects in practical industrial applications, such as poor stability, easy inactivation, difficulty in separation and recovery, and inability to be reused, which severely restricts their industrial application. Therefore, lipase immobilization technology has become an effective way to improve these defects, achieve easy separation and recovery of enzyme catalysts, and enhance stability, attracting widespread attention from academia and industry. Currently, lipase immobilization methods mainly include adsorption, covalent bonding, and encapsulation. Among them, adsorption is simple to operate and has mild conditions, but the binding force between the enzyme and the carrier is weak, and enzyme molecules are easy to detach from the carrier surface; encapsulation can encapsulate enzyme molecules in a carrier grid, but often suffers from problems such as high substrate diffusion resistance, incomplete encapsulation of enzyme molecules, and insufficient mechanical strength. In contrast, covalent bonding, through the formation of stable covalent bonds between the amino and carboxyl functional groups on the enzyme molecule surface and the active groups on the carrier surface, creates a strong interaction between the enzyme and the carrier. This effectively prevents enzyme molecule detachment and significantly improves the stability and reusability of immobilized enzymes, making it a highly favored method.

[0003] Among covalent bonding methods, resin covalent bonding has become a popular method for lipase immobilization due to its advantages such as simple operation, high retention of enzyme catalytic activity, and mild reaction conditions. Epoxy resins are one of the most commonly used carriers in resin covalent bonding methods. The abundant epoxy groups on their surface can undergo ring-opening reactions with the amino groups on the enzyme molecule surface to form stable covalent bonds. However, existing epoxy resin immobilized lipase technology still faces the following key challenges: First, the contradiction between immobilization efficiency and enzyme activity. Epoxy resins react slowly with lipases under mild conditions, while harsh conditions can easily damage the lipases. Furthermore, high ionic strength environments and the hydrophobic properties of the carrier may impact the protein structure of some lipases, altering their native conformation and leading to partial or complete inactivation. Second, pH conditions have a dual impact on both immobilization efficiency and enzyme activity. Alkaline pH (8.0-9.0) is beneficial for improving immobilization efficiency, but near-neutral pH (6.0-7.0) is more conducive to maintaining the native conformation and catalytic activity of lipases. Third, how to precisely control reaction conditions to effectively open the active sites (i.e., the "cap" structure) of lipases, allowing the active center to be fully exposed to the reaction interface, is also a key challenge for improving the catalytic efficiency of immobilized enzymes.

[0004] Sodium alginate, a natural high-molecular-weight polysaccharide, is commonly used as an encapsulating agent in enzyme immobilization. By cross-linking with calcium ions to form a semi-permeable hydrogel network structure, it allows small-molecule substrates and products to pass freely while restricting enzyme molecule leakage, thus maintaining enzyme bioactivity and enabling reuse. As a natural polysaccharide, sodium alginate has advantages such as being green and non-toxic, having good biocompatibility, and being widely available, making it a safe immobilization carrier material. Furthermore, the surface of sodium alginate molecules contains a large number of hydroxyl (-OH) and carboxyl (-COOH) groups, which can form a porous hydrogel network structure on the surface of epoxy-immobilized lipases. This allows for the free diffusion of substrates and products while reducing enzyme molecule leakage through steric hindrance and electrostatic repulsion. Furthermore, sodium alginate possesses negative electrical properties, while the amino groups on the surface of lipases are partially positively charged at appropriate pH levels. The two can form ionic bonds through electrostatic interactions. This non-covalent bonding helps increase the initial adsorption of enzymes on the carrier surface. Sodium alginate can act like a "molecular glue," enriching a large number of enzyme molecules near the resin surface, increasing the local enzyme concentration, thereby accelerating and enhancing the subsequent covalent reaction between the enzyme and epoxy groups, theoretically improving immobilization efficiency. However, when sodium alginate is used alone as an immobilized enzyme carrier, it suffers from significant drawbacks such as poor mechanical strength, incomplete enzyme molecule encapsulation, and insufficient chemical stability, making it difficult to meet the requirements of continuous industrial operation. Moreover, the aforementioned synergistic mechanism still faces multiple bottlenecks in practical applications, including high ionic strength impacting enzyme conformation, insufficient mechanical strength of sodium alginate, and difficulty in achieving pH balance, thus a mature and reliable industrial technology solution has not yet been developed.

[0005] Meanwhile, the proportion of lipase effectively loaded on the carrier in existing immobilization methods is low. In order to ensure immobilization efficiency, excessive enzyme solution usually needs to be added, resulting in a large amount of free enzyme that cannot be effectively immobilized and is wasted. The utilization rate of lipase is low, which further increases the production cost.

[0006] In conclusion, developing a novel method for immobilizing lipases using a domestically produced composite carrier that offers high stability, low cost, high immobilization efficiency, high enzyme activity retention, and high lipase utilization is of great theoretical and practical significance for breaking through foreign technological monopolies, reducing production costs, and promoting the industrial application of lipase catalysis technology. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a method for preparing and applying sodium alginate-modified immobilized Aspergillus niger lipase. The method involves placing the enzyme, sodium alginate, and epoxy resin in the same system to simultaneously achieve multi-point covalent anchoring of the enzyme and grafting of sodium alginate onto the carrier surface, thereby improving the stability of the immobilized enzyme.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing Aspergillus niger lipase immobilized by in-situ interface modification with sodium alginate, comprising the following steps: (1) Dissolve sodium alginate in first phosphate buffer to prepare sodium alginate solution; (2) Dissolve the free lipase in the sodium alginate solution to obtain an enzyme-sodium alginate mixture; (3) Add pretreated epoxy resin to the enzyme-sodium alginate mixture and carry out the first reaction under the first pH and shaking conditions; (4) Add second phosphate buffer to the reaction system to adjust the pH of the system to the second pH, and continue to shake the reaction for 3 hours; (5) Stop shaking, let stand for 12-18 hours, wash the reaction product until no protein is detected in the supernatant, and then dry to obtain immobilized lipase.

[0009] Furthermore, the free lipase is Aspergillus niger lipase powder.

[0010] Furthermore, the pretreatment method for the pretreated epoxy resin is as follows: wash the epoxy resin with a first phosphate buffer solution, vortex, repeat 4-5 times, and then filter.

[0011] Furthermore, the first phosphate buffer is a 0.2 M phosphate buffer with a pH of 7.0.

[0012] Furthermore, the amount of sodium alginate added is 1% of the volume of the first phosphate buffer solution.

[0013] Furthermore, the conditions for the first reaction are: temperature 25°C, rotation speed 150 rpm, and reaction time 9 hours.

[0014] Furthermore, the second phosphate buffer is a 1 M phosphate buffer with a pH of 9.5, and the second pH is 8.5-9.0.

[0015] Furthermore, the drying in step (5) is vacuum drying, and the drying time is 12 hours.

[0016] This invention also provides a method for preparing immobilized Aspergillus niger lipase by in-situ interface modification with sodium alginate.

[0017] This invention also provides the application of immobilized Aspergillus niger lipase in the preparation of medium- and long-chain triglycerides.

[0018] Furthermore, the medium- and long-chain triglycerides are prepared by enzymatic transesterification of immobilized Aspergillus niger lipase with high-oleic peanut oil and coconut oil.

[0019] Compared with the prior art, the present invention has at least the following advantages and technical effects: This invention utilizes the epoxy groups (COC) on the surface of epoxy resin to undergo ring-opening nucleophilic addition reactions with the amino groups on the surface of Aspergillus niger lipase and the hydroxyl or carboxyl groups on the sodium alginate molecular chain, respectively. This achieves a strong covalent bond between the enzyme molecule and the carrier, as well as in-situ covalent immobilization of sodium alginate on the resin surface. Through this dual covalent mechanism, a hydrophilic porous hydration layer is formed around the enzyme molecule. This hydration layer effectively ensures free mass transfer between the substrate and product, reduces diffusion resistance, and significantly reduces enzyme leakage through steric hindrance and electrostatic repulsion. This greatly improves the mechanical, chemical, and operational stability of the immobilized enzyme, effectively overcoming the shortcomings of the single sodium alginate embedding method, such as poor mechanical strength and easy enzyme leakage, and the single resin covalent binding method, such as easy enzyme detachment.

