High-stability immobilized candida antarctica lipase B as well as preparation method and application thereof

Abstract

The invention discloses high-stability immobilized candida antarctica lipase B as well as a preparation method and application thereof, and belongs to the technical field of enzyme engineering and biological catalysis. The preparation method comprises the following steps: modifying resin by adopting a silane coupling agent to obtain silanized resin; carrying out aldehyde functional modification on the silanized resin by using dialdehyde cellulose to obtain alkyl-aldehyde modified resin; candida antarctica lipase B is immobilized by using alkyl-aldehyde modified resin, and after the Candida antarctica lipase B is subjected to interfacial activation on the surface of the alkyl-aldehyde modified resin, the Candida antarctica lipase B is covalently bound with aldehyde on the surface of the alkyl-aldehyde modified resin; the prepared immobilized Candida antarctica lipase B has relatively high activity and stability. The method is simple to operate, the prepared immobilized lipase is high in stability, and the method can be used for preparing the high-stability immobilized lipase on a large scale and has the potential of industrial popularization and application.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering and biocatalysis, and specifically relates to a high-stability immobilized Antarctic Candida lipase B and a preparation method and application thereof. Background Art

[0002] As an important industrial enzyme, lipase is widely used in food processing, oil modification, biodiesel preparation, chiral compound separation and other fields. It has the characteristics of high selectivity, mild catalytic conditions and environmental friendliness. Although lipase has great application potential in industry, most free lipases have high preparation costs, low operational stability, and are easy to agglomerate in the organic phase, which greatly reduces the utilization rate of lipase. In addition, free lipases are difficult to recover and reuse, resulting in high application costs of lipase. The immobilized form of lipase is superior to the free state in many properties. After being immobilized, the enzyme has the advantages of high stability, easy recovery, repeated use and low cost.

[0003] Candida antarctica lipase B (CALB) shows high catalytic activity for many lipid substances. It is the most widely used lipase and the most successful commercial lipase. At present, the most widely used immobilized CALB is Novozym 435 from Novozymes of Denmark. However, Novozym 435 is expensive (more than 15,000 yuan / kg), and its preparation technology has been monopolized by Novozymes, which seriously restricts the development of bio-enzyme catalysis technology in my country. In view of the current situation that China's independent research and development capabilities of immobilized enzymes are insufficient, there is a lack of immobilized enzyme preparation technology with independent intellectual property rights, and immobilized lipases are heavily dependent on imports, it is urgent to carry out research on the preparation technology of immobilized lipases.

[0004] Studies have shown that most lipases are interfacially activated enzymes, and their active centers are covered by a "lid" structure. The inside of the "lid" is hydrophobic and the outside is hydrophilic. Therefore, when the lipase comes into contact with a hydrophobic surface, the conformation of the lipase changes, the "lid" opens, and the active center is exposed to the reaction medium, thereby promoting the interaction between the active center and the substrate, greatly increasing the catalytic activity of the lipase. On this basis, the activity and stability of the lipase can be significantly improved by immobilizing the lipase on a carrier with a hydrophobic surface. In early studies, it was believed that CALB was a special lipase that did not show obvious interfacial activation. However, later studies have shown that CALB has a small "lid" at its active site. Even if this small "lid" cannot completely close the active center, CALB is still an interfacial enzyme. Therefore, by changing the hydrophilicity of the carrier, the affinity between the carrier and the enzyme can be improved, which is beneficial to improving the activity and stability of the enzyme.

[0005] Covalent bonding is one of the most widely used immobilization methods. It is a method of connecting the functional groups on the surface of the enzyme molecule with the active groups of the carrier material through covalent bonds. The covalent bond makes the enzyme and the carrier firmly bonded, so that the immobilized enzyme prepared by this method has high stability. Usually, in order to make these reactions occur, the surface of the carrier must be modified or functionalized. The most common modifier is glutaraldehyde (GA). However, GA itself is toxic and will not only cause environmental pollution, but also excessive doses may inhibit lipase activity. Cellulose is a green and non-toxic plant polysaccharide with a wide range of sources. It has good stability and biocompatibility and can be prepared into aldehyde derivatives for enzyme cross-linking and immobilization.

[0006] CN 116463327 A discloses an immobilized lipase with an acyl chloride-modified amino resin as a carrier, and its preparation method and application. Acid chloride is coupled to the amino resin to obtain an acyl chloride-modified amino resin, lipase is adsorbed on the modified resin, and genipin is used as a cross-linking agent to prepare the immobilized enzyme. The immobilized enzyme catalyzes the reaction of oleic acid and glycerol to generate 1,3-diacerol. When it is reused 10 times, the content of 1,3-diacerol is 89% of the initial value. CN 110331139 A discloses a method for immobilizing Antarctic Candida lipase B. Dimethyl imidazole aqueous solution, zinc nitrate aqueous solution and CALB are mixed, and immobilized CALB is prepared using a macroporous adsorption resin as an immobilization carrier and polyethyleneimine as a cross-linking agent. The immobilized enzyme catalyzes the reaction of oleic acid and methanol for 10 batches, and the relative activity is maintained at about 85%.

[0007] In conclusion, the activity of immobilized CALB prepared in the laboratory is comparable to or higher than that of Novozym 435, but the stability is still far behind that of Novozym 435. Therefore, we proposed a preparation method to obtain immobilized CALB with good stability and activity. Summary of the invention

[0008] The present invention aims to improve the stability of immobilized lipase and to provide a highly stable immobilized Antarctic Candida lipase B and a preparation method and application thereof. The present invention modifies the resin of the immobilization carrier by using a silane coupling agent and dialdehyde cellulose, and uses the resin to immobilize the Antarctic Candida lipase B, thereby obtaining an Antarctic Candida lipase B with higher stability and activity.

[0009] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for preparing high-stability immobilized Antarctic Candida lipase B, comprising the following steps: Adding a silane coupling agent to a mixed solution of deionized water and anhydrous ethanol and mixing uniformly to obtain a blended solution, adding a resin to the blended solution and mixing uniformly to obtain a suspension, heating the suspension until the liquid is completely evaporated, adding anhydrous ethanol to the product obtained after evaporation and evaporating again to obtain a silanized resin; The dialdehyde fiber is added to a sodium hydroxide-urea solution, and then deionized water and a silanized resin are added and mixed evenly, the mixed solution is subjected to a constant temperature oscillation treatment, and after the constant temperature oscillation treatment, the pH is adjusted to neutral to obtain a crude resin product, and the crude resin product is washed and dried to obtain an alkyl-aldehyde modified resin; The alkyl-aldehyde modified resin is added to a mixed solution of Antarctic Candida lipase B enzyme solution and a buffer solution and mixed evenly. The mixed solution is placed in a constant temperature environment to solidify the Antarctic Candida lipase B. After the solidification treatment, the immobilized enzyme is separated, and the immobilized enzyme is washed with a buffer solution until no protein is detected in the filtrate, and then dried to obtain the immobilized Antarctic Candida lipase B.

[0010] The volume ratio of deionized water to anhydrous ethanol in the mixed solution of deionized water and anhydrous ethanol is 1:2 to 1:4; The silane coupling agent is one of phenyltriethoxysilane, octyltriethoxysilane and dodecyltriethoxysilane, and the added amount of the silane coupling agent is 5% to 10% of the total volume of deionized water and anhydrous ethanol.

[0011] The resin is ECR1030 macroporous adsorption resin, and 0.02 g to 0.05 g of the resin is added to each milliliter of the blended solution.

[0012] The resin is added to the blended solution and mixed evenly to obtain a suspension, the suspension is heated until the liquid is completely evaporated, anhydrous ethanol is added to the product obtained after evaporation and then evaporated to obtain a silanized resin, specifically: Add resin to the blended solution, and obtain a suspension after ultrasonic dispersion treatment for 0.5 h to 1 h. Heat the suspension in an environment at a temperature of 80°C to 95°C until the liquid is completely evaporated. Add anhydrous ethanol to the product obtained after evaporation and evaporate it again, repeating 2 to 3 times. Wash with anhydrous ethanol and deionized water, and dry in an environment at a temperature of 40°C to 45°C for 8 h to 10 h to obtain a silanized resin.

[0013] The sodium hydroxide-urea aqueous solution is 7% sodium hydroxide and 12% urea aqueous solution, the mass ratio of dialdehyde cellulose to silylated resin is 1:6-1:7, and the amount of deionized water added is 15-20 times the volume of the sodium hydroxide-urea aqueous solution.

[0014] The dialdehyde fiber is added to a sodium hydroxide-urea solution, and then deionized water and a silanized resin are added and mixed evenly. The mixed solution is subjected to a constant temperature oscillation treatment. After the constant temperature oscillation treatment, the pH is adjusted to neutral to obtain a crude resin product. The crude resin product is washed and dried to obtain an alkyl-aldehyde modified resin, specifically: Take dialdehyde cellulose and add it to the sodium hydroxide-urea aqueous solution and mix it evenly. After adding deionized water and silanized resin, ultrasonicate it for 10 min~30 min, and oscillate it at a constant temperature for 2 h~4 h at a temperature of 40℃~45℃ and a rotation speed of 120 rpm~150 rpm. After the constant temperature oscillation treatment, adjust the pH to neutral with a hydrochloric acid solution to obtain a crude resin product. Wash the crude resin product with anhydrous ethanol and deionized water, and dry it at a temperature of 40℃~45℃ for 8 h~10 h to obtain an alkyl-aldehyde modified resin.

[0015] The buffer is a 0.02 mol / L phosphate buffer with a pH of 7, the volume ratio of the Antarctic Candida lipase B enzyme solution to the buffer is 1:1, and the added amount of the alkyl-aldehyde modified resin is 80 mg / g~120 mg / g, where mg / g represents the amount of the alkyl-aldehyde modified resin added per gram of enzyme protein in the Antarctic Candida lipase B enzyme solution.

[0016] The alkyl-aldehyde modified resin is added to a mixed solution of an Antarctic Candida lipase B enzyme solution and a buffer solution and mixed evenly, the mixed solution is placed in a constant temperature environment to solidify the Antarctic Candida lipase B, the immobilized enzyme is separated after the solidification treatment, the immobilized enzyme is washed with a buffer solution until no protein is detected in the filtrate, and then dried to obtain the immobilized Antarctic Candida lipase B, specifically: After thoroughly mixing the Antarctic Candida lipase B enzyme solution and the buffer solution, alkyl-aldehyde modified resin was added and mixed evenly. The mixed solution was oscillated in a constant temperature gas bath for 6 h~10 h at a temperature of 30℃~40℃ and a rotation speed of 110 rpm~130 rpm to solidify the Antarctic Candida lipase B. After solidification, the immobilized enzyme was separated by filtration, and the immobilized enzyme was washed with buffer solution until no protein was detected in the filtrate. The washed immobilized enzyme was dried at a temperature of 40℃~45℃ for 8 h~10 h to obtain immobilized Antarctic Candida lipase B.

[0017] The present invention also provides a high-stability immobilized Antarctic Candida lipase B prepared according to the above preparation method, wherein the amino groups of the Antarctic Candida lipase B are covalently bonded to the aldehyde groups on the surface of the alkyl-aldehyde modified resin.

[0018] The present invention also provides the application of the above-mentioned high-stability immobilized Candida antarctica lipase B in the fields of functional oil preparation, high acid value oil deacidification and biofuel preparation.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a method for preparing a highly stable immobilized Antarctic Candida lipase B. The method comprises modifying a resin as an immobilization carrier, and applying the modified resin to the immobilization of Antarctic Candida lipase B to improve the stability of the immobilized Antarctic Candida lipase B. First, a silane coupling agent is used to modify the resin to obtain a silanized resin; then, the silanized resin is further modified by dialdehyde cellulose, and the aldehyde group is connected to the surface of the silanized resin to obtain an alkyl-aldehyde modified resin. Subsequently, the alkyl-aldehyde modified resin is applied to the immobilization of Antarctic Candida lipase B. Since the Antarctic Candida lipase B is interfacially activated on the surface of the alkyl-aldehyde modified resin, a part of the Antarctic Candida lipase B is covalently bonded to an appropriate amount of aldehyde groups on the surface of the alkyl-aldehyde modified resin when immobilized, so that the prepared immobilized Antarctic Candida lipase B has high activity and stability. The immobilized Antarctic Candida lipase B prepared by the preparation method of the present invention has an enzyme activity of up to 9567.93 U / g, a thermal stability of about 97% in application, an operational stability of about 99%, a storage stability of about 97%, and repeated use of 50 batches with almost no loss of activity. The present invention is simple to operate, and the immobilized Antarctic Candida lipase B prepared by the method has high stability, and has the potential for industrial promotion and application.

