Preparation method of immobilized lipase and application of immobilized lipase in catalysis of grease hydrolysis
By immobilizing Candida Antarctic lipase A on porous ceramic carrier, the problems of its stability and recycling were solved, and efficient catalytic hydrolysis of oil and fats were achieved, the acid value of the product reached 180-195mgKOH/g, and the production cost was significantly reduced. , and the enzyme activity is maintained well, suitable for industrial applications.
Patent Information
- Application Number
- CN202411638670.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-01-14
AI Technical Summary
Candida Antarctic lipase A has poor stability, making it difficult to obtain high acid-valent products in oil and fat hydrolysis, and cannot be recycled, resulting in high production costs and limited acid resistance, making it difficult to maintain activity in a high acidity environment.
Using porous ceramics as immobilization carrier, Candida Antarctic lipase A is mixed with phosphate buffer, and after stirring and drying, immobilized lipase is prepared and used in catalytic oil and fat hydrolysis reaction, by controlling the temperature and pH value. , improve the acid resistance and thermal stability of enzymes and achieve efficient catalysis.
It significantly improves the catalytic activity and reusability of the enzyme, can maintain 91% of the initial hydrolase activity under the same conditions, reduces the technical cost, and the acid price of the catalytic product can reach 180-195mgKOH/g, which is easy to operate and green and environmentally friendly. .
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Figure CN119307488A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological enzymes, and in particular to a preparation method of immobilized lipase and application thereof in catalyzing oil hydrolysis. Background Art
[0002] Lipase exists in animals, plants and various microorganisms. It is a stable natural catalyst and is widely used in the fields of bioenzymatic oil modification such as oil hydrolysis, esterification and transesterification. Antarctic Candida lipase A is a heat-resistant lipase that has great advantages in the fields of polyunsaturated fatty acid enrichment, biodiesel preparation, and zero trans fatty acid oil preparation. However, the stability of free lipase is poor and cannot be recycled. After lipase is immobilized, the stability and reusability of lipase can be significantly improved, thereby reducing production costs and facilitating industrialization.
[0003] Oil hydrolysis is a common process for obtaining fatty acids in the oil and fat industry. Compared with chemical catalysis, enzymatic hydrolysis has the advantages of low temperature, environmental protection, and greenness. However, as the hydrolysis proceeds, the acidity of the system gradually increases, and lipase gradually becomes inactive due to its limited ability to tolerate acid, making it difficult to obtain oil hydrolyzate with a higher acid value.
[0004] Antarctic Candida lipase A has good hydrolysis activity. However, as mentioned above, free Antarctic Candida lipase A cannot be recycled; in addition, although Antarctic Candida lipase A has a certain acid resistance, it is difficult for its catalytic system to obtain products with an acid value of up to 180-195 mgKOH / g. Therefore, immobilizing Antarctic Candida lipase A is intended to improve its acid resistance, heat stability, and reusability, which is of great significance for reducing production costs and obtaining oil hydrolyzates with high acid values. Summary of the invention
[0005] Based on the existing technical problems, the present invention proposes a preparation method of immobilized lipase and its application in catalyzing oil hydrolysis.
[0006] The present invention provides a method for preparing an immobilized lipase, comprising the following steps: step 1, selecting materials, selecting Antarctic Candida lipase A, porous ceramics and phosphate buffer; Step 2: Mixing the porous ceramic, Candida antarctica lipase A, and phosphate buffer, stirring the mixture at a set temperature for a period of time, and obtaining an immobilized system mixture; Step 3: Then the obtained immobilized system mixture is subjected to solid-liquid separation and dried to obtain the immobilized lipase.
[0007] Preferably, the content of the porous ceramic in step 1 is 8-15 g / L, and the concentration of the Candida antarctica lipase A is 250-450 μg / mL.
[0008] Preferably, the temperature of the immobilization system mixture in step 2 is 25-30° C., and the stirring time is 30-55 min.
[0009] Preferably, the pH of the phosphate buffer in step 1 is 4.8-5.2, wherein the concentration of phosphate in the phosphate buffer is 25 mM.
[0010] A method for preparing an immobilized lipase, wherein the prepared immobilized lipase is used in catalyzing the hydrolysis of oils and fats, and oils and water are mixed with the prepared immobilized lipase, and reacted at 40-50° C. for 8-15 hours to obtain a hydrolysis product with an acid value of 180-195 mgKOH / g.
[0011] Preferably, the mass ratio of oil to water is 1:(0.9-1.1), and the amount of immobilized lipase used is 1.5-4.0wt% of the total mass of oil and water.
[0012] Preferably, the oil is selected from one or more of soybean oil, palm oil, rapeseed oil, cottonseed oil, corn oil, peanut oil, and camellia oil.
[0013] The beneficial effects of the present invention are: The present invention uses porous ceramics as an immobilized carrier to immobilize Antarctic Candida lipase A; the obtained immobilized lipase is used to catalyze the oil hydrolysis reaction, and the acid value of the product after catalyzing the oil hydrolysis can reach 180-195 mgKOH / g. Compared with the free enzyme, the immobilized enzyme significantly improves the catalytic activity of the enzyme under the same conditions; in addition, the immobilized enzyme has high thermal stability and pH tolerance; the immobilized enzyme has good reusability and is easy to recycle: after the immobilized lipase is reused 15 times to catalyze the soybean oil hydrolysis reaction, it can maintain 91% of its initial hydrolytic enzyme activity, significantly reducing the technical cost, and is easy to separate from the product, easy to operate, and green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 The present invention is a structural data parameter diagram of the immobilized lipase prepared by a method for preparing the immobilized lipase and its application in catalyzing the hydrolysis of oils and fats. DETAILED DESCRIPTION
[0015] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Example
[0016] A method for preparing immobilized lipase comprises the following steps: step 1, selecting materials, selecting Antarctic Candida lipase A, porous ceramics and phosphate buffer; the content of the porous ceramics in step 1 is 8-15 g / L, and the concentration of the Antarctic Candida lipase A is 250-450 μg / mL; the pH of the phosphate buffer in step 1 is 4.8-5.2, wherein the concentration of phosphate in the phosphate buffer is 25 mM.
[0017] Step 2: Mix the porous ceramic, Antarctic Candida lipase A, and phosphate buffer, and stir them at a set temperature for a period of time to obtain an immobilized system mixture; the temperature of the immobilized system mixture in step 2 is 25-30°C, and the stirring time is 30-55min.
[0018] Step 3: Then the obtained immobilized system mixture is subjected to solid-liquid separation and dried to obtain the immobilized lipase. Example
[0019] A method for preparing an immobilized lipase, and application of the prepared immobilized lipase in catalyzing the hydrolysis of oils and fats; mixing oils and water with the prepared immobilized lipase, reacting at 40-50°C for 8-15h to obtain a hydrolysis product with an acid value of 180-195mgKOH / g, wherein the mass ratio of the oils and fats to water is 1:(0.9-1.1), the amount of the immobilized lipase used is 1.5-4.0wt% of the total mass of the oils and fats and water, and the oils and fats are selected from one or more of soybean oil, palm oil, rapeseed oil, cottonseed oil, corn oil, peanut oil, and camellia oil. Example
[0020] S1: 1.0 g of porous ceramic was dispersed in 100 mL of phosphate buffer (25 mM, pH 5.0), and 35 mg of Antarctic Candida lipase A protein was added; the system was stirred at 25 °C for 40 min; then filtered to obtain the immobilized enzyme, and then vacuum dried at 30 °C for 6 h to obtain the porous ceramic immobilized enzyme.
[0021] S2: Take 25 g of soybean oil and 25 g of water, then add 1.0 g of the immobilized enzyme obtained in step S1 of the above embodiment 3, and stir the system at 45° C. for 12 h to obtain a soybean oil hydrolyzate with an acid value of 188 mgKOH / g. Example
[0022] S1: 5.0 g of porous ceramics were dispersed in 600 mL of phosphate buffer (25 mM, pH 5.1), and 180 mg of Antarctic Candida lipase A protein was added; the system was stirred at 30 °C for 30 min; then filtered to obtain the immobilized enzyme, and then vacuum dried at 30 °C for 6 h to obtain the porous ceramic immobilized enzyme.
[0023] S2: Take 100 g of rapeseed oil and 100 g of water, then add 4.5 g of the immobilized enzyme obtained in step S1 of the above embodiment 4, and stir the system at 45° C. for 10 h to obtain a rapeseed oil hydrolyzate with an acid value of 185 mgKOH / g. Example
[0024] S1: 24.0 g of porous ceramics were dispersed in 2000 mL of phosphate buffer (25 mM, pH 5.2), and 800 mg of Antarctic Candida lipase A protein was added; the system was stirred at 28 °C for 35 min; then filtered to obtain the immobilized enzyme, and then vacuum dried at 30 °C for 6 h to obtain the porous ceramic immobilized enzyme.
[0025] S2: 300 g peanut oil and 305 g water were taken, and then 18.15 g of the immobilized enzyme obtained in step S1 of the above embodiment 5 was added. The system was stirred and reacted at 45° C. for 13 h to obtain a peanut oil hydrolyzate with an acid value of 190 mgKOH / g. Example
[0026] S1: 15 g of porous ceramics were dispersed in 1500 mL of phosphate buffer (25 mM, pH 4.9), and 525 mg of Antarctic Candida lipase A protein was added; the system was stirred at 25 °C for 40 min; then filtered to obtain the immobilized enzyme, and then vacuum dried at 30 °C for 6 h to obtain the porous ceramic immobilized enzyme.
[0027] S2: Take 250 g of camellia oil and 230 g of water, then add 14.4 g of the immobilized enzyme obtained in step S1 of the above Example 6, and stir the system at 43° C. for 14 h to obtain a camellia oil hydrolyzate with an acid value of 183 mgKOH / g. Example
[0028] S1: 22 g of porous ceramics were dispersed in 2000 mL of phosphate buffer (25 mM, pH 5.0), and 700 mg of Antarctic Candida lipase A protein was added; the system was stirred at 30 °C for 30 min; then filtered to obtain the immobilized enzyme, and then vacuum dried at 30 °C for 6 h to obtain the porous ceramic immobilized enzyme.
[0029] S2: 300 g soybean oil and 310 g water were taken, and then 18.6 g of the immobilized enzyme obtained in step S1 of the above embodiment 7 was added. The system was stirred and reacted at 45° C. for 13 h to obtain a soybean oil hydrolyzate with an acid value of 195 mgKOH / g. Example
[0030] like Figure 1As shown, the system after the reaction in the above Example 7 is filtered to obtain the immobilized enzyme, which is then used in step S2 of Example 7 to catalyze the hydrolysis of soybean oil; after the catalytic reaction, the immobilized enzyme is filtered again to be recovered, and then used again in step S2 of Example 7 to catalyze the hydrolysis product of soybean oil; the immobilized enzyme is repeatedly used in this way; and this is repeated 15 times.
[0031] The present invention uses porous ceramics as an immobilized carrier to immobilize Antarctic Candida lipase A; the obtained immobilized lipase is used to catalyze the oil hydrolysis reaction, and the acid value of the product after catalyzing the oil hydrolysis can reach 180-195 mgKOH / g. Compared with the free enzyme, the immobilized enzyme significantly improves the catalytic activity of the enzyme under the same conditions; in addition, the immobilized enzyme has high thermal stability and pH tolerance; the immobilized enzyme has good reusability and is easy to recycle: after the immobilized lipase is reused 15 times to catalyze the soybean oil hydrolysis reaction, it can maintain 91% of its initial hydrolytic enzyme activity, significantly reducing the technical cost, and is easy to separate from the product, easy to operate, and green and environmentally friendly.
[0032] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A method for preparing immobilized lipase, characterized in that: The method comprises the following steps: step 1, selecting materials, selecting Antarctic Candida lipase A, porous ceramics and phosphate buffer; Step 2: Mixing the porous ceramic, Candida antarctica lipase A, and phosphate buffer, stirring the mixture at a set temperature for a period of time, and obtaining an immobilized system mixture; Step 3: Then the obtained immobilized system mixture is subjected to solid-liquid separation and dried to obtain the immobilized lipase.
2. The method for preparing an immobilized lipase according to claim 1, characterized in that: The content of the porous ceramic in the step 1 is 8-15 g / L, and the concentration of the Antarctic Candida lipase A is 250-450 μg / mL.
3. The method for preparing an immobilized lipase according to claim 1, characterized in that: The temperature of the immobilization system mixture in step 2 is 25-30° C., and the stirring time is 30-55 min.
4. The method for preparing an immobilized lipase according to claim 1, characterized in that: The pH of the phosphate buffer in step 1 is 4.8-5.2, wherein the concentration of phosphate in the phosphate buffer is 25 mM.
5. The method for preparing an immobilized lipase according to claim 1, wherein the immobilized lipase prepared therefrom is used for catalyzing the hydrolysis of fats and oils, characterized in that: The prepared immobilized lipase is mixed with oil and water, and reacted at 40-50° C. for 8-15 hours to obtain a hydrolyzate with an acid value of 180-195 mgKOH / g.
6. The method for preparing an immobilized lipase according to claim 5, wherein the immobilized lipase prepared therefrom is used for catalyzing the hydrolysis of fats and oils, characterized in that: The mass ratio of oil to water is 1:(0.9-1.1), and the amount of immobilized lipase used is 1.5-4.0wt% of the total mass of oil and water.
7. The method for preparing an immobilized lipase according to claim 5, wherein the immobilized lipase prepared therefrom is used for catalyzing the hydrolysis of fats and oils, characterized in that: The oil is selected from one or more of soybean oil, palm oil, rapeseed oil, cottonseed oil, corn oil, peanut oil and camellia oil.