Phospholipase-lipase complex solidified enzyme, its preparation and use in diacylglycerol production

Abstract

This invention belongs to the field of immobilized enzymes, specifically relating to a phospholipase-lipase composite immobilized enzyme, its preparation, and its application in diglyceride preparation. The preparation method involves: modifying a magnetic carrier with a modifier of formula 1() to obtain a modified carrier; then mixing the modified carrier and phospholipase for a first-stage immobilization to obtain a phospholipase-immobilized material; adding an auxiliary agent to the first-stage immobilization system for aging treatment; then adding a histidine-modified lipase and performing a second-stage immobilization to obtain the aforementioned phospholipase-lipase composite immobilized enzyme; the auxiliary agent is a water-soluble salt of nickel. Research in this invention shows that the combination of the aforementioned one-pot, two-stage immobilization method and means can achieve synergy, enabling the composite immobilization of phospholipase and lipase, avoiding problems such as site competition and active site shielding inherent in composite immobilization, thereby achieving synergistic effects between the phospholipase and lipase in the same carrier, enhancing the conversion rate and selectivity of diglycerides.

Description

Technical Field

[0001] This invention belongs to the field of enzymes, specifically to the field of phospholipase-lipase immobilization enzymes. Background Technology

[0002] Diacylglycerols are structural lipids in which one fatty acid of a triglyceride is replaced by a hydroxyl group. They are functional oils prepared from edible oils through modification. Related studies have shown that they can slow weight gain, inhibit fat accumulation, and lower blood lipids, making diglycerides a popular and safe functional oil.

[0003] Currently, the main methods for preparing diglycerides are chemical methods and enzymatic methods. However, existing diglyceride preparation technologies still have many shortcomings. For example, chemical methods typically require high temperature, high pressure, and strong acid or strong base catalysis conditions for the reaction. The preparation process can lead to side reactions such as oil oxidation and polymerization, affecting product quality. In addition, the product separation process is complex, generating large amounts of wastewater and resulting in high energy consumption. Compared with chemical methods, enzymatic methods for preparing diglycerides are milder, and the active ingredients in the resulting product are better preserved.

[0004] Traditional enzymatic methods use free lipases and phospholipases to prepare diglycerides. However, enzymes have poor stability in the reaction system, and free enzymes are difficult to reuse, resulting in high costs. Moreover, the catalytic efficiency of a single enzyme is limited, and its substrate specificity is high, so the yield of diglycerides is generally low, usually only reaching 30% to 40%.

[0005] In addition, to achieve cyclic enzyme catalysis, existing technologies have also reported some methods for immobilizing phospholipases or lipases. For example, patent document CN102199634A discloses a method for preparing functional oils rich in diglycerides, which includes the following steps: (1) immobilization of phospholipase Lecitase Ultra; (2) catalysis of glycerolization of vegetable oil by the immobilized enzyme; and (3) product separation. In addition, patent document US6162623A discloses a method for preparing diglycerides by immobilizing lipase. Most existing technologies require the physical interaction between the carrier and the enzyme to achieve enzyme immobilization. However, these methods have low mass transfer efficiency, unreasonable pore structure, and hindered contact between the enzyme and the substrate, thus leading to a decrease in catalytic activity.

[0006] To address the shortcomings of existing adsorption and immobilization technologies, some covalent modification schemes have been proposed. For example, patent document BRPI2226032A2 discloses the preparation of a lipase immobilization enzyme, which modifies an amino group on a support and then crosslinks it with glutaraldehyde and the enzyme.

[0007] In summary, although existing technologies disclose some methods for immobilizing lipases or phospholipases, there is still a lack of immobilization methods for lipase-phospholipase complex enzymes. Summary of the Invention

[0008] To address the problems existing in the prior art, the primary objective of this invention is to provide a method for preparing a phospholipase-lipase composite solidified enzyme, aiming to obtain a composite solidified enzyme that simultaneously solidifies phospholipase and lipase.

[0009] The second objective of this invention is to provide a phospholipase-lipase complex immobilized enzyme prepared by the aforementioned method.

[0010] A third objective of this invention is to provide the application of the aforementioned phospholipase-lipase complex immobilizing enzyme in the preparation of diglycerides.

[0011] Existing technologies rarely offer solutions for the simultaneous and efficient covalent immobilization of phospholipase and lipase on the same carrier. The main challenge lies in the fact that phospholipase and lipase have different structures, leading to site competition during co-immobilization and potential issues such as active site shielding, making it difficult to achieve synergistic performance of the two enzymes. To address the difficulty of co-immobilizing phospholipase and lipase, this invention, after in-depth research, provides the following improved solution:

[0012] A method for preparing a phospholipase-lipase composite solidified enzyme involves modifying a magnetic carrier with a modifier of Formula 1 to obtain a modified carrier; then mixing the modified carrier and phospholipase for a first-stage solidification process to obtain a phospholipase solidified material.

[0013] Add an auxiliary agent to the first stage of the solidification reaction system for aging treatment, then add a histidine-modified lipase and carry out the second stage of solidification to obtain the phospholipase-lipase complex solidified enzyme.

[0014] Formula 1;

[0015] In Formula 1, R1~R3 ​​are individually H, C1~C6 alkyl or C1~C6 alkoxy; