Graphene oxide immobilized lipase as well as preparation method and application thereof in preparation of diglyceride oil

Abstract

The invention discloses graphene oxide immobilized lipase as well as a preparation method and application thereof in preparation of diglyceride oil, and the preparation method comprises the following steps: dissolving graphene oxide powder in a phosphate buffer solution, and carrying out ultrasonic treatment and centrifugation to obtain pretreated graphene oxide; the preparation method comprises the following steps: adding pretreated graphene oxide and lipase into a phosphate buffer solution for primary incubation, then adding a cross-linking agent for secondary incubation, and carrying out centrifugal separation, precipitate washing, secondary centrifugal separation and precipitate drying to obtain the graphene oxide immobilized lipase. According to the preparation method, the stability of the lipase and the yield of diglyceride oil are improved, the cost is reduced, the prepared graphene oxide immobilized lipase can be applied to preparation of the diglyceride oil, the content of diglyceride in the obtained product is larger than or equal to 50 wt%, the content of monoglyceride is smaller than or equal to 1.5 wt%, the content of free fatty acid is smaller than or equal to 0.5 wt%, and the yield of the diglyceride oil is increased. The enzyme activity is greater than or equal to 90% after the graphene oxide immobilized lipase is repeatedly used for more than 100 times.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme engineering and oil and fat chemical engineering, and particularly relates to an oxidized graphene immobilized lipase, a preparation method thereof and application thereof in preparing diglyceride oil. BACKGROUND

[0002] Diglyceride is a trace component of natural vegetable oil and fat, is a recognized safe food ingredient, has similar physicochemical properties to triglyceride, but has a metabolic pathway different from triglyceride. In recent years, it has been found that diglyceride has effects of reducing lipogenesis, reducing visceral fat content, inhibiting weight gain, reducing blood lipids, promoting digestion and enhancing immunity, and thus has attracted widespread attention. Early production methods of diglyceride mostly adopt chemical synthesis, which usually needs to use a large amount of organic solvent, has a complicated and harsh reaction procedure, has high energy consumption, poor reaction specificity, many by-products and easy environmental pollution. The later developed biological enzymatic hydrolysis method for preparing diglyceride under normal temperature and pressure has advantages of mild reaction conditions, environmental friendliness, strong specificity, few by-products, low energy consumption and the like, and becomes an important direction for industrial development of diglyceride.

[0003] In the biological enzymatic hydrolysis method for preparing diglyceride, the selection of immobilized lipase is a key factor determining the production efficiency of diglyceride. The immobilized lipase can improve the stability of the enzyme and realize recycling, thereby reducing the production cost of diglyceride. However, in the process of catalyzing glycerolysis reaction by the existing immobilized lipase to prepare diglyceride, the cost of the immobilized lipase is high, the catalytic efficiency decreases rapidly when repeatedly used, and the contents of monoglyceride and fatty acid generated are high, thereby greatly affecting the quality of the product and reducing the yield. Therefore, it is urgent to further develop an immobilized lipase with high catalytic efficiency, good stability and good effect of repeated use, so as to obtain high content of diglyceride and meet the requirements of the contents of monoglyceride and fatty acid, thereby reducing the pressure of separation and purification and improving the yield. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the shortcomings of the prior art, and to provide an oxidized graphene immobilized lipase, a preparation method thereof and application thereof in preparing diglyceride oil. The content of diglyceride prepared by the catalysis of the oxidized graphene immobilized lipase in glycerolysis reaction is ≥50wt%, the content of monoglyceride is ≤1.5wt%, the content of free fatty acid is ≤0.5%, and the enzyme activity of the oxidized graphene immobilized lipase can still be maintained at more than 90% after catalyzing glycerolysis reaction for more than 100 times.

[0005] To solve the above technical problems, the following technical solutions are adopted in the present application.

[0006] A preparation method of an oxidized graphene immobilized lipase, comprising the following steps:

[0007] (1) dissolving graphene oxide powder in a phosphate buffer solution, performing ultrasonic treatment to exfoliate and uniformly disperse the graphene oxide, and then centrifuging to remove unexfoliated graphene oxide, to obtain pretreated graphene oxide, the Zeta potential of the pretreated graphene oxide being -18 mV to -30 mV, and the average flake diameter being 1 μm to 3.5 μm;

[0008] (2) adding the pretreated graphene oxide and lipase to a phosphate buffer solution, performing primary incubation at 25°C to 40°C to allow the lipase to adsorb to the pretreated graphene oxide, then adding a crosslinking agent, performing secondary incubation at 25°C to 35°C to allow the lipase to be covalently immobilized on the graphene oxide, and after incubation, performing centrifugal separation, washing the obtained precipitate with a phosphate buffer solution, again performing centrifugal separation, and drying the obtained precipitate, to obtain graphene oxide immobilized lipase.

[0009] The preparation method of graphene oxide immobilized lipase described above, preferably, in step (1), the pH value of the phosphate buffer solution is 6.8 to 7.5, the power of the ultrasonic treatment is 200 W to 300 W, the time of the ultrasonic treatment is 3 h to 5 h, the rotation speed of the centrifugation is 2500 rpm to 3000 rpm, and the time of the centrifugation is 10 min to 25 min.

[0010] The preparation method of graphene oxide immobilized lipase described above, preferably, in step (2), the mass ratio of the pretreated graphene oxide to the lipase is 5 to 15:1.

[0011] The preparation method of graphene oxide immobilized lipase described above, preferably, in step (2), the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and the mass of the crosslinking agent is 10% to 15% of the mass of the pretreated graphene oxide.

[0012] The preparation method of graphene oxide immobilized lipase described above, preferably, in step (2), the time of the primary incubation is 0.5 h to 1 h, the time of the secondary incubation is 12 h to 24 h, the pH value of the phosphate buffer solution is 6.8 to 7.2, the rotation speed of the centrifugal separation is 2000 rpm to 2800 rpm, the time of the centrifugal separation is 10 min to 15 min, the temperature of the drying is 40°C to 50°C, and the time of the drying is 12 h to 24 h.

[0013] As a general technical concept, the present application also provides graphene oxide immobilized lipase prepared by the preparation method of graphene oxide immobilized lipase described above.

[0014] As a general technical concept, the present application also provides an application of the above-mentioned graphene oxide immobilized lipase in preparing diacylglycerol oil.

[0015] The above-mentioned application, preferably, comprises catalyzing edible oil to prepare diacylglycerol oil by glycerolysis reaction using the graphene oxide immobilized lipase.

[0016] The above-mentioned application, preferably, comprises the following steps: mixing glycerol, edible oil and the graphene oxide immobilized lipase, performing glycerolysis reaction at 50-75℃, and obtaining diacylglycerol oil after solid-liquid separation and purification.

[0017] The above-mentioned application, preferably, the molar ratio of glycerol to edible oil is 1-4:1, and the mass of the graphene oxide immobilized lipase is 0.5%-15% of the total mass of edible oil and glycerol.

[0018] The edible oil comprises one or more of camellia oil, rapeseed oil, palm oil, flaxseed oil, corn oil and peanut oil.

[0019] The glycerolysis reaction time is 6-12h.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] (1) The preparation method of the present application uses graphene oxide as a lipase immobilization carrier, which has good biological affinity, better acid-base stability and thermal stability, and a large specific surface area. The Zeta potential of the pretreated graphene oxide is-18mV--30mV, and the average flake diameter is 1-3.5μm. The pretreated graphene oxide is further covalently cross-linked and immobilized with lipase through physical adsorption, which enhances the immobilization and reduces the damage to the three-dimensional structure of lipase, thereby significantly improving the stability of the immobilized lipase and ensuring the catalytic activity of the lipase.

[0022] (2) In the application of the present application, the graphene oxide immobilized lipase is used to catalyze edible oil glycerolysis reaction to prepare diacylglycerol oil, the diacylglycerol content is ≥50wt%, the monoglyceride content is ≤1.5wt%, and the free fatty acid content is ≤0.5%. The graphene oxide immobilized lipase of the present application can be reused for more than 100 times, and the enzyme activity is ≥90%. The graphene oxide immobilized lipase used for catalyzing glycerolysis reaction to prepare diacylglycerol oil can simplify the separation process, significantly improve the stability of the lipase, increase the yield of the product diacylglycerol oil, and reduce the preparation cost of the diacylglycerol oil. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The transmission electron microscope image of the pretreated graphene oxide in Example 1 of the present application. DETAILED DESCRIPTION

[0024] The application is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the scope of the protection of the application is not limited thereby. The Candida rugosa lipase B used in the following examples can be commercially available or prepared by synthetic biology means, with an enzyme activity of about 80000 U / g, the graphene oxide powder is purchased from Jiangsu Xianfeng Nanometer Material Technology Co., Ltd. (Nanjing, Jiangsu), the phosphate buffer is purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and other materials and instruments are commercially available.

