Novel lipase for efficiently preparing sterol ester and preparation method thereof

Abstract

The invention provides novel lipase for efficiently preparing sterol ester and a preparation method thereof, and relates to the technical field of biochemical engineering. The novel lipase comprises the following raw materials in parts by weight: 20-30 parts of a porous carrier material, 10-15 parts of an enzyme immobilization reagent, 5-10 parts of an active modifier, 30-40 parts of a buffer solution, 8-12 parts of free lipase, 3-8 parts of a synergist and 4-8 parts of a stabilizer. A compound of graphene and zeolite is used as a porous carrier material, so that the loading capacity and the stability of the lipase are remarkably improved. The nano gold particles modified by polyethylene glycol are used as an active modifier, so that the activity and the stability of lipase are effectively improved. And by adding the synergist and the stabilizer, the catalytic efficiency and the stability of the lipase are further improved.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, in particular to a novel lipase for efficiently preparing sterol esters and a preparation method thereof. Background Art

[0002] Sterol esters have wide application value in the fields of food, medicine, etc. The traditional method for preparing sterol esters has problems such as low efficiency, high cost, and environmental pollution. Therefore, it is of great practical significance to develop an efficient and environmentally friendly method for preparing sterol esters.

[0003] As a biocatalyst, lipase has the advantages of high efficiency, specificity and environmental protection, and has broad application prospects in the preparation of sterol esters. However, current lipases still have some shortcomings in terms of stability and activity, which limits their application in the preparation of sterol esters. Summary of the invention

[0004] The object of the present invention is to provide a novel lipase for efficiently preparing sterol esters. The lipase has the advantages of high activity, high stability, easy recovery, etc., and can effectively improve the preparation efficiency and quality of sterol esters.

[0005] Another object of the present invention is to provide a method for preparing the novel lipase, which is simple, low-cost and suitable for large-scale production.

[0006] Technical Solution

[0007] A novel lipase for efficiently preparing sterol esters comprises the following raw materials in parts by weight:

[0008] The porous carrier material contains 20 to 30 parts of a composite material of graphene and zeolite, which combines the high specific surface area of ​​graphene and the good stability of zeolite, and provides more attachment sites and a more stable environment for lipase.

[0009] 10-15 parts of enzyme immobilization reagent, glutaraldehyde is used to immobilize lipase on the porous carrier material.

[0010] The active modifier is 5 to 10 parts, which is nano-gold particles modified with polyethylene glycol, and improves the activity and stability of lipase.

[0011] 30 to 40 parts of buffer solution are phosphate buffer solution.

[0012] 8-12 parts of free lipase.

[0013] The synergist is 3 to 8 parts of tris(hydroxymethyl)aminomethane, which improves the catalytic efficiency of lipase.

[0014] The stabilizer is 4 to 8 parts of trehalose, which enhances the stability of the lipase.

[0015] A method for preparing a novel lipase for efficiently preparing sterol esters:

[0016] S1. Carrier pretreatment: Place the porous carrier material in a container containing a dilute acid solution (prepared by dilute hydrochloric acid and water), treat it under ultrasonic conditions for 20 to 30 minutes, then wash it with deionized water until it is neutral, and dry it for later use. This step removes impurities on the surface of the porous carrier material and activates its surface.

[0017] S2. Enzyme immobilization: Add the pretreated porous carrier material to a buffer solution containing an enzyme immobilization reagent, and stir the reaction at 30-40°C for 1-2 hours to allow the enzyme immobilization reagent to react with the active groups on the surface of the porous carrier material. Subsequently, add free lipase, and continue to stir the reaction at the same temperature for 2-3 hours to fix the lipase on the porous carrier material. After the reaction is completed, filter and wash with a buffer solution to obtain immobilized lipase.

[0018] S3. Activity modification: Add the immobilized lipase to a solution containing an activity modifier, and stir the reaction at 25-35°C for 1-2 hours to allow the activity modifier to combine with the immobilized lipase to improve the activity and stability of the lipase. After the reaction is completed, filter and wash with a buffer solution to obtain the immobilized lipase after activity modification.

[0019] S4. Enhancement and stabilization: Add the active modified immobilized lipase to a buffer solution containing a synergist and a stabilizer, and stir the reaction at 35-45°C for 30-60 minutes to allow the synergist and stabilizer to fully combine with the lipase, thereby improving the catalytic efficiency and stability of the lipase. After the reaction is completed, filter and wash with a buffer solution to obtain a new lipase for the efficient preparation of sterol esters.

[0020] S5. Filling and storage: The prepared novel lipase is placed in a sealed container and stored under low temperature conditions.

[0021] Beneficial Effects

[0022] 1. The present invention significantly improves the loading capacity and stability of lipase by using a composite of graphene and zeolite as a porous carrier material.

[0023] 2. The present invention uses polyethylene glycol-modified gold nanoparticles as an active modifier to effectively improve the activity and stability of lipase.

[0024] 3. The present invention further improves the catalytic efficiency and stability of lipase by adding synergists and stabilizers.

[0025] 4. The preparation method of the present invention is simple, easy to operate, low-cost and suitable for large-scale production. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Embodiment 1:

[0028] The embodiment of the present invention provides a novel lipase for efficiently preparing sterol esters, comprising the following raw materials in parts by weight: 20 parts of porous carrier material, 10 parts of enzyme immobilization agent, 5 parts of activity modifier, 30 parts of buffer solution, 8 parts of free lipase, 3 parts of enhancer, and 4 parts of stabilizer.

[0029] The porous carrier material is a composite of graphene and zeolite; the enzyme immobilization reagent is glutaraldehyde; the activity modifier is polyethylene glycol-modified nano-gold particles; the buffer solution is phosphate buffer solution; the enhancer is tris(hydroxymethyl)aminomethane; and the stabilizer is trehalose.

[0030] A method for preparing a novel lipase for efficiently preparing sterol esters comprises the following steps:

[0031] S1. Carrier Pretreatment

[0032] The porous carrier material is placed in a container containing a dilute acid solution, treated under ultrasonic conditions for 20 minutes, then washed with deionized water until neutral, and dried for later use; the dilute acid solution is prepared by mixing dilute hydrochloric acid with water;

[0033] S2. Enzyme Immobilization

[0034] The pretreated porous carrier material is added to a buffer solution containing an enzyme immobilization reagent, and stirred at 30°C for 1 hour to allow the enzyme immobilization reagent to react with the active groups on the surface of the porous carrier material. Free lipase is then added, and the reaction is continued at the same temperature for 2 hours to allow the lipase to be fixed on the porous carrier material. After the reaction is completed, the material is filtered and washed with a buffer solution to obtain an immobilized lipase.

[0035] S3. Active Modification

[0036] The immobilized lipase is added to a solution containing an activity modifier, and the mixture is stirred and reacted at 25°C for 1 hour to allow the activity modifier to bind to the immobilized lipase and improve the activity and stability of the lipase. After the reaction is completed, the mixture is filtered and washed with a buffer solution to obtain an activity-modified immobilized lipase.

[0037] S4. Efficiency and stability

[0038] The immobilized lipase after activity modification is added to a buffer solution containing a synergist and a stabilizer, and the reaction is stirred at 35°C for 30 minutes to allow the synergist and the stabilizer to fully combine with the lipase, thereby improving the catalytic efficiency and stability of the lipase. After the reaction is completed, the lipase is filtered and washed with a buffer solution to obtain a new lipase for efficiently preparing sterol esters.

[0039] S5. Filling and storage

[0040] The prepared novel lipase is packed into a sealed container and stored under low temperature conditions.

[0041] The present embodiment adopts the raw material formula of specific ratio, makes the novel lipase have higher catalytic activity in the process of preparing sterol ester.The porous carrier material is a composite of graphene and zeolite, combines the high specific surface area of ​​graphene and the good stability of zeolite, provides more attachment sites for lipase, thereby improves the loading capacity of enzyme, and then improves catalytic efficiency.Through multi-step processing, including enzyme immobilization, activity modification, synergism and stability, the activity of lipase is fully exerted.For example, the activity modifier is the nano gold particles modified by polyethylene glycol, which can significantly improve the activity and stability of lipase, and further accelerate the preparation speed of sterol ester.

[0042] The selection of porous carrier materials and the use of enzyme immobilization agents enable lipase to be firmly fixed on the carrier and not easily inactivated or detached during the reaction. Glutaraldehyde, as an enzyme immobilization agent, reacts with the active groups on the surface of the porous carrier material to ensure the stability of the lipase. The addition of stabilizer trehalose provides a stable environment for the lipase to prevent it from losing its activity due to environmental factors during storage and use. At the same time, in each step of the preparation process, impurities that may affect the stability of the lipase are removed through operations such as filtration and washing.

[0043] The immobilized lipase can be separated from the reaction system by a simple filtration operation, which is convenient for recycling and reuse. This not only reduces production costs, but also reduces environmental pollution. Since the lipase is fixed on a porous carrier, it can be easily separated and stored after the reaction is completed, ready for the next use.

[0044] The preparation method of the novel lipase includes the steps of carrier pretreatment, enzyme immobilization, activity modification, synergism and stabilization, and filling and storage. The operation is relatively simple and does not require complex equipment and harsh reaction conditions. The reagents and materials used in each step are relatively common, easy to obtain, and relatively low in cost. This makes the preparation method suitable for large-scale production and is conducive to promotion and application.

[0045] Most of the reagents used in the entire preparation process are environmentally friendly materials, such as phosphate buffer solution, polyethylene glycol-modified gold nanoparticles, etc. Due to the efficient catalytic effect of lipase, the use of chemical catalysts can be reduced, the risk of environmental pollution can be reduced, and it meets the development requirements of green chemistry.

[0046] Embodiment 2:

[0047] The embodiment of the present invention provides a novel lipase for efficiently preparing sterol esters, comprising the following raw materials in parts by weight: 30 parts of porous carrier material, 15 parts of enzyme immobilization agent, 10 parts of activity modifier, 40 parts of buffer solution, 12 parts of free lipase, 8 parts of synergist, and 8 parts of stabilizer.

[0048] The porous carrier material is a composite of graphene and zeolite; the enzyme immobilization reagent is glutaraldehyde; the activity modifier is polyethylene glycol-modified nano-gold particles; the buffer solution is phosphate buffer solution; the enhancer is tris(hydroxymethyl)aminomethane; and the stabilizer is trehalose.

[0049] A method for preparing a novel lipase for efficiently preparing sterol esters comprises the following steps:

[0050] S1. Carrier Pretreatment

[0051] The porous carrier material is placed in a container containing a dilute acid solution, treated under ultrasonic conditions for 30 minutes, then washed with deionized water until neutral, and dried for later use; the dilute acid solution is prepared by mixing dilute hydrochloric acid with water;

[0052] S2. Enzyme Immobilization

[0053] The pretreated porous carrier material is added to a buffer solution containing an enzyme immobilization reagent, and stirred at 40°C for 2 hours to allow the enzyme immobilization reagent to react with the active groups on the surface of the porous carrier material. Free lipase is then added, and the reaction is continued at the same temperature for 3 hours to allow the lipase to be fixed on the porous carrier material. After the reaction is completed, the material is filtered and washed with a buffer solution to obtain an immobilized lipase.

[0054] S3. Active Modification

[0055] The immobilized lipase is added to a solution containing an activity modifier, and the mixture is stirred and reacted at 35°C for 2 hours to allow the activity modifier to bind to the immobilized lipase and improve the activity and stability of the lipase. After the reaction is completed, the mixture is filtered and washed with a buffer solution to obtain an activity-modified immobilized lipase.

[0056] S4. Efficiency and stability

[0057] The immobilized lipase after activity modification is added to a buffer solution containing a synergist and a stabilizer, and the reaction is stirred at 45°C for 60 minutes to allow the synergist and the stabilizer to fully combine with the lipase, thereby improving the catalytic efficiency and stability of the lipase. After the reaction is completed, the lipase is filtered and washed with a buffer solution to obtain a new lipase for efficiently preparing sterol esters.

[0058] S5. Filling and storage

[0059] The prepared novel lipase is packed into a sealed container and stored under low temperature conditions.

[0060] The present embodiment adopts the raw material formula of specific ratio, makes the novel lipase have higher catalytic activity in the process of preparing sterol ester.The porous carrier material is a composite of graphene and zeolite, combines the high specific surface area of ​​graphene and the good stability of zeolite, provides more attachment sites for lipase, thereby improves the loading capacity of enzyme, and then improves catalytic efficiency.Through multi-step processing, including enzyme immobilization, activity modification, synergism and stability, the activity of lipase is fully exerted.For example, the activity modifier is the nano gold particles modified by polyethylene glycol, which can significantly improve the activity and stability of lipase, and further accelerate the preparation speed of sterol ester.

[0061] The selection of porous carrier materials and the use of enzyme immobilization agents enable lipase to be firmly fixed on the carrier and not easily inactivated or detached during the reaction. Glutaraldehyde, as an enzyme immobilization agent, reacts with the active groups on the surface of the porous carrier material to ensure the stability of the lipase. The addition of stabilizer trehalose provides a stable environment for the lipase to prevent it from losing its activity due to environmental factors during storage and use. At the same time, in each step of the preparation process, impurities that may affect the stability of the lipase are removed through operations such as filtration and washing.

[0062] The immobilized lipase can be separated from the reaction system by a simple filtration operation, which is convenient for recycling and reuse. This not only reduces production costs, but also reduces environmental pollution. Since the lipase is fixed on a porous carrier, it can be easily separated and stored after the reaction is completed, ready for the next use.

[0063] The preparation method of the novel lipase includes the steps of carrier pretreatment, enzyme immobilization, activity modification, synergism and stabilization, and filling and storage. The operation is relatively simple and does not require complex equipment and harsh reaction conditions. The reagents and materials used in each step are relatively common, easy to obtain, and relatively low in cost. This makes the preparation method suitable for large-scale production and is conducive to promotion and application.

[0064] Most of the reagents used in the entire preparation process are environmentally friendly materials, such as phosphate buffer solution, polyethylene glycol-modified gold nanoparticles, etc. Due to the efficient catalytic effect of lipase, the use of chemical catalysts can be reduced, the risk of environmental pollution can be reduced, and it meets the development requirements of green chemistry.

[0065] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A novel lipase for efficiently preparing sterol esters, characterized in that: The invention comprises the following raw materials in parts by weight: 20-30 parts of porous carrier material, 10-15 parts of enzyme immobilization reagent, 5-10 parts of activity modifier, 30-40 parts of buffer solution, 8-12 parts of free lipase, 3-8 parts of synergist and 4-8 parts of stabilizer.

2. A novel lipase for efficiently preparing sterol esters according to claim 1, characterized in that: The porous carrier material is a composite of graphene and zeolite; and the enzyme immobilization reagent is glutaraldehyde.

3. The novel lipase for efficiently preparing sterol esters according to claim 1, characterized in that: The active modifier is polyethylene glycol-modified nanogold particles; and the buffer solution is a phosphate buffer solution.

4. The novel lipase for efficiently preparing sterol esters according to claim 1, characterized in that: The synergist is tris(hydroxymethyl)aminomethane; and the stabilizer is trehalose.

5. A method for preparing a novel lipase for efficiently preparing sterol esters according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Carrier Pretreatment The porous carrier material is placed in a container containing a dilute acid solution, treated under ultrasonic conditions for 20 to 30 minutes, then washed with deionized water until neutral, and dried for later use; S2. Enzyme Immobilization The pretreated porous carrier material is added to a buffer solution containing an enzyme immobilization reagent, and the reaction is stirred at 30-40°C for 1-2 hours to allow the enzyme immobilization reagent to react with the active groups on the surface of the porous carrier material, and then free lipase is added, and the reaction is continued at the same temperature for 2-3 hours to immobilize the lipase on the porous carrier material. After the reaction is completed, the lipase is filtered and washed with a buffer solution to obtain an immobilized lipase; S3. Active Modification The immobilized lipase is added to a solution containing an activity modifier, and the reaction is stirred at 25 to 35°C for 1 to 2 hours to allow the activity modifier to bind to the immobilized lipase and improve the activity and stability of the lipase. After the reaction is completed, the solution is filtered and washed with a buffer solution to obtain an activity-modified immobilized lipase; S4. Efficiency and stability The immobilized lipase after activity modification is added to a buffer solution containing a synergist and a stabilizer, and stirred at 35-45° C. for 30-60 minutes to allow the synergist and the stabilizer to fully combine with the lipase, thereby improving the catalytic efficiency and stability of the lipase. After the reaction is completed, the lipase is filtered and washed with a buffer solution to obtain a new lipase for efficiently preparing sterol esters. S5. Filling and storage The prepared novel lipase is packed into a sealed container and stored under low temperature conditions.