Immobilized lipase as well as preparation method and application thereof

Abstract

The invention relates to the technical field of preparation of immobilized lipase, in particular to immobilized lipase as well as a preparation method and application thereof. The preparation method of the immobilized lipase comprises the following steps: grafting an amino compound on Fe3O4 to obtain an aminated Fe3O4 carrier; mixing the aminated Fe3O4 carrier, an aldehyde group-containing cross-linking agent and lipase, and carrying out cross-linking immobilization to obtain a cross-linked enzyme; and carrying out embedding treatment on the cross-linked enzyme by using an embedding medium to obtain the immobilized lipase. According to the method for preparing the immobilized lipase, the problems that the lipase immobilized by a direct adsorption method is easy to fall off and poor in reusability are solved, and meanwhile, the immobilized lipase is easy to separate by adopting a magnetic separation technology.

Description

Technical Field

[0001] The present invention relates to the technical field of preparing immobilized lipase, and particularly relates to an immobilized lipase, a preparation method thereof and uses thereof. Background Art

[0002] Lipase (EC 3.1.1.3) is a triacylglycerol hydrolase that can catalyze the hydrolysis of triacylglycerol into glycerol and fatty acids, and has a wide range of uses in the fields of food, pharmaceuticals, textiles, leather making and light industry, and occupies an important position in the world enzyme preparation market.

[0003] Free lipase has disadvantages such as being easily inactivated and not being easily recycled, which increases the cost of using enzymes. The technology of immobilized enzymes has advantages such as being easy to store and transport, reusable, and easy to separate and purify, and has received extensive attention from scientific and technological workers. At present, there are various methods for immobilizing lipase. Among them, the adsorption method has been widely studied because of its simplicity, high enzyme loading, and high immobilization yield of enzyme activity. However, the enzyme immobilized by the adsorption method has disadvantages such as being easily detached from the carrier and poor reusability; although the adsorption cross-linking method can significantly improve the stability of the immobilized enzyme, the reaction between the enzyme and the cross-linking agent is violent, and the enzyme protein is exposed in the organic phase solution, which is greatly affected by the solution environment and is easily caused by chemical modification or conformational change of the enzyme protein to inactivate, resulting in a low immobilization yield of enzyme activity.

[0004] In the prior art, Fe 3 O 4 is used as a carrier, and after modification, lipase is immobilized. However, the lipase immobilized by this method still has the disadvantages of low immobilization yield and poor reusability. Therefore, there is an urgent need for a method that can improve the immobilization yield of enzyme activity and the stability of enzyme activity of the immobilized enzyme. Summary of the Invention

[0005] To improve the stability of the immobilized lipase, the present invention provides a method for preparing an immobilized lipase. This method uses amino-functionalized Fe 3 O 4 as the "core" of the immobilized enzyme, and immobilizes lipase on the amino-functionalized Fe 3 O 4 by a cross-linking method, and then embeds it, which not only solves the problem of poor ability of Fe 3 O 4 and the modified material to directly cross-link and cause poor immobilized lipase ability, but also improves the stability of the immobilized lipase and the immobilization yield of enzyme activity.

[0006] A method for preparing an immobilized lipase, comprising the following steps: Graft an amino compound onto Fe 3 O 4 to obtain an amino-functionalized Fe 3 O 4 carrier; Mix the amino-functionalized Fe 3 O 4 carrier, aldehyde group-containing crosslinking agent and lipase for crosslinking immobilization to obtain a crosslinked enzyme; Use an embedding agent to embed the crosslinked enzyme to obtain the immobilized lipase.

[0007] In the present invention, Fe 3 O 4 is used as the magnetic core, and by grafting a multi-amino compound, an amino-functionalized Fe 3 O 4 carrier is constructed. Then, a crosslinked-embedded immobilized lipase with high stability and high enzyme activity recovery is prepared by combining the crosslinking method and the embedding method. The amino-functionalized Fe 3 O 4 carrier provided by the present invention has a large number of amino (-NH 2 ) groups on its surface, which is conducive to the acetalamine reaction with the aldehyde group (-CHO) of the crosslinking agent and is more conducive to the immobilization of lipase. At the same time, it also provides convenience for the separation of the immobilized enzyme; in addition, the crosslinked-embedded immobilized lipase prepared by the present invention not only overcomes the problems of easy detachment and poor reusability of directly adsorbing and immobilizing lipase, but also improves the activity of the immobilized lipase.