[0020] Furthermore, silane coupling agents of different chain lengths have different hydrophilicity and hydrophobicity. By connecting organic groups on the resin surface through silanization reaction, the hydrophilicity and hydrophobicity of the resin surface can be regulated. The type and amount of silane coupling agent added can ensure that the water contact angle of the silanized resin is controlled within the range of 110°~120°, preventing the silane coupling agent from over-modifying the resin, which is beneficial to the subsequent improvement of the stability of immobilized Antarctic Candida lipase B.

[0021] Furthermore, the type and amount of the resin added can ensure appropriate hydrophobicity and an appropriate aldehyde group concentration on the resin surface. Furthermore, the amount of dialdehyde cellulose added and the time of resin treatment can ensure that the aldehyde content on the resin is in the range of 1.0 mmol / g~3.0 mmol / g, thereby preventing excessive cross-linking of Antarctic Candida lipase B during immobilization, which is beneficial to maintaining the activity and stability of immobilized Antarctic Candida lipase B.

[0022] Furthermore, the amount of Antarctic Candida lipase B enzyme solution added during immobilization can ensure that the Antarctic Candida lipase B is fully immobilized on the surface and inside of the resin, thereby obtaining an immobilized Antarctic Candida lipase B that takes into account economy, activity and stability.

[0023] Furthermore, the drying conditions of the immobilized lipase can not only ensure a lower water activity of the immobilized lipase Candida antarctica lipase B, but also help maintain the activity and stability of the immobilized Candida antarctica lipase B. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for use in the embodiment: Figure 1 The flowchart of the preparation method of the high-stability immobilized Antarctic Candida lipase B of the present invention is as follows; Figure 2 This is a schematic diagram of the principle of resin modification according to the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific examples, which are intended to explain the present invention rather than to limit it. Unless otherwise specified, the lipase solutions described in the following examples are all Novozymes Antarctic Candida lipase B.

[0026] like Figure 1 As shown, the present invention provides a method for preparing high-stability immobilized Antarctic Candida lipase B, comprising the following steps: Deionized water and anhydrous ethanol are added to a conical flask, mixed evenly, and then a silane coupling agent is added and mixed evenly to obtain a blended solution, resin is added to the blended solution, and after ultrasonic dispersion treatment at room temperature for 0.5 h~1 h, it is heated in an environment at a temperature of 80°C~95°C until the liquid is almost evaporated to obtain an alkyl functionalized product; the alkyl functionalized product is washed with anhydrous ethanol and then evaporated, and the process is repeated 2~3 times; then it is washed with anhydrous ethanol and deionized water, and dried in an environment at a temperature of 40°C~45°C for 8 h~10 h to obtain a silanized resin.

[0027] Take dialdehyde cellulose (DAC) and add it to a sodium hydroxide-urea aqueous solution and mix it evenly. After adding deionized water and silanized resin, ultrasonicate it for 10 min~30 min. The mixed solution is placed in a constant temperature oscillator at a temperature of 40℃~45℃ and a rotation speed of 120 rpm~150 rpm for 2 h~4 h. Then, the pH is adjusted to neutral with a hydrochloric acid solution. The aldehyde functionalized product is washed with anhydrous ethanol and deionized water, and dried at a temperature of 40℃~45℃ for 8 h~10 h to obtain a silanized resin, thereby obtaining an alkyl-aldehyde modified resin. After thoroughly mixing the Antarctic Candida lipase B enzyme solution and the buffer solution, alkyl-aldehyde modified resin was added, and the mixed solution was placed in a constant temperature oscillator. At a temperature of 30°C~40°C and a rotation speed of 110 rpm~130 rpm, the constant temperature oscillation was performed for 6 h~10 h. The immobilized product was separated by filtration, and the immobilized product was washed with buffer solution until no protein was detected in the filtrate. The washed immobilized product was dried in an environment of 40°C~45°C for 8 h~10 h to obtain immobilized Antarctic Candida lipase B.

[0028] Wherein, the volume ratio of deionized water to anhydrous ethanol is 1:2-1:4; The silane coupling agent is one of phenyltriethoxysilane, octyltriethoxysilane and dodecyltriethoxysilane, and the added amount of the silane coupling agent is 5% to 10% of the total volume of deionized water and anhydrous ethanol.

[0029] The resin is ECR1030 macroporous adsorption resin, and 0.002 g to 0.005 g of the resin is added to each milliliter of the blended solution.

[0030] The sodium hydroxide-urea aqueous solution is 7% sodium hydroxide and 12% urea aqueous solution, the mass ratio of dialdehyde cellulose to silylated resin is 1:6-1:7, and the amount of deionized water added is 15-20 times the volume of the sodium hydroxide-urea aqueous solution.

[0031] The buffer is a 0.02 mol / L phosphate buffer with a pH of 7, the volume ratio of the Antarctic Candida lipase B enzyme solution to the buffer is 1:1, and the added amount of the alkyl-aldehyde modified resin is 80 mg / g~120 mg / g, where mg / g represents the amount of the alkyl-aldehyde modified resin added per gram of enzyme protein in the Antarctic Candida lipase B enzyme solution.

[0032] like Figure 2 As shown, the principle of modifying the resin in the present invention is: first, the resin is modified by a silane coupling agent. Due to the presence of alkoxy groups in the silane coupling agent, the alkyl-functionalized silanized resin has moderate hydrophobicity; then, the silanized resin is further functionalized by aldehyde groups using dialdehyde cellulose. Dialdehyde cellulose is an aldehyde derivative of cellulose, which exists in the form of hemiacetal in an aqueous medium and is usually produced by oxidation of cellulose with sodium periodate; after modification by dialdehyde cellulose, the alkyl-aldehyde modified resin surface is connected to aldehyde groups.