[0025] Example 1

[0026] A method for preparing the graphene oxide immobilized lipase of the application, comprising the following steps:

[0027] (1) 10 g of graphene oxide powder (with an average flake diameter of about 5 μm) is dissolved in 200 mL of phosphate buffer with a pH of 7.0, and is treated with an ultrasonic cell disruptor for 3 h at an ultrasonic power of 200 W, so as to exfoliate and uniformly disperse the graphene oxide, and then is centrifuged at a speed of 2500 rpm for 15 min to remove the unexfoliated graphene oxide, so as to obtain pretreated graphene oxide with a Zeta potential of -27 mV and an average flake diameter of 2.5 μm, as shown in FIG. 1. Figure 1

[0028] (2) 5 g of the pretreated graphene oxide and 1 g of Candida rugosa lipase B are added to 100 mL of phosphate buffer with a pH of 7.0, and are incubated in a constant-temperature shaker at 30 °C for 1 h, so as to mix uniformly and allow the lipase to be adsorbed on the pretreated graphene oxide, and then 0.5 g of a crosslinking agent, i.e., 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), is added, and the incubation is continued in the constant-temperature shaker at 30 °C for 12 h, so as to connect the carboxyl groups on the graphene oxide to the amino groups on the lipase through the crosslinking agent, thereby allowing the lipase to be covalently immobilized on the graphene oxide, and after the incubation, the mixture is centrifuged at 2400 rpm for 10 min to separate, the precipitate is washed with phosphate buffer with a pH of 7.0, and then is centrifuged at 2400 rpm for 10 min to separate, and the obtained precipitate is dried at 40 °C for 24 h, so as to obtain graphene oxide immobilized lipase.

[0029] Example 2

[0030] ​A preparation method of the graphene oxide immobilized lipase of the present application is basically the same as that of Example 1, with the difference that in step (1), 10 g of graphene oxide powder (average flake diameter of about 5 μm) is dissolved in 200 mL of a phosphate buffer solution with a pH of 7.0, and an ultrasonic cell disrupter is used for ultrasonic treatment for 5 h at a power of 200 W, so as to exfoliate and uniformly disperse the graphene oxide, and then centrifugation is performed at a speed of 3000 rpm for 15 min to remove the unexfoliated graphene oxide, so as to obtain a pretreated graphene oxide carrier with a Zeta potential of -24 mV and an average flake diameter of about 1.5 μm.

[0031] Example 3

[0032] A preparation method of the graphene oxide immobilized lipase of the present application is basically the same as that of Example 1, with the difference that in step (2), 5 g of the pretreated graphene oxide and 0.35 g of Candida rugosa lipase B are added into 100 mL of a phosphate buffer solution with a pH of 7.0, and incubation is performed in a constant-temperature shaker at 40 °C for 1 h, so as to uniformly mix and adsorb the lipase on the pretreated graphene oxide, 0.5 g of a crosslinking agent, i.e., 1-ethyl-(3-dimethylaminopropyl) carbodiimide, is added, and incubation is continued in a constant-temperature shaker at 30 °C for 12 h, so as to connect the carboxyl groups on the graphene oxide with the amino groups on the lipase through the crosslinking agent, thereby covalently immobilizing the lipase on the graphene oxide, and after the incubation, centrifugation is performed at 2000 rpm for 10 min to separate, the precipitate is collected, and after washing the precipitate with a phosphate buffer solution with a pH of 7.0, centrifugation is again performed at 2000 rpm for 10 min to separate, and the obtained precipitate is dried at 40 °C for 24 h, so as to obtain the graphene oxide immobilized lipase.

[0033] Example 4

[0034] An application of the graphene oxide immobilized lipase of the present application in the preparation of a diglyceride oil, which uses the graphene oxide immobilized lipase prepared in Example 1, and specifically refers to the graphene oxide immobilized lipase catalyzing the glycerolysis reaction of edible oil (camellia oil) to prepare a diglyceride oil, which comprises the following steps:

[0035] Glycerol, camellia oil and the graphene oxide immobilized lipase prepared in Example 1 are mixed, the molar ratio of glycerol to camellia oil is 2:1, and the mass of the graphene oxide immobilized lipase added is 4.5 wt% of the total mass of the camellia oil and glycerol, and reaction is performed at 60 °C for 8 h, and after solid-liquid separation and purification, a diglyceride oil is obtained. The product is analyzed by high performance liquid chromatography, and the content of diglyceride is 52.9 wt%, the content of triglyceride is 43.4 wt%, the content of monoglyceride is 1.4 wt%, and the content of free fatty acid is 0.5 wt%.