[0008] Furthermore, the amino compound is 3-aminopropyltriethoxysilane, γ-mercaptopropyltrimethoxysilane or amino-functionalized cellulose, and the molar ratio of Fe 3 O 4 to the amino compound is 1:1 to 10.

[0009] Furthermore, the grafting reaction is carried out at 40°C to 50°C and 380 r / min to 400 r / min for 4 h to 8 h.

[0010] Furthermore, the lipase is derived from porcine pancreas, Candida antarctica, Candida rugosa, Pseudomonas alcaligenes, Mucor miehei or Thermomyces lanuginosus, and the mass ratio of the amino-functionalized Fe 3 O 4 carrier to the lipase is 1:0.5 to 2.0.

[0011] Furthermore, the crosslinking immobilization is carried out at 20°C to 45°C and 180 r / min to 220 r / min for 2 h to 7 h under the action of an aldehyde group-containing crosslinking agent.

[0012] Furthermore, the crosslinking agent is glutaraldehyde, succinaldehyde, terephthalaldehyde or glyoxal, and the volume fraction of the crosslinking agent is 0.5% to 2.5%.

[0013] Furthermore, the embedding agent is selected from sodium alginate, carrageenan, polyvinyl alcohol, gelatin or gum arabic.

[0014] Further, the embedding is to mix the embedding agent with the cross-linked enzyme, and then drop the mixture into an aqueous solution of calcium chloride for curing to obtain the immobilized lipase.

[0015] Furthermore, the mass concentration of the aqueous calcium chloride solution is 0.5% - 2.5%, and the mass concentration of the embedding agent is 0.5% - 2.5%.

[0016] Further, the curing is carried out at room temperature for 0.5 h - 2.0 h.

[0017] The present invention also provides an immobilized lipase prepared by the above method.

[0018] The present invention also provides the use of the above immobilized lipase in catalyzing the hydrolysis of triglyceride.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses amino-functionalized Fe 3 O 4 as the "core" of the immobilized enzyme, and immobilizes lipase on amino-functionalized Fe 3 O 4 by the cross-linking method, and then embeds it. The prepared immobilized enzyme not only improves the stability of the immobilized lipase and the fixed yield of enzyme activity, can be reused multiple times, reduces costs, but also improves the activity of the immobilized lipase.

[0020] The fixed yield of enzyme activity of the immobilized lipase prepared by the cross-linking method of the present invention is 66.3%. After repeating the catalysis for 5 batches, the relative enzyme activity is 50.5%; the fixed yield of enzyme activity of the cross-linking-embedding method for immobilizing lipase is above 82.1%. After repeating the catalysis for 5 batches, the relative enzyme activity is 67.8%; compared with the cross-linking method, the fixed yield of enzyme activity of the immobilized lipase prepared by the cross-linking-embedding method adopted by the present invention is increased by at least 23.8%, and the enzyme activity stability is increased by 34.3%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 It is a schematic diagram of the preparation process of the immobilized lipase.

[0023] Figure 2 It is the reuse effect of the immobilized enzyme.

[0024] Figure 3 Effect of immobilized enzyme on the synthesis yield of biodiesel. Detailed implementation manners

[0025] The following is a detailed description of the specific implementation manners of the present invention. However, it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0026] Lipase is a triacylglycerol hydrolase that can catalyze the hydrolysis of triacylglycerol into glycerol and fatty acids, and has a wide range of uses in fields such as food, pharmaceuticals, textiles, leather making, and light industry.

[0027] Free lipase has disadvantages such as being easily inactivated and not being easily recycled, which increases the cost of using enzymes. The immobilized enzyme technology has advantages such as being easy to store and transport, reusable, and easy to separate and purify, and has received extensive attention from scientific and technological workers. At present, there are various methods for immobilizing lipase. Among them, the adsorption method has been widely studied because of its simplicity, high enzyme loading, and high immobilization yield of enzyme activity. However, the enzyme immobilized by the adsorption method has disadvantages such as being easily detached from the carrier and poor reusability; while the adsorption cross-linking method can significantly improve the stability of the immobilized enzyme, but there are disadvantages such as violent reaction between the enzyme and the cross-linking agent, and the enzyme protein is exposed in the organic phase solution and is greatly affected by the solution environment, which may cause chemical modification or conformational change of the enzyme protein and inactivation, resulting in a low immobilization yield of enzyme activity.