[0033] When alkyl-aldehyde modified resin is used for lipase immobilization, due to the moderate hydrophobicity of the alkyl-aldehyde modified resin and the presence of appropriate aldehyde groups on the surface and inside the alkyl-aldehyde modified resin, after the Antarctic Candida lipase B is interfacially activated on the surface of the alkyl-aldehyde modified resin, the amino group of the Antarctic Candida lipase B reacts with the aldehyde group on the surface of the alkyl-aldehyde modified resin to form a covalent bond, thereby greatly improving the stability of the immobilized Antarctic Candida lipase B.

[0034] The present invention also discloses an immobilized Antarctic Candida lipase B prepared by the above-mentioned preparation method. The immobilized Antarctic Candida lipase B prepared by the present invention has good stability and high activity. During the test, the immobilized Antarctic Candida lipase B prepared was reused for 50 batches without losing any activity. The prepared immobilized Antarctic Candida lipase B can be used in the fields of functional oil preparation, high acid value oil deacidification and biofuel preparation.

[0035] Example 1 In a 150 mL conical flask, 15 mL of deionized water and 30 mL of anhydrous ethanol were added and mixed evenly, then 2.25 mL of phenyltriethoxysilane was added, stirred evenly, and 1 g of ECR1030 resin was added. After ultrasonic dispersion treatment at room temperature for 0.5 h, the mixture was heated at 80 °C until the liquid was almost evaporated to obtain an alkyl functionalized product. Then, 10 mL of anhydrous ethanol was added to wash the alkyl functionalized product and evaporated again. The operation was repeated twice. Finally, the alkyl functionalized product was washed with anhydrous ethanol and deionized water, and the washed alkyl functionalized product was dried at 40 °C for 8 h to obtain a silanized resin (water contact angle of 114°). 0.2 g of dialdehyde cellulose was added to 10 mL of sodium hydroxide-urea aqueous solution, followed by 150 mL of deionized water and 1.2 g of silanized resin. After ultrasonic treatment for 10 min, the mixture was placed in a 40℃ constant temperature oscillator and oscillated at 120 rpm for 2 h. The pH was then adjusted to neutral with hydrochloric acid solution, and the aldehyde functionalized product was washed with anhydrous ethanol and deionized water. The washed aldehyde functionalized product was dried at 40℃ with air blast for 8 h to obtain an alkyl-aldehyde modified resin (aldehyde content of 2.6 mmol / g). 9.6 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, and 1 g of alkyl-aldehyde modified resin was added. After being fixed in a constant temperature air bath oscillator at 30℃ and 110 rpm for 6 h, the immobilized product was separated by suction filtration, and the immobilized product was washed with phosphate buffer until there was no protein residue in the filtrate. The washed immobilized product was dried at 40℃ with air blast for 8 h.

[0036] The enzymatic properties of the immobilized CALB were characterized. The results showed that the activity of the immobilized CALB was 7955.72±148.30 U / g. After being incubated at 65℃ for 6 h, it could still retain 97.62% of the initial enzyme activity. Novozym 435 retained 84.06% of the initial enzyme activity under the same conditions. The immobilized CALB was used for deacidification of high acid value rice bran oil and was reused for 50 batches. It could still retain 99.40% of the initial enzyme activity. Novozym 435 only retained 86.25% of the initial enzyme activity after being reused for 50 batches. After being sealed and stored at 4℃ for 60 days, the enzyme activity of the immobilized CALB was maintained at about 98.83% of the initial enzyme activity, while Novozym 435 could only maintain 90.27% of the initial enzyme activity.

[0037] Example 2 In a 150 mL conical flask, 15 mL of deionized water and 60 mL of anhydrous ethanol were added and mixed evenly, then 7.5 mL of phenyltriethoxysilane was added, stirred evenly, and 4.2 g of ECR1030 resin was added. After ultrasonic dispersion treatment at room temperature for 1 h, the mixture was heated at 95 °C until the liquid was almost evaporated to obtain an alkyl functionalized product. Then, 10 mL of anhydrous ethanol was added to wash the mixture to obtain an alkyl functionalized product, which was then evaporated again. The operation was repeated 3 times. Finally, the alkyl functionalized product was washed with anhydrous ethanol and deionized water, and the washed alkyl functionalized product was dried at 45 °C with air for 10 h to obtain a silanized resin (water contact angle of 117°). 0.2 g of dialdehyde cellulose was added to 10 mL of sodium hydroxide-urea aqueous solution, followed by 200 mL of deionized water and 1.4 g of silanized resin. After ultrasonic treatment for 30 min, the mixture was placed in a 45°C constant temperature oscillator and shaken at 150 rpm for 4 h. The pH was then adjusted to neutral with hydrochloric acid solution, and the aldehyde functionalized product was washed with anhydrous ethanol and deionized water. The washed aldehyde functionalized product was dried at 45°C with air blast for 10 h to obtain an alkyl-aldehyde modified resin (aldehyde content of 2.2 mmol / g). 14.5 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, and 1 g of alkyl-aldehyde modified resin was added. After being fixed in a constant temperature air bath oscillator at 40°C and 130 rpm for 10 h, the immobilized product was separated by suction filtration, and the immobilized product was washed with phosphate buffer until there was no protein residue in the filtrate. The washed immobilized product was dried at 45°C with air blast for 10 h.

[0038] The enzymatic properties of the immobilized CALB were characterized. The results showed that the activity of the immobilized CALB was 8022.38±141.20 U / g. After being incubated at 65℃ for 6 h, it could still retain 97.28% of the initial enzyme activity. Novozym 435 retained 84.06% of the initial enzyme activity under the same conditions. The immobilized CALB was used for deacidification of high acid value rice bran oil and was reused for 50 batches. It could still retain 99.52% of the initial enzyme activity. Novozym 435 could only retain 86.25% of the initial enzyme activity after being reused for 50 batches. After being sealed and stored at 4℃ for 60 days, the enzyme activity of the immobilized enzyme was maintained at about 96.22% of the initial enzyme activity, while Novozym 435 could only maintain 90.27% of the initial enzyme activity.