[0036] Example 5

[0037] The application of the graphene oxide immobilized lipase of the present application in preparing diacylglycerol oil is basically the same as that in Example 4, except that the molar ratio of glycerol to camellia oil is 3:1, and the mass of the graphene oxide immobilized lipase added is 10wt% of the total mass of the camellia oil and glycerol. The product is analyzed by high performance liquid chromatography, and the content of diacylglycerol is 57.4wt%, the content of triacylglycerol is 39.2wt%, the content of monoacylglycerol is 1.3wt%, and the content of free fatty acid is 0.4wt%.

[0038] Example 6

[0039] The application of the graphene oxide immobilized lipase of the present application in preparing diacylglycerol oil is basically the same as that in Example 4, except that the molar ratio of glycerol to camellia oil is 4:1, and the mass of the graphene oxide immobilized lipase added is 15wt% of the total mass of the camellia oil and glycerol. The product is analyzed by high performance liquid chromatography, and the content of diacylglycerol is 65.8wt%, the content of triacylglycerol is 31.4wt%, the content of monoacylglycerol is 0.8wt%, and the content of free fatty acid is 0.2wt%.

[0040] The graphene oxide immobilized lipase after the reaction for preparing diacylglycerol oil in this example is continuously used for the glycerolysis reaction of camellia oil, the molar ratio of glycerol to camellia oil is 4:1, the mass of the graphene oxide immobilized lipase added is 15wt% of the total mass of the camellia oil and glycerol, and the reaction is carried out at 60℃ for 8h. After solid-liquid separation and purification, diacylglycerol oil is obtained. The product is analyzed by high performance liquid chromatography, and the graphene oxide immobilized lipase is repeatedly used. After 101 times of repetition, the graphene oxide immobilized lipase prepared in Example 1 catalyzes the hydrolysis reaction of camellia oil for 102 times. The analysis of the product of the 102th time shows that the content of diacylglycerol is 60.6wt%, the content of triacylglycerol is 35.9wt%, the content of monoacylglycerol is 1.2wt%, and the content of free fatty acid is 0.4wt%. This example shows that the graphene oxide immobilized lipase of the present application has an excellent stability, and the enzyme activity is ≥90% after 100 times of repetition.

[0041] Example 7

[0042] The application of the graphene oxide immobilized lipase of the present application in preparing diacylglycerol oil is basically the same as that in Example 4, except that the edible oil is rapeseed oil, and the reaction is carried out at 70℃ for 10h. The product is analyzed by high performance liquid chromatography, and the content of diacylglycerol is 62.2wt%, the content of triacylglycerol is 34.2wt%, the content of monoacylglycerol is 2.1wt%, and the content of free fatty acid is 0.2wt%.

[0043] Example 8

[0044] An application of the graphene oxide immobilized lipase of the present application in preparing diacylglycerol oil, which is basically the same as that in Example 4, with the exception that the graphene oxide immobilized lipase prepared in Example 2 is used. The product is analyzed by high performance liquid chromatography, and the content of diacylglycerol is 60.8wt%, the content of triglyceride is 36.4wt%, the content of monoglyceride is 0.9wt%, and the content of free fatty acid is 0.1wt%.

[0045] Comparative Example 1

[0046] A preparation method of graphene oxide immobilized lipase, which is basically the same as that in Example 1, with the exception that no 1-ethyl-(3-dimethylaminopropyl) carbodiimide is added in step (2) for covalent cross-linking reaction, and Candida rugosa lipase is simply adsorbed on the pretreated graphene oxide by physical interaction, and after subsequent incubation, centrifugal separation and washing, most of the Candida rugosa lipase cannot be firmly immobilized on the graphene oxide.

[0047] Comparative Example 2

[0048] An application of graphene oxide immobilized lipase in preparing diacylglycerol oil, which is basically the same as that in Example 4, with the exception that the graphene oxide immobilized lipase prepared in Comparative Example 1 is used instead of that in Example 4, and specifically comprising the following steps:

[0049] Glycerol, camellia oil and the graphene oxide immobilized lipase prepared in Comparative Example 1 are mixed, the molar ratio of glycerol to camellia oil is 2:1, and the mass of the graphene oxide immobilized lipase added is 4.5wt% of the total mass of camellia oil and glycerol, and the reaction is carried out at 60℃ for 8h, and then diacylglycerol oil is obtained through solid-liquid separation and purification. Since most of the Candida rugosa lipase in Comparative Example 2 cannot be firmly immobilized on the graphene oxide, the enzyme amount is insufficient, and the glycerolysis reaction yield is low. The product is analyzed by high performance liquid chromatography, and the content of diacylglycerol is 22.7wt%, the content of triglyceride is 69.9wt%, the content of monoglyceride is 3.5wt%, and the content of free fatty acid is 2.1wt%.