[0020] This invention employs a precise pH control strategy to significantly enhance the nucleophilic ability of amino groups on the surface of the free enzyme while protecting the native conformational stability of Aspergillus niger lipase molecules. This enables multi-point anchoring and efficient covalent binding of the enzyme molecule on the carrier surface. This technical solution effectively resolves the technical contradiction in traditional immobilization processes where immobilization efficiency and enzyme activity are difficult to balance. It avoids the impact and damage to the enzyme protein conformation caused by high ionic strength or violent reaction conditions. While ensuring high immobilization efficiency, it maximizes the preservation of lipase catalytic activity and facilitates the precise opening of the "cap" structure of the lipase active site, significantly improving the catalytic efficiency of the immobilized enzyme.

[0021] This invention further enhances the immobilization effect and improves process controllability by synergistically optimizing the amount of sodium alginate added and the timing of pH adjustment. An appropriate concentration of sodium alginate can form a uniform and dense hydrogel network structure on the resin surface. If the concentration is too low, the interface modification effect is not obvious and enzyme leakage cannot be effectively inhibited; if the concentration is too high, the system viscosity will be too high, which will hinder the sufficient contact and mass transfer between enzyme molecules and resin active sites, reducing immobilization efficiency. Simultaneously, by strictly controlling the timing of pH adjustment, the adverse effects of premature pH adjustment on enzyme conformational stability are avoided, while the weakening effect of late adjustment on the promoting effect of covalent binding reactions is prevented. This achieves a synergistic effect between the enzyme immobilization process and the interface modification process, ensuring the stability and repeatability of the process. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0023] Figure 1 This is a schematic diagram illustrating the principle of in-situ interface modification and immobilization of Aspergillus niger lipase using sodium alginate according to the present invention. Figure 2 The yield of MLCT prepared by immobilized Aspergillus niger lipase in Example 1 and Comparative Examples 4-9; Figure 3 The yield of MLCT prepared by immobilized Aspergillus niger lipase in Example 1 and Comparative Examples 1-3; Figure 4 The yield of MLCT prepared by immobilized Aspergillus niger lipase in Example 1 and Comparative Examples 10-12 is shown. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] This invention provides a method for preparing Aspergillus niger lipase immobilized by in-situ interface modification with sodium alginate, comprising the following steps: (1) Dissolve sodium alginate in first phosphate buffer to prepare sodium alginate solution; (2) Dissolve the free lipase in the sodium alginate solution to obtain an enzyme-sodium alginate mixture; (3) Add pretreated epoxy resin to the enzyme-sodium alginate mixture and carry out the first reaction under the first pH and shaking conditions; (4) Add second phosphate buffer to the reaction system to adjust the pH of the system to the second pH, and continue to shake the reaction for 3 hours; (5) Stop shaking, let stand for 12-18 hours, wash the reaction product until no protein is detected in the supernatant, and then dry to obtain immobilized lipase.

[0030] In some embodiments of the present invention, the free lipase is Aspergillus niger lipase powder.

[0031] In some embodiments of the present invention, the pretreatment method for the pretreated epoxy resin is as follows: washing the epoxy resin with a first phosphate buffer solution, vortexing, repeating 4 to 5 times, and then filtering.

[0032] In some embodiments of the present invention, the first phosphate buffer is a 0.2 M, pH 7.0 phosphate buffer.

[0033] In some embodiments of the present invention, the amount of sodium alginate added is 1% (w / v) of the volume of the first phosphate buffer solution.

[0034] In some embodiments of the present invention, the conditions for the first reaction are: temperature 25°C, rotation speed 150 rpm, and reaction time 9 hours.

[0035] In some embodiments of the present invention, the second phosphate buffer is a 1 M phosphate buffer with pH 9.5, and the second pH is 8.5-9.0.

[0036] In some embodiments of the present invention, the drying in step (5) is vacuum drying, and the drying time is 12 hours.

[0037] This invention also provides a method for preparing immobilized Aspergillus niger lipase by in-situ interface modification with sodium alginate.

[0038] This invention also provides an application of immobilized Aspergillus niger lipase in the preparation of medium- and long-chain triglycerides.

[0039] In some embodiments of the present invention, the medium- and long-chain triglycerides are prepared by immobilized Aspergillus niger lipase by enzymatic transesterification of high-oleic peanut oil and coconut oil.