[0039] Example 3 In a 150 mL conical flask, 15 mL of deionized water and 30 mL of anhydrous ethanol were added and mixed evenly, then 2.25 mL of octyltriethoxysilane was added, stirred evenly, and 1 g of ECR1030 resin was added. After ultrasonic dispersion treatment at room temperature for 0.5 h, the mixture was heated at 80 °C until the liquid was almost evaporated to obtain an alkyl functionalized product. Then, 10 mL of anhydrous ethanol was added to wash the alkyl functionalized product and evaporated again. The operation was repeated twice. Finally, the alkyl functionalized product was washed with anhydrous ethanol and deionized water, and the washed alkyl functionalized product was dried at 40 °C for 8 h to obtain a silanized resin (water contact angle of 118°). 0.2 g of dialdehyde cellulose was added to 10 mL of sodium hydroxide-urea aqueous solution, followed by 150 mL of deionized water and 1.2 g of silanized resin. After ultrasonic treatment for 10 min, the mixture was placed in a 40℃ constant temperature oscillator and shaken at 120 rpm for 2 h. The pH was then adjusted to neutral with hydrochloric acid solution, and the resin aldehyde functionalized product was washed with anhydrous ethanol and deionized water. The washed aldehyde functionalized product was dried at 40℃ with air blast for 8 h to obtain an alkyl-aldehyde modified resin (aldehyde content of 2.6 mmol / g). 9.6 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, and 1 g of alkyl-aldehyde modified resin was added. After being fixed in a constant temperature air bath oscillator at 30℃ and 110 rpm for 6 h, the immobilized product was separated by suction filtration, and the immobilized product was washed with phosphate buffer until there was no protein residue in the filtrate. The washed immobilized product was dried at 40℃ with air blast for 8 h.

[0040] The enzymatic properties of the immobilized CALB were characterized. The results showed that the activity of the immobilized CALB was 9326.72±86.90 U / g. After incubation at 65℃ for 6 h, it could still retain 95.17% of the initial enzyme activity. Novozym 435 retained 84.06% of the initial enzyme activity under the same conditions. The immobilized CALB was used for deacidification of high acid value rice bran oil and was reused for 50 batches. It could still retain 99.28% of the initial enzyme activity. Novozym 435 could only retain 86.25% of the initial enzyme activity after being reused for 50 batches. After the immobilized enzyme was sealed and stored at 4℃ for 60 days, the enzyme activity was maintained at about 97.16% of the initial enzyme activity, while Novozym 435 could only maintain 90.27% of the initial enzyme activity.

[0041] Example 4 In a 150 mL conical flask, 15 mL of deionized water and 60 mL of anhydrous ethanol were added and mixed evenly, then 7.5 mL of octyltriethoxysilane was added, and after stirring evenly, 4.2 g of ECR1030 resin was added. After ultrasonic dispersion treatment at room temperature for 1 h, the mixture was heated at 95 °C until the liquid was almost evaporated to obtain an alkyl functionalized product. Then, 10 mL of anhydrous ethanol was added to wash the alkyl functionalized product and evaporated again. The operation was repeated 3 times. Finally, the alkyl functionalized product was washed with anhydrous ethanol and deionized water, and the washed alkyl functionalized product was dried at 45 °C for 10 h to obtain a silanized resin (water contact angle of 116°). 0.2 g of dialdehyde cellulose was added to 10 mL of sodium hydroxide-urea aqueous solution, followed by 200 mL of deionized water and 1.4 g of silanized resin. After ultrasonic treatment for 30 min, the mixture was placed in a 45℃ constant temperature oscillator and shaken at 150 rpm for 4 h. The pH was then adjusted to neutral with hydrochloric acid solution, and the aldehyde functionalized product was washed with anhydrous ethanol and deionized water. The washed aldehyde functionalized product was dried at 45℃ for 10 h to obtain an alkyl-aldehyde modified resin (aldehyde content of 2.3 mmol / g). 14.5 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, and 1 g of alkyl-aldehyde modified resin was added. After being fixed in a constant temperature air bath oscillator at 40℃ and 130 rpm for 10 h, the immobilized product was separated by suction filtration, and the immobilized product was washed with phosphate buffer until there was no protein residue in the filtrate. The washed immobilized product was dried at 45℃ for 10 h.

[0042] The enzymatic properties of the immobilized CALB were characterized. The results showed that the activity of the immobilized CALB was 9567.93±228.57 U / g. After being incubated at 65℃ for 6 h, it could still retain 97.20% of the initial enzyme activity. Novozym 435 retained 84.06% of the initial enzyme activity under the same conditions. The immobilized CALB was used for deacidification of high acid value rice bran oil. After being reused for 50 batches, it could still retain 98.92% of the initial enzyme activity. After being reused for 50 batches, Novozym 435 could only retain 86.25% of the initial enzyme activity. After being sealed and stored at 4℃ for 60 days, the enzyme activity of the immobilized enzyme was maintained at about 95.69% of the initial enzyme activity, while Novozym 435 could only maintain 90.27% of the initial enzyme activity.

[0043] Example 5 In a 150 mL conical flask, 15 mL of deionized water and 30 mL of anhydrous ethanol were added and mixed evenly, then 2.25 mL of dodecyltriethoxysilane was added, and after stirring evenly, 1 g of ECR1030 resin was added. After ultrasonic dispersion treatment at room temperature for 0.5 h, the mixture was heated at 80 °C until the liquid was almost evaporated to obtain an alkyl functionalized product. Then, 10 mL of anhydrous ethanol was added to wash the alkyl functionalized product and evaporated again. The operation was repeated twice. Finally, the alkyl functionalized product was washed with anhydrous ethanol and deionized water, and the washed alkyl functionalized product was dried at 40 °C for 8 h to obtain a silanized resin (water contact angle of 115°). 0.2 g of dialdehyde cellulose was added to 10 mL of sodium hydroxide-urea aqueous solution, followed by 150 mL of deionized water and 1.2 g of silanized resin. After ultrasonic treatment for 10 min, the mixture was placed in a 40℃ constant temperature oscillator and oscillated at 120 rpm for 2 h. The pH was then adjusted to neutral with hydrochloric acid solution, and the aldehyde functionalized product was washed with anhydrous ethanol and deionized water. The washed aldehyde functionalized product was dried at 40℃ with air blast for 8 h to obtain an alkyl-aldehyde modified resin (aldehyde content of 2.7 mmol / g). 9.6 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, and 1 g of alkyl-aldehyde modified resin was added. After being fixed in a constant temperature air bath oscillator at 30℃ and 110 rpm for 6 h, the immobilized product was separated by suction filtration, and the immobilized product was washed with phosphate buffer until there was no protein residue in the filtrate. The washed immobilized product was dried at 40℃ with air blast for 8 h.