[0050] Comparative Example 3

[0051] An application of graphene oxide immobilized lipase in preparing diacylglycerol oil, which is basically the same as that in Example 4, with the exception that the glycerolysis reaction time is shortened to 2h at 60℃, and then diacylglycerol oil is obtained through solid-liquid separation and purification. The product is analyzed by high performance liquid chromatography, and the content of diacylglycerol is 38.6wt%, the content of triglyceride is 56.2wt%, the content of monoglyceride is 2.2wt%, and the content of free fatty acid is 1.3wt%.

[0052] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art, without departing from the spirit and technical solutions of the present application, can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solutions of the present application, still falls within the scope of protection of the technical solutions of the present application.

Claims

1. A method for preparing graphene oxide immobilized lipase, characterized by, The method comprises the following steps: (1) dissolving graphene oxide powder in a phosphate buffer solution, performing ultrasonic treatment to exfoliate and uniformly disperse graphene oxide, and then centrifuging to remove unexfoliated graphene oxide, to obtain pretreated graphene oxide, wherein the zeta potential of the pretreated graphene oxide is -18 mV to -30 mV, and the average flake size is 1 μm to 3.5 μm; (2) adding the pretreated graphene oxide and lipase into a phosphate buffer solution, performing first incubation at 25°C to 40°C to allow the lipase to be adsorbed on the pretreated graphene oxide, then adding a crosslinking agent, performing second incubation at 25°C to 35°C to allow the lipase to be covalently immobilized on the graphene oxide, and then performing centrifugal separation, washing the obtained precipitate with a phosphate buffer solution, performing centrifugal separation again, and drying the obtained precipitate to obtain graphene oxide immobilized lipase.

2. The method of preparing graphene oxide-immobilized lipase according to claim 1, wherein, In step (1), the pH value of the phosphate buffer solution is 6.8 to 7.5, the power of the ultrasonic treatment is 200 W to 300 W, the time of the ultrasonic treatment is 3 h to 5 h, the rotation speed of the centrifugal separation is 2500 rpm to 3000 rpm, and the time of the centrifugal separation is 10 min to 25 min.

3. The method of preparing graphene oxide-immobilized lipase according to claim 1, wherein, In step (2), the mass ratio of the pretreated graphene oxide to the lipase is 5 to 15:

1.

4. The method for preparing graphene oxide-immobilized lipase according to any one of claims 1 to 3, characterized by, In step (2), the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and the mass of the crosslinking agent is 10% to 15% of the mass of the pretreated graphene oxide.

5. The method for preparing graphene oxide-immobilized lipase according to any one of claims 1 to 3, characterized by, In step (2), the time of the first incubation is 0.5 h to 1 h, the time of the second incubation is 12 h to 24 h, the pH value of the phosphate buffer solution is 6.8 to 7.2, the rotation speed of the centrifugal separation is 2000 rpm to 2800 rpm, the time of the centrifugal separation is 10 min to 15 min, the drying temperature is 40°C to 50°C, and the drying time is 12 h to 24 h.

6. Graphene oxide immobilized lipase prepared by the method of any one of claims 1 to 5.

7. Use of the graphene oxide immobilized lipase of claim 6 in the preparation of diglyceride oil.

8. Use according to claim 7, characterized in that, The use comprises using the graphene oxide immobilized lipase to catalyze edible oil to prepare diglyceride oil through glycerolysis reaction.

9. Use according to claim 8, characterized in that, The method comprises the following steps: mixing glycerol, edible oil and the graphene oxide immobilized lipase, and performing glycerolysis reaction at 50°C to 75°C, and then performing solid-liquid separation and purification to obtain diglyceride oil.

10. Use according to claim 9, characterized in that, The molar ratio of the glycerol to the edible oil is 1 to 4:1, and the mass of the graphene oxide immobilized lipase is 0.5% to 15% of the total mass of the edible oil and the glycerol. The edible oil comprises one or more of camellia oil, rapeseed oil, palm oil, flaxseed oil, corn oil and peanut oil. The time of the glycerolysis reaction is 6 h to 12 h.

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