[0028] In the prior art, Fe 3 O 4 is used as a carrier, and after modification, lipase is immobilized. However, the lipase immobilized by this method has disadvantages such as low immobilization yield and poor reusability effect.

[0029] Based on this, the present invention provides a method for preparing immobilized lipase, including the following steps: Graft an amino compound onto Fe 3 O 4 to obtain an amino-functionalized Fe 3 O 4 carrier; Mix the amino-functionalized Fe 3 O 4 carrier with lipase for cross-linking immobilization to obtain a cross-linked enzyme; Use an embedding agent to embed the cross-linked enzyme to obtain immobilized lipase.

[0030] The present invention uses amino-functionalized Fe3 O 4 serves as the "core" of the immobilized enzyme. The lipase is immobilized on the aminated Fe through the cross-linking method 3 O 4 and then embedded. The prepared immobilized enzyme not only improves the stability of the immobilized lipase and the fixed yield of enzyme activity, can be reused multiple times, reduces costs, but also improves the activity of the immobilized lipase.

[0031] The lipase involved in the present invention can be derived from porcine pancreas ( Porcine pancreatic ), Candida antarctica ( C. Antarctica ), Candida rugosa ( C. rugosa ), Pseudomonas alcaligenes ( P. pseudoalcaligenes ), Mucor michei ( R. miehei ), Thermomyces lanuginosus ( T. lanuginosus ), etc. However, it is not limited to lipase.

[0032] The lipase used in the following examples of the present invention is all derived from porcine pancreas.

[0033] Example 1 A method for preparing immobilized lipase, as Figure 1 shown, includes the following steps: Preparation of immobilized enzyme carrier: Weigh 2.4 g of magnetic Fe 3 O 4 (MNPs) and add them to 800 mL of an ethanol aqueous solution (ethanol: water = 4:1, v / v), ultrasonically disperse for 20 min, slowly add 8 mL of ammonia water, continue ultrasonic treatment for 30 min, then add 8 mL of tetraethyl orthosilicate (TEOS), continuously stir with a magnetic stirrer for 2 h, finally dropwise add 8 mL of 3-aminopropyltriethoxysilane (APTMS), react at 45 °C and 390 rpm for 6 h, separate with a magnet, and wash 5 times with ethanol and deionized water, collect the solid, dry at 60 °C for 6 h, and then store it in a refrigerator at 4 °C for later use to obtain the aminated Fe 3 O 4 carrier.

[0034] Preparation of cross-linked and embedded immobilized enzyme: Weigh 10 g of sodium alginate and add it to 100 mL of distilled water, heat to dissolve, cool to room temperature, and prepare a 10% sodium alginate solution mother liquor for later use; Dissolve glutaraldehyde in a phosphate buffer solution with a pH of 7 and a concentration of 0.2 mol / L to prepare a cross-linking agent solution with a volume fraction of 1%; take 50 mL of the cross-linking agent solution and add 50 mg of aminated Fe 3 O 4A carrier and 50 mg of lipase were placed in a constant temperature incubator at 40 °C and 200 rpm for 5 h of immobilization. After the immobilization was completed, 7.5 mL of 10% sodium alginate solution was added to make the concentration of sodium alginate reach 1.5%. After mixing evenly, it was slowly dropped into 1.0% CaCl 2 solution with room temperature curing for 2.0 h. After the curing was completed, the immobilized enzyme was separated by a strong magnet, and the immobilized enzyme was washed 3 times with 0.2 mol / L phosphate buffer solution to obtain cross-linked-entrapped enzyme.

[0035] Example 2 A method for preparing immobilized lipase, comprising the following steps: Preparation of immobilized enzyme carrier: Weigh 2.4 g of magnetic Fe 3 O 4 (MNPs) and add them to 800 mL of ethanol aqueous solution (ethanol: water = 4:1, v / v), ultrasonically disperse for 20 min, slowly add 8 mL of ammonia water, continue ultrasonic for 30 min, then add 8 mL of tetraethyl orthosilicate (TEOS), continuously stir with a magnetic stirrer for 2 h, and finally dropwise add 8 mL of γ-mercaptopropyltrimethoxysilane, react at 40 °C and 390 rpm for 4 h, separate with a magnet, and wash 5 times with ethanol and deionized water, collect the solid, dry at 60 °C for 6 h, and then store in a refrigerator at 4 °C for later use to obtain amino-functionalized Fe 3 O 4 carrier.