[0040] Example 1 A method for preparing Aspergillus niger lipase immobilized by in-situ interface modification with sodium alginate includes the following steps: Raw materials: epoxy resin, Aspergillus niger lipase, sodium alginate, phosphate buffer (0.2M, pH 7), phosphate buffer (1M, pH 9.5).

[0041] Preparation method: First, add 1% sodium alginate (0.1g by buffer volume) to 10 mL of phosphate buffer (0.2M, pH 7) and stir until completely dissolved. Then, dissolve 5 g of Aspergillus niger lipase (powder) in the above solution (the concentration of Aspergillus niger lipase in the solution is 500 mg / mL). Add 1 g of pretreated epoxy resin (pretreatment method: wash the resin with first phosphate buffer, vortex, repeat 4-5 times and then filter). Shake at 25℃ and 150 rpm for 9 h. Then add a small amount of second phosphate buffer to adjust the immobilization environment to slightly alkaline (pH 8.5). Continue to fix on the shaker for 3 hours, then remove and let stand for 18 h. After washing, until no protein is detected in the supernatant, place in a vacuum drying oven and dry for 12 h to obtain immobilized lipase.

[0042] The immobilized lipase prepared above was added to high-oleic peanut oil and coconut oil for enzymatic transesterification to prepare medium- and long-chain triglycerides (MLCT).

[0043] High-oleic peanut oil and coconut oil were weighed in a 1:1 molar ratio, and 2 g of the total amount was placed in a 25 mL round-bottom flask. Immobilized lipase (10%, w / w) was added, and the reaction was carried out in a constant temperature silicone oil magnetic stirrer at 350 r / min. The reaction was sealed, and after 4 h of reaction, the enzyme was separated by centrifugation to obtain the crude MLCT product after transesterification.

[0044] Example 1 yielded a product with an MLCT content of 51.16%, achieving the same catalytic effect as the imported lipase Novozymes RMC. The catalytic activity of this immobilized lipase remained unaffected after one month of storage. Furthermore, compared to the conventional covalent cross-linking immobilization method, the immobilized lipase obtained by covalent grafting with sodium alginate and pH-responsive adjustment showed less loss of catalytic activity after multiple cycles, indicating improved lipase stability.

[0045] Comparative Example 1 The amount of sodium alginate added was limited to 0.5% (w / v) of the volume of the phosphate buffer solution, and the remaining steps were the same as in Example 1.

[0046] Comparative Example 2 The amount of sodium alginate added was limited to 1.5% (w / v) of the volume of the phosphate buffer solution, and the remaining steps were the same as in Example 1.

[0047] Comparative Example 3 The amount of sodium alginate added was limited to 2% (w / v) of the volume of the phosphate buffer solution, and the remaining steps were the same as in Example 1.

[0048] Comparative Example 4 The concentration of Aspergillus niger lipase was limited to 100 mg / mL, and the remaining steps were the same as in Example 1.

[0049] Comparative Example 5 The concentration of Aspergillus niger lipase was limited to 200 mg / mL, and the remaining steps were the same as in Example 1.

[0050] Comparative Example 6 The concentration of Aspergillus niger lipase was limited to 300 mg / mL, and the remaining steps were the same as in Example 1.

[0051] Comparative Example 7 The concentration of Aspergillus niger lipase was limited to 400 mg / mL, and the remaining steps were the same as in Example 1.

[0052] Comparative Example 8 The concentration of Aspergillus niger lipase was limited to 600 mg / mL, and the remaining steps were the same as in Example 1.

[0053] Comparative Example 9 The concentration of Aspergillus niger lipase was limited to 900 mg / mL, and the remaining steps were the same as in Example 1.

[0054] Comparative Example 10 The second phosphate buffer solution was added 3 hours after the epoxy resin was added and the shaker was shaken for another 12 hours. The remaining steps were the same as in Example 1.

[0055] Comparative Example 11 The second phosphate buffer solution was added 6 hours after the epoxy resin was added and the shaker was shaken for another 12 hours. The remaining steps were the same as in Example 1.

[0056] Comparative Example 12 The second phosphate buffer solution was added 12 hours after the epoxy resin was added to the shaker, and the remaining steps were the same as in Example 1.

[0057] Figure 2To obtain the MLCT content for Examples 1 and Comparative Examples 4-9, the immobilization time refers to the time when the pretreated resin is added to the enzyme-sodium alginate mixture to begin immobilization. It can be seen that after the immobilization time reaches 12 hours, the MLCT yield no longer increases, indicating that when the immobilization synthesis time is 12 hours, the immobilized lipase obtained can be used to synthesize MLCT with the highest yield, which is the optimal immobilization time parameter for the experiment. Furthermore, as the concentration of Aspergillus niger lipase solution increases, the MLCT yield first increases and then decreases, indicating that when the concentration of Aspergillus niger lipase is 500 mg / mL, the MLCT yield is the highest, which is the optimal lipase concentration parameter for MLCT synthesis.

[0058] Figure 3 The MLCT content was obtained in Example 1 and Comparative Examples 1-3, and the MLCT content in the transesterification product was used as the evaluation index. It can be seen that when the enzyme concentration is 500 mg / mL and the sodium alginate addition is 1% under the immobilization conditions, the immobilized lipase has the highest yield for MLCT synthesis.

[0059] Figure 4 The MLCT content was obtained for Examples 1 and Comparative Examples 10-12. It can be seen that adding the second phosphate buffer at 9 hours after adding the epoxy resin and shaking the enzyme, followed by a full 12 hours of shaking fixation, resulted in the immobilized enzyme exhibiting the best catalytic activity in the synthesis of MLCT. Adjusting the time too early is detrimental to enzyme conformational stability, while adjusting the time too late does not promote covalent bonding. Therefore, adding the second phosphate buffer at 9 hours after adding the epoxy resin and shaking the enzyme is the optimal adjustment time.

[0060] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing Aspergillus niger lipase immobilized by in-situ interface modification with sodium alginate, characterized in that, Includes the following steps: (1) Dissolve sodium alginate in first phosphate buffer to prepare sodium alginate solution; (2) Dissolve the free lipase in the sodium alginate solution to obtain an enzyme-sodium alginate mixture; (3) Add pretreated epoxy resin to the enzyme-sodium alginate mixture and carry out the first reaction under the first pH and shaking conditions; (4) Add second phosphate buffer to the reaction system to adjust the pH of the system to the second pH, and continue to shake the reaction for 3 hours; (5) Stop shaking, let stand for 12-18 hours, wash the reaction product until no protein is detected in the supernatant, and then dry to obtain immobilized lipase.

2. The method for preparing Aspergillus niger lipase by in-situ interface modification with sodium alginate according to claim 1, characterized in that, The free lipase is Aspergillus niger lipase powder; the pretreatment method for the pretreated epoxy resin is as follows: wash the epoxy resin with first phosphate buffer, vortex, repeat 4-5 times, and then filter.

3. The method for preparing Aspergillus niger lipase by in-situ interface modification with sodium alginate according to claim 1, characterized in that, The first phosphate buffer is a 0.2 M phosphate buffer with a pH of 7.

0.

4. The method for preparing Aspergillus niger lipase by in-situ interface modification with sodium alginate according to claim 1, characterized in that, The amount of sodium alginate added is 1% of the volume of the first phosphate buffer solution.

5. The method for preparing Aspergillus niger lipase by in-situ interface modification with sodium alginate according to claim 1, characterized in that, The conditions for the first reaction were: temperature 25℃, rotation speed 150 rpm, and reaction time 9 hours.

6. The method for preparing Aspergillus niger lipase by in-situ interface modification with sodium alginate according to claim 1, characterized in that, The second phosphate buffer is a 1 M phosphate buffer with a pH of 9.5, and the second pH is 8.5-9.

0.

7. The method for preparing Aspergillus niger lipase by in-situ interface modification with sodium alginate according to claim 1, characterized in that, In step (5), the drying is vacuum drying, and the drying time is 12 hours.

8. An immobilized Aspergillus niger lipase prepared by the method of sodium alginate in situ interface modification according to any one of claims 1-7.

9. The use of the immobilized Aspergillus niger lipase of claim 8 in the preparation of medium- and long-chain triglycerides.

10. The application according to claim 9, characterized in that, The medium- and long-chain triglycerides are prepared by enzymatic transesterification of the immobilized Aspergillus niger lipase described in claim 8 with high-oleic peanut oil and coconut oil.

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