[0044] The enzymatic properties of the immobilized CALB were characterized. The results showed that the activity of the immobilized CALB was 8847.16±116.43 U / g. After incubation at 65°C for 6 h, it could still retain 98.13% of the initial enzyme activity. Novozym 435 retained 84.06% of the initial enzyme activity under the same conditions. The immobilized CALB was used for deacidification of high acid value rice bran oil. After repeated use for 50 batches, it could still retain 98.84% of the initial enzyme activity. After repeated use for 50 batches, Novozym 435 could only retain 86.25% of the initial enzyme activity. After the immobilized enzyme was sealed and stored at 4°C for 60 days, the enzyme activity was maintained at about 96.51% of the initial enzyme activity, while Novozym 435 could only maintain 90.27% of the initial enzyme activity.

[0045] Example 6 In a 150 mL conical flask, 15 mL of deionized water and 60 mL of anhydrous ethanol were added and mixed evenly, then 7.5 mL of dodecyltriethoxysilane was added, stirred evenly, and 4.2 g of ECR1030 resin was added. After ultrasonic dispersion treatment at room temperature for 1 h, the mixture was heated at 95 °C until the liquid was almost evaporated to obtain an alkyl functionalized product. Then, 10 mL of anhydrous ethanol was added to wash the alkyl functionalized product and evaporated again. The operation was repeated 3 times. Finally, the alkyl functionalized product was washed with anhydrous ethanol and deionized water, and the washed alkyl functionalized product was dried at 45 °C for 10 h to obtain a silanized resin (water contact angle of 116°). 0.2 g of dialdehyde cellulose was added to 10 mL of sodium hydroxide-urea aqueous solution, followed by 200 mL of deionized water and 1.4 g of silanized resin. After ultrasonic treatment for 30 min, the mixture was placed in a constant temperature oscillator at 45°C and oscillated at 150 rpm for 4 h. The pH was then adjusted to neutral with hydrochloric acid solution, and the aldehyde functionalized product was washed with anhydrous ethanol and deionized water. The washed aldehyde functionalized product was dried at 45°C with air blast for 10 h to obtain an alkyl-aldehyde modified resin (aldehyde content of 2.2 mmol / g). 14.5 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, and 1 g of alkyl-aldehyde modified resin was added. After being fixed in a constant temperature air bath oscillator at 40°C and 130 rpm for 10 h, the immobilized product was separated by suction filtration, and the immobilized product was washed with phosphate buffer until there was no protein residue in the filtrate. The washed immobilized product was dried at 45°C with air blast for 10 h.

[0046] The enzymatic properties of the immobilized CALB were characterized. The results showed that the activity of the immobilized CALB was 8211.54±135.50 U / g. After incubation at 65℃ for 6 h, it could still retain 96.46% of the initial enzyme activity. Novozym 435 retained 84.06% of the initial enzyme activity under the same conditions. The immobilized CALB was used for deacidification of high acid value rice bran oil. After repeated use for 50 batches, it could still retain 99.65% of the initial enzyme activity. After repeated use for 50 batches, Novozym 435 could only retain 86.25% of the initial enzyme activity. After the immobilized enzyme was sealed and stored at 4℃ for 60 days, the enzyme activity was maintained at about 95.94% of the initial enzyme activity, while Novozym 435 could only maintain 90.27% of the initial enzyme activity.

[0047] Comparative Example 1 In a 150 mL conical flask, 15 mL of deionized water and 30 mL of anhydrous ethanol were added and mixed evenly, then 2.25 mL of phenyltriethoxysilane was added, stirred evenly, and 1 g of ECR1030 resin was added. After ultrasonic dispersion treatment at room temperature for 0.5 h, the mixture was heated at 80 °C until the liquid was almost evaporated to obtain an alkyl functionalized product. Then, 10 mL of anhydrous ethanol was added to wash the alkyl functionalized product and evaporated again. The operation was repeated twice. Finally, the alkyl functionalized product was washed with anhydrous ethanol and deionized water, and the washed alkyl functionalized product was dried at 40 °C with air for 8 h to obtain a silanized resin (water contact angle of 114°). After 9.6 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, 1 g of silanized resin was added, and the mixture was fixed in a constant temperature air bath oscillator at 30°C and 110 rpm for 6 h. The immobilized product was separated by filtration, and the immobilized product was washed with phosphate buffer until no protein remained in the filtrate. The washed immobilized product was dried at 40°C for 8 h.

[0048] The enzymatic properties of the immobilized CALB were characterized, and the results showed that the activity of the immobilized CALB was 8063.74±272.80 U / g, and 70.15% of the initial enzyme activity was retained after 6 h of thermal incubation at 65°C; the immobilized CALB prepared was used for deacidification of high acid value rice bran oil, and 52.08% of the initial enzyme activity was retained after repeated use for 50 batches; after sealed storage at 4°C for 60 days, the enzyme activity was maintained at about 78.63% of the initial enzyme activity. Comparative Example 1 CALB was immobilized under the same silane coupling agent modified resin conditions as Example 1. Compared with Example 1, the stability of the immobilized enzyme prepared in Comparative Example 1 was poor.

[0049] Comparative Example 2 0.2 g of dialdehyde cellulose was added to 10 mL of sodium hydroxide-urea aqueous solution, followed by 150 mL of deionized water and 1.2 g of ECR1030 resin. After ultrasonic treatment for 10 min, the mixture was placed in a 40℃ constant temperature oscillator and oscillated at 120 rpm for 2 h. The pH was then adjusted to neutral with hydrochloric acid solution, and the aldehyde functionalized product was washed with anhydrous ethanol and deionized water. The washed aldehyde functionalized product was dried at 40℃ with air blast for 8 h to obtain aldehyde modified resin (aldehyde content of 2.2 mmol / g). 9.6 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, and 1 g of aldehyde modified resin was added. After being fixed in a constant temperature air bath oscillator at 30℃ and 110 rpm for 6 h, the immobilized product was separated by suction filtration, and the immobilized product was washed with phosphate buffer until there was no protein residue in the filtrate. The washed immobilized product was dried at 40℃ with air blast for 8 h.

[0050] The enzymatic properties of the immobilized CALB were characterized, and the results showed that the activity of the immobilized CALB was 7348.55±179.63 U / g, and 72.62% of the initial enzyme activity was retained after 6 h of thermal incubation at 65°C. The prepared immobilized CALB was used for deacidification of high acid value rice bran oil, and 70.87% of the initial enzyme activity was retained after repeated use for 50 batches. After sealed storage at 4°C for 60 days, the enzyme activity was maintained at about 81.98% of the initial enzyme activity. Comparative Example 2 immobilized CALB under the same DAC modified resin conditions as Example 1. Compared with Example 1, the stability of the immobilized enzyme prepared in Comparative Example 2 was poor.

[0051] Comparative Example 3 In a 150 mL conical flask, 15 mL of deionized water and 30 mL of anhydrous ethanol were added and mixed evenly, then 10 mL of hexadecyltriethoxysilane was added, stirred evenly, and 1 g of ECR1030 resin was added. After ultrasonic dispersion treatment at room temperature for 0.5 h, the mixture was heated at 80°C until the liquid was almost evaporated to obtain an alkyl functionalized product. Then, 10 mL of anhydrous ethanol was added to wash the alkyl functionalized product and evaporate it again. The operation was repeated twice. Finally, the alkyl functionalized product was washed with anhydrous ethanol and deionized water, and the washed alkyl functionalized product was dried at 40°C for 8 h to obtain a silanized resin (water contact angle of 129°). 0.2 g of dialdehyde cellulose was added to 10 mL of sodium hydroxide-urea aqueous solution, followed by 150 mL of deionized water and 1.2 g of silanized resin. After ultrasonic treatment for 10 min, the mixture was placed in a 40℃ constant temperature oscillator and oscillated at 120 rpm for 2 h. The pH was then adjusted to neutral with hydrochloric acid solution, and the aldehyde functionalized product was washed with anhydrous ethanol and deionized water. The washed aldehyde functionalized product was dried at 40℃ with air blast for 8 h to obtain an alkyl-aldehyde modified resin (aldehyde content of 2.6 mmol / g). 9.6 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, and 1 g of alkyl-aldehyde modified resin was added. After being fixed in a constant temperature air bath oscillator at 30℃ and 110 rpm for 6 h, the immobilized product was separated by suction filtration, and the immobilized product was washed with phosphate buffer until there was no protein residue in the filtrate. The washed immobilized product was dried at 40℃ with air blast for 8 h.

[0052] The enzymatic properties of the immobilized CALB were characterized, and the results showed that the activity of the immobilized CALB was 6246.27±163.10 U / g, and 44.52% of the initial enzyme activity was retained after 6 h of thermal incubation at 65°C; the immobilized CALB prepared was used for deacidification of high acid value rice bran oil, and 56.31% of the initial enzyme activity was retained after repeated use for 50 batches; after sealed storage at 4°C for 60 days, the enzyme activity was maintained at about 68.79% of the initial enzyme activity. Comparative Example 3 Compared with Example 1, the silane coupling agent over-modified the resin, resulting in reduced stability of the immobilized enzyme.

[0053] Comparative Example 4 In a 150 mL conical flask, 15 mL of deionized water and 30 mL of anhydrous ethanol were added and mixed evenly, then 2.25 mL of phenyltriethoxysilane was added, stirred evenly, and 1 g of ECR1030 resin was added. After ultrasonic dispersion treatment at room temperature for 0.5 h, the mixture was heated at 80 °C until the liquid was almost evaporated to obtain an alkyl functionalized product. Then, 10 mL of anhydrous ethanol was added to wash the alkyl functionalized product and evaporated again. The operation was repeated twice. Finally, the alkyl functionalized product was washed with anhydrous ethanol and deionized water, and the washed alkyl functionalized product was dried at 40 °C for 8 h to obtain a silanized resin (water contact angle of 114°). 3 g of dialdehyde cellulose was added to 10 mL of sodium hydroxide-urea aqueous solution, followed by 150 mL of deionized water and 1.2 g of silanized resin. After ultrasonic treatment for 10 min, the mixture was placed in a constant temperature oscillator at 40°C and oscillated at 120 rpm for 8 h. The pH was then adjusted to neutral with hydrochloric acid solution, and the aldehyde functionalized product was washed with anhydrous ethanol and deionized water. The washed aldehyde functionalized product was dried at 40°C with air blast for 8 h to obtain an alkyl-aldehyde modified resin (aldehyde content of 4.5 mmol / g). 9.6 mL of CALB enzyme solution was thoroughly mixed with an equal volume of 0.02 mol / L, pH 7 phosphate buffer, and 1 g of alkyl-aldehyde modified resin was added. After being fixed in a constant temperature air bath oscillator at 30°C and 110 rpm for 6 h, the immobilized product was separated by suction filtration, and the immobilized product was washed with phosphate buffer until there was no protein residue in the filtrate. The washed immobilized product was dried at 40°C with air blast for 8 h.

[0054] The enzymatic properties of the immobilized CALB were characterized, and the results showed that the activity of the immobilized CALB was 5203.79±202.13 U / g, and 66.35% of the initial enzyme activity was retained after 6 h of thermal incubation at 65°C; the immobilized CALB prepared was used for deacidification of high acid value rice bran oil, and 74.27% of the initial enzyme activity was retained after repeated use for 50 batches; after sealed storage at 4°C for 60 days, the enzyme activity was maintained at about 71.43% of the initial enzyme activity. Comparative Example 4 Compared with Example 1, DAC over-modified the resin, resulting in reduced stability of the immobilized enzyme.

[0055] The following table shows the type and amount of silane coupling agent used in each example and comparative example, the ratio of dialdehyde cellulose to resin, the activity, thermal stability, operational stability and storage stability of immobilized lipase:

[0056] Note: Thermal stability, operational stability and storage stability are all percentages of the initial enzyme activity. “-” means that the reagent is not added.

[0057] The above is only part of the embodiments of the present invention. Although some terms are used in the present invention, the possibility of using other terms is not excluded. These terms are used only to conveniently describe and explain the essence of the present invention. It is contrary to the spirit of the present invention to interpret them as any additional restrictions. The above is only to further illustrate the content of the present invention with examples for easier understanding, but it does not mean that the embodiments of the present invention are limited to this. Any technical extension or re-creation made according to the present invention is protected by the present invention.

Claims

1. A method for preparing highly stable immobilized Antarctic Candida lipase B, characterized in that: The following steps are involved: Adding a silane coupling agent to a mixed solution of deionized water and anhydrous ethanol and mixing uniformly to obtain a blended solution, adding a resin to the blended solution and mixing uniformly to obtain a suspension, heating the suspension until the liquid is completely evaporated, adding anhydrous ethanol to the product obtained after evaporation and evaporating again to obtain a silanized resin; The dialdehyde fiber is added to a sodium hydroxide-urea solution, and then deionized water and a silanized resin are added and mixed evenly, the mixed solution is subjected to a constant temperature oscillation treatment, and after the constant temperature oscillation treatment, the pH is adjusted to neutral to obtain a crude resin product, and the crude resin product is washed and dried to obtain an alkyl-aldehyde modified resin; The alkyl-aldehyde modified resin is added to a mixed solution of Antarctic Candida lipase B enzyme solution and a buffer solution and mixed evenly. The mixed solution is placed in a constant temperature environment to solidify the Antarctic Candida lipase B. After the solidification treatment, the immobilized enzyme is separated, and the immobilized enzyme is washed with a buffer solution until no protein is detected in the filtrate, and then dried to obtain the immobilized Antarctic Candida lipase B.

2. The method for preparing a highly stable immobilized Antarctic Candida lipase B according to claim 1, characterized in that: The volume ratio of deionized water to anhydrous ethanol in the mixed solution of deionized water and anhydrous ethanol is 1:2 to 1:4; The silane coupling agent is one of phenyltriethoxysilane, octyltriethoxysilane and dodecyltriethoxysilane, and the added amount of the silane coupling agent is 5% to 10% of the total volume of deionized water and anhydrous ethanol.

3. The method for preparing a highly stable immobilized Antarctic Candida lipase B according to claim 1, characterized in that: The resin is ECR1030 macroporous adsorption resin, and 0.02 g to 0.05 g of the resin is added to each milliliter of the blended solution.

4. The method for preparing a highly stable immobilized Antarctic Candida lipase B according to claim 1, characterized in that: The resin is added to the blended solution and mixed evenly to obtain a suspension, the suspension is heated until the liquid is completely evaporated, anhydrous ethanol is added to the product obtained after evaporation and then evaporated to obtain a silanized resin, specifically: Add resin to the blended solution, and obtain a suspension after ultrasonic dispersion treatment for 0.5 h to 1 h. Heat the suspension in an environment at a temperature of 80°C to 95°C until the liquid is completely evaporated. Add anhydrous ethanol to the product obtained after evaporation and evaporate it again, repeating 2 to 3 times. Wash with anhydrous ethanol and deionized water, and dry in an environment at a temperature of 40°C to 45°C for 8 h to 10 h to obtain a silanized resin.

5. The method for preparing a highly stable immobilized Antarctic Candida lipase B according to claim 1, characterized in that: The sodium hydroxide-urea aqueous solution is 7% sodium hydroxide and 12% urea aqueous solution, the mass ratio of dialdehyde cellulose to silylated resin is 1:6-1:7, and the amount of deionized water added is 15-20 times the volume of the sodium hydroxide-urea aqueous solution.

6. The method for preparing a highly stable immobilized Candida antarctica lipase B according to claim 1, characterized in that: The dialdehyde fiber is added to a sodium hydroxide-urea solution, and then deionized water and a silanized resin are added and mixed evenly. The mixed solution is subjected to a constant temperature oscillation treatment. After the constant temperature oscillation treatment, the pH is adjusted to neutral to obtain a crude resin product. The crude resin product is washed and dried to obtain an alkyl-aldehyde modified resin, specifically: Take dialdehyde cellulose and add it to the sodium hydroxide-urea aqueous solution and mix it evenly. After adding deionized water and silanized resin, ultrasonicate it for 10 min~30 min, and oscillate it at a constant temperature for 2 h~4 h at a temperature of 40℃~45℃ and a rotation speed of 120 rpm~150 rpm. After the constant temperature oscillation treatment, adjust the pH to neutral with a hydrochloric acid solution to obtain a crude resin product. Wash the crude resin product with anhydrous ethanol and deionized water, and dry it at a temperature of 40℃~45℃ for 8 h~10 h to obtain an alkyl-aldehyde modified resin.

7. The method for preparing a highly stable immobilized Candida antarctica lipase B according to claim 1, characterized in that: The buffer is a 0.02 mol / L phosphate buffer with a pH of 7, the volume ratio of the Antarctic Candida lipase B enzyme solution to the buffer is 1:1, and the added amount of the alkyl-aldehyde modified resin is 80 mg / g~120 mg / g, where mg / g represents the amount of the alkyl-aldehyde modified resin added per gram of enzyme protein in the Antarctic Candida lipase B enzyme solution.

8. The method for preparing a highly stable immobilized Candida antarctica lipase B according to claim 1, characterized in that: The alkyl-aldehyde modified resin is added to a mixed solution of an Antarctic Candida lipase B enzyme solution and a buffer solution and mixed evenly, the mixed solution is placed in a constant temperature environment to solidify the Antarctic Candida lipase B, the immobilized enzyme is separated after the solidification treatment, the immobilized enzyme is washed with a buffer solution until no protein is detected in the filtrate, and then dried to obtain the immobilized Antarctic Candida lipase B, specifically: After thoroughly mixing the Antarctic Candida lipase B enzyme solution and the buffer solution, alkyl-aldehyde modified resin was added and mixed evenly. The mixed solution was oscillated in a constant temperature gas bath for 6 h to 10 h at a temperature of 30 ° C to 40 ° C and a rotation speed of 110 rpm to 130 rpm to solidify the Antarctic Candida lipase B. After solidification, the immobilized enzyme was separated by filtration, and the immobilized enzyme was washed with buffer until no protein was detected in the filtrate. The washed immobilized enzyme was dried in an environment of a temperature of 40 ° C to 45 ° C for 8 h to 10 h to obtain immobilized Antarctic Candida lipase B.

9. The highly stable immobilized Antarctic Candida lipase B prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The amino group of Candida antarctica lipase B covalently binds to the aldehyde group on the surface of the alkyl-aldehyde modified resin. 10 . Use of the high-stability immobilized Candida antarctica lipase B according to claim 9 in the fields of functional oil preparation, high acid value oil deacidification and biofuel preparation.

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