[0036] Preparation of cross-linked-entrapped immobilized enzyme: Weigh 10 g of sodium alginate and add it to 100 mL of distilled water, heat to dissolve, cool to room temperature, and prepare a 10% sodium alginate solution mother liquor for later use; Dissolve glutaraldehyde in phosphate buffer solution with pH 7 and concentration 0.2 mol / L to prepare a cross-linking agent solution with a volume fraction of 1%; take 50 mL of the cross-linking agent solution, and add 50 mg of amino-functionalized Fe 3 O 4 carrier and 50 mg of lipase, place them in a constant temperature incubator at 40 °C and 200 rpm for 5 h of immobilization. After the immobilization is completed, add 7.5 mL of 10% sodium alginate solution to make the concentration of sodium alginate reach 1.5%. After mixing evenly, it is slowly dropped into 1.0% CaCl 2 solution, cure at room temperature for 2.5 h. After the curing is completed, the immobilized enzyme is separated by a strong magnet, and the immobilized enzyme is washed 3 times with 0.2 mol / L phosphate buffer solution to obtain cross-linked-entrapped enzyme.

[0037] Example 3 A method for preparing immobilized lipase, comprising the following steps: Preparation of immobilized enzyme carrier: Weigh 2.4 g of magnetic Fe 3 O 4 (MNPs) and add them to 800 mL of an ethanol-water solution (ethanol: water = 4:1, v / v). Ultrasonically disperse for 20 min, slowly add 8 mL of ammonia water, continue ultrasonic treatment for 30 min, then add 8 mL of tetraethyl orthosilicate (TEOS), continuously stir with a magnetic stirrer for 2 h, and finally dropwise add 8 mL of amino-functionalized cellulose. React at 50 °C and 400 rpm for 8 h, separate with a magnet, and wash 5 times with ethanol and deionized water. Collect the solid, dry at 60 °C for 6 h, and then store in a refrigerator at 4 °C for later use to obtain the amino-functionalized Fe 3 O 4 carrier.

[0038] Preparation of crosslinked-entrapped immobilized enzyme: Weigh 10 g of sodium alginate and add it to 100 mL of distilled water. Heat to dissolve and cool to room temperature to prepare a 10% sodium alginate solution mother liquor for later use; Dissolve glutaraldehyde in a phosphate buffer solution with a pH of 7 and a concentration of 0.2 mol / L to prepare a crosslinking agent solution with a volume fraction of 1%. Take 50 mL of the crosslinking agent solution, and add 50 mg of the amino-functionalized Fe 3 O 4 carrier and 50 mg of lipase respectively. Place them in a constant temperature incubator at 40 °C and a rotation speed of 200 rpm for immobilization for 5 h. After the immobilization is completed, add 7.5 mL of the 10% sodium alginate solution to make the concentration of sodium alginate reach 1.5%. Mix well, and then slowly drip it into a 1.0% CaCl 2 solution. Cure at room temperature for 1.5 h. After the curing is completed, separate the immobilized enzyme with a strong magnet. Wash the immobilized enzyme 3 times with a 0.2 mol / L phosphate buffer solution to obtain the crosslinked-entrapped enzyme.

[0039] Example 4 A method for preparing immobilized lipase, comprising the following steps: Preparation of immobilized enzyme carrier: Weigh 2.4 g of magnetic Fe 3 O 4Magnetic nanoparticles (MNPs) were added to 800 mL of an ethanol-water solution (ethanol: water = 4:1, v / v), and ultrasonically dispersed for 20 min. Then, 8 mL of ammonia water was slowly added, and ultrasonic treatment was continued for 30 min. Next, 8 mL of tetraethyl orthosilicate (TEOS) was added, and the mixture was continuously stirred with a magnetic stirrer for 2 h. Finally, 8 mL of 3-aminopropyltriethoxysilane (APTMS) was added dropwise, and the reaction was carried out at 45 °C and 390 rpm for 6 h. The product was separated by a magnet, washed 5 times with ethanol and deionized water, the solid was collected, dried at 60 °C for 6 h, and then stored in a refrigerator at 4 °C for later use to obtain the amino-functionalized Fe 3 O 4 carrier.

[0040] Preparation of crosslinked-entrapped immobilized enzyme: Weigh 10 g of sodium alginate and 5 mL of polyvinyl alcohol, add them to 100 mL of distilled water, heat to dissolve, and cool to room temperature to prepare a stock solution of sodium alginate-polyvinyl alcohol solution for later use; Glutaraldehyde was dissolved in a phosphate buffer with a pH of 7 and a concentration of 0.2 mol / L to prepare a crosslinking agent solution with a volume fraction of 1%. Take 50 mL of the crosslinking agent solution, add 50 mg of the amino-functionalized Fe 3 O 4 carrier and 50 mg of lipase, place them in a constant temperature incubator at 40 °C and 200 rpm for immobilization for 5 h. After immobilization, add 7.5 mL of the sodium alginate-polyvinyl alcohol solution to make the mass concentration of sodium alginate reach 1.5%, mix well, and then slowly drip it into a 1.0% boric acid solution of CaCl 2 to cure at room temperature for 2.0 h. After curing, the immobilized enzyme was separated by a strong magnet, and the immobilized enzyme was washed 3 times with 0.2 mol / L phosphate buffer to obtain the crosslinked-entrapped enzyme.

[0041] Example 5 A method for preparing immobilized lipase, comprising the following steps: Preparation of immobilized enzyme carrier: Weigh 2.4 g of magnetic Fe 3 O 4 Magnetic nanoparticles (MNPs) were added to 800 mL of an ethanol-water solution (ethanol: water = 4:1, v / v), and ultrasonically dispersed for 20 min. Then, 8 mL of ammonia water was slowly added, and ultrasonic treatment was continued for 30 min. Next, 8 mL of tetraethyl orthosilicate (TEOS) was added, and the mixture was continuously stirred with a magnetic stirrer for 2 h. Finally, 8 mL of 3-aminopropyltriethoxysilane (APTMS) was added dropwise, and the reaction was carried out at 45 °C and 390 rpm for 6 h. The product was separated by a magnet, washed 5 times with ethanol and deionized water, the solid was collected, dried at 60 °C for 6 h, and then stored in a refrigerator at 4 °C for later use to obtain the amino-functionalized Fe 3 O 4Carrier.

[0042] Preparation of crosslinked-embedded immobilized enzyme: Weigh 10 g of sodium alginate and add it to 100 mL of distilled water, heat to dissolve, cool to room temperature, and prepare a 10% sodium alginate solution mother liquor for standby; Dissolve glutaraldehyde in a phosphate buffer solution with a pH of 7 and a concentration of 0.2 mol / L to prepare a crosslinking agent solution with a volume fraction of 1%; Take 50 mL of the crosslinking agent solution, and add 50 mg of amino-functionalized Fe 3 O 4 carrier and 50 mg of lipase, place them in a constant temperature incubator at 20 °C and a rotation speed of 180 rpm for 2 h of immobilization. After the immobilization is completed, add 7.5 mL of 10% sodium alginate solution to make the mass concentration of sodium alginate reach 1.5%, mix well, and then slowly drip it into 1.0% CaCl 2 solution, solidify at room temperature for 2.0 h. After the solidification is completed, separate the immobilized enzyme by a strong magnet, and wash the immobilized enzyme 3 times with 0.2 mol / L phosphate buffer solution to obtain crosslinked-embedded enzyme.

[0043] Example 6 A method for preparing immobilized lipase, comprising the following steps: Preparation of immobilized enzyme carrier: Weigh 2.4 g of magnetic Fe 3 O 4 (MNPs) and add it to 800 mL of ethanol aqueous solution (ethanol: water = 4:1, v / v), ultrasonically disperse for 20 min, slowly add 8 mL of ammonia water, continue ultrasonic for 30 min, then add 8 mL of tetraethyl orthosilicate (TEOS), continuously stir with a magnetic stirrer for 2 h, and finally dropwise add 8 mL of 3-aminopropyltriethoxysilane (APTMS), react at 45 °C and 390 rpm for 6 h, separate with a magnet, and wash 5 times with ethanol and deionized water, collect the solid, dry at 60 °C for 6 h, and then store it in a refrigerator at 4 °C for standby to prepare amino-functionalized Fe 3 O 4 carrier.

[0044] Preparation of crosslinked-embedded immobilized enzyme: Weigh 10 g of sodium alginate and add it to 100 mL of distilled water, heat to dissolve, cool to room temperature, and prepare a 10% sodium alginate solution mother liquor for standby; Dissolve glutaraldehyde in a phosphate buffer solution with a pH of 7 and a concentration of 0.2 mol / L to prepare a crosslinking agent solution with a volume fraction of 1%; Take 50 mL of the crosslinking agent solution, and add 50 mg of amino-functionalized Fe 3 O 4The carrier and 50 mg of lipase were placed in a constant temperature incubator at 30 °C with a rotation speed of 220 rpm and fixed for 7 h. After the fixation, 7.5 mL of 10% sodium alginate solution was added to make the mass concentration of sodium alginate reach 1.5%. After mixing evenly, it was slowly dropped into 1.0% CaCl 2 solution at room temperature for 2.0 h of solidification. After the solidification, the immobilized enzyme was separated by a strong magnet, and the immobilized enzyme was washed 3 times with 0.2 mol / L phosphate buffer solution to obtain the cross-linked and entrapped enzyme.

[0045] Comparative Example 1 A method for preparing immobilized lipase includes the following steps: Preparation of immobilized enzyme carrier: Weigh 2.4 g of magnetic Fe 3 O 4 (MNPs) and add them to 800 mL of ethanol aqueous solution (ethanol: water = 4:1, v / v), ultrasonically disperse for 20 min, slowly add 8 mL of ammonia water, continue ultrasonic treatment for 30 min, then add 8 mL of tetraethyl orthosilicate (TEOS), continuously stir with a magnetic stirrer for 2 h, and finally dropwise add 8 mL of 3-aminopropyltriethoxysilane (APTMS), react at 45 °C and 390 rpm for 6 h, separate with a magnet, and wash 5 times with ethanol and deionized water, collect the solid, dry at 60 °C for 6 h, and then store it in a refrigerator at 4 °C for later use to obtain the amino-functionalized Fe 3 O 4 carrier.

[0046] Preparation of cross-linked enzyme: Glutaraldehyde was dissolved in a phosphate buffer solution with a pH of 6 and a concentration of 0.2 mol / L to prepare a cross-linking agent solution with a volume fraction of 1%. Take 50 mL of the solution and add 50 mg of amino-functionalized Fe 3 O 4 carrier and 50 mg of lipase, place them in a constant temperature incubator at 45 °C with a rotation speed of 200 rpm and fix for 5 h. After the fixation, the immobilized enzyme was separated by a strong magnet, and the immobilized enzyme was washed 3 times with 0.2 mol / L phosphate buffer solution to remove the cross-linking agent and free lipase to obtain the cross-linked enzyme.

[0047] Comparative Example 2 A method for preparing immobilized lipase includes the following steps: Preparation of immobilized enzyme carrier: Weigh 2.4 g of magnetic Fe 3 O 4(MNPs) were added to 800 mL of an ethanol aqueous solution (ethanol: water = 4:1, v / v), ultrasonically dispersed for 20 min, 8 mL of ammonia water was slowly added, and ultrasonic treatment was continued for 30 min. Then, 8 mL of tetraethyl orthosilicate (TEOS) was added, and the mixture was continuously stirred with a magnetic stirrer for 2 h. Finally, 8 mL of 3-aminopropyltriethoxysilane (APTMS) was added dropwise, and the reaction was carried out at 45 °C and 390 rpm for 6 h. The product was separated by a magnet, washed 5 times with ethanol and deionized water, the solid was collected, dried at 60 °C for 6 h, and then stored in a refrigerator at 4 °C for later use to obtain the amino-functionalized Fe 3 O 4 carrier.

[0048] Preparation of entrapped immobilized enzyme: Weigh 10 g of sodium alginate and add it to 100 mL of distilled water, heat to dissolve, and cool to room temperature to prepare a 10% sodium alginate solution mother liquor; Take 7.5 mL of the 10% sodium alginate solution, add 50 mg of the amino-functionalized Fe 3 O 4 carrier and 50 mg of lipase, mix well, and then slowly drop it into a 1.0% CaCl 2 solution with a syringe, and solidify at room temperature for 2.0 h. After the solidification is completed, the immobilized enzyme is separated by a strong magnet, and the immobilized enzyme is washed 3 times with 0.2 mol / L phosphate buffer to obtain the entrapped enzyme.

[0049] Comparative Example 3 A method for preparing immobilized lipase, comprising the following steps: Preparation of immobilized carrier: Weigh 2.4 g of magnetic Fe 3 O 4 (MNPs) were added to 800 mL of an ethanol aqueous solution (ethanol: water = 4:1, v / v), ultrasonically dispersed for 20 min, separated by a magnet, and washed 5 times with ethanol and deionized water, the solid was collected, dried at 60 °C for 6 h, and then stored in a refrigerator at 4 °C for later use to obtain Fe 3 O 4 carrier.

[0050] Preparation of immobilized enzyme: Take 50 mL of a phosphate buffer with a pH of 6 and a concentration of 0.2 mol / L, add 50 mg of Fe 3 O 4 carrier and 50 mg of lipase, place them in a constant temperature incubator at 45 °C and a rotation speed of 200 rpm for 5 h. After the immobilization is completed, the immobilized enzyme is separated by a strong magnet, and the immobilized enzyme is washed 3 times with 0.2 mol / L phosphate buffer to remove free lipase to obtain immobilized lipase.

[0051] 6. Determination of lipase activity 7. Test Method (1) Preparation of reaction solution: Solution A (substrate): Take 333 mg of p-nitrophenyl laurate and make up the volume to 100 mL with isopropanol solution to prepare a substrate solution with a concentration of 3.33 mg / mL.

[0052] Solution B (buffer solution): Prepare a 0.1 M phosphate buffer solution with a pH value of 8 and add 0.4% Triton X-100 to it.

[0053] (8) Determination of enzyme activity: Take 0.1 mL of Solution A and 1.5 mL of Solution B, mix them evenly, preheat at 40 °C for 5 min, add 0.1 mL of free enzyme (10 mg of immobilized enzyme), react at 40 °C for 6 min, then quickly add 2 mL of ethanol and terminate the reaction in an ice bath. Measure the absorbance at 410 nm with a UV spectrophotometer; the blank control group is the reaction of an equal amount of free enzyme (immobilized enzyme) that has been inactivated by boiling. The unit of lipase activity (U) is defined as: the amount of enzyme required for 1 mL of free enzyme (1 mg of immobilized enzyme) to catalyze the hydrolysis of the substrate to produce 1 umoL of p-nitrophenol in 1 min under the conditions of 40 °C and pH 8. Enzyme activity calculation formula:

[0054] 。

[0055] In the formula: X is the lipase activity (U), C is the pNP concentration (umol / L), V 1 is the final volume of the reaction solution (mL), T is the reaction time (min), V 2 is the volume of the enzyme solution used (mL).

[0056] Fixed recovery rate of lipase activity: The ratio of the total immobilized enzyme activity to the total free enzyme activity.

[0057] Table 1 Determination of the fixed recovery rate of the lipase activity of the immobilized enzymes prepared in different examples and comparative examples II. Reusability of the immobilized enzyme 1. Test method: Mix 0.1 mL of Solution A and 1.5 mL of Solution B above, preheat at 40 °C for 5 min, and add 0.1 mL of free enzyme with a concentration of 1 mg / mL and 10 mg of the immobilized enzyme prepared in Example 1 (since the properties and effects of the immobilized enzymes prepared in Examples 1 - 6 are basically the same, only Example 1 is taken as an example for illustration here). React at 40 °C for 6 min, then quickly add 2 mL of ethanol and terminate the reaction in an ice bath. Measure the absorbance at 410 nm with a UV - visible spectrophotometer; the blank control group is the reaction of an equal amount of free enzyme or immobilized enzyme inactivated by boiling. After the catalysis, separate the immobilized enzyme with a strong magnetic magnet and add it to the fresh mixed solution of Solution A and Solution B to continue catalysis, and so on. Taking the first - catalysis enzyme activity as 100%, obtain the relative retained enzyme activities of each batch.

[0058] 2. Test results: As Figure 2 shown, after continuous catalysis for 5 times, the relative enzyme activity retention of the cross - linked enzyme provided in Comparative Example 1 is 50.5%; the relative enzyme activity retentions of the entrapped enzymes prepared in Comparative Example 2 and Comparative Example 3 are 64.2% and 63.5% respectively, and the relative enzyme activity retention of the cross - linked - entrapped enzyme prepared in Example 1 is 67.8%; compared with the cross - linked enzyme in Comparative Example 1, the entrapped enzyme significantly improves the reusability stability, and the enzyme activity stability of the cross - linked - entrapped enzyme is even increased by 34.3%.

[0059] III. Application of the immobilized enzyme in biodiesel synthesis 1. Test method Take 200 mg of the immobilized enzymes prepared in Example 1 and Comparative Example 1 (since the properties and effects of the immobilized enzymes prepared in Examples 1 - 6 are basically the same, only Example 1 is taken as an example for illustration here), and add them to the mixed reaction solution containing 1.5 mL of methanol, 1 mL of soybean oil, and 0.1 mL of deionized water respectively. After reacting at 40 °C for 2 h, add 1.5 mL of methanol, react until 4 h, then add 1 mL of methanol, and continue to react until 14 h. Every 2 h, use gas chromatography to measure the content of fatty acid methyl esters in the reaction solution and calculate the biodiesel yield.

[0060] 2. Test results The results are as Figure 3 shown. Although the catalytic rate of the cross - linked enzyme prepared in Comparative Example 1 per unit mass is higher than that of the cross - linked - entrapped enzyme, the final biodiesel yields are all about 75.5%. Considering that the reusability and immobilization yield of the cross - linked - entrapped enzyme are significantly higher than those of the cross - linked enzyme, the cross - linked - entrapped enzyme provided by the present invention has greater market application value.

[0061] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A method for preparing immobilized lipase, characterized in that: The following steps are involved: Grafting amino compounds onto Fe3O4 to obtain an amination Fe3O4 carrier; The aminated Fe3O4 carrier, the aldehyde-containing cross-linking agent and the lipase are mixed for cross-linking and fixation to obtain a cross-linked enzyme; The cross-linking enzyme is embedded in an embedding agent to obtain the immobilized lipase.

2. The method for preparing immobilized lipase according to claim 1, characterized in that: The amino compound is 3-aminopropyltriethoxysilane, γ-mercaptopropyltrimethoxysilane or amino cellulose, and the molar ratio of Fe3O4 to the amino compound is 1:1-10.

3. The method for preparing immobilized lipase according to claim 2, characterized in that: The grafting is carried out at 40°C to 50°C and 380 r / min to 400 r / min for 4 h to 8 h.

4. The method for preparing immobilized lipase according to claim 1, characterized in that: The lipase is derived from porcine pancreas, Candida antarctica, Candida rugosa, Pseudomonas alcaligenes, Rhizomucor manhei or Thermomyces lanuginosus, and the mass ratio of the aminated Fe3O4 carrier to the lipase is 1:0.5-2.

0.

5. The method for preparing immobilized lipase according to claim 4, characterized in that: The cross-linking fixation is performed under the action of an aldehyde-containing cross-linking agent at 20°C to 45°C and 180 r / min to 220 r / min for 2 h to 7 h.

6. The method for preparing immobilized lipase according to claim 1, characterized in that: The embedding agent is selected from sodium alginate, carrageenan, polyvinyl alcohol, gelatin or gum arabic.

7. The method for preparing immobilized lipase according to claim 6, characterized in that: The embedding is to mix the embedding agent with the cross-linking enzyme, and then drop the mixed solution into a calcium chloride aqueous solution for solidification to obtain the immobilized lipase.

8. The method for preparing immobilized lipase according to claim 7, characterized in that: The mass concentration of the aqueous solution of calcium chloride is 0.5% to 2.5%, and the curing is carried out at room temperature for 0.5 h to 2.0 h.

9. An immobilized lipase prepared according to the method according to any one of claims 1 to 8.

10. Use of the immobilized lipase according to claim 9 in catalyzing the hydrolysis of triacylglycerides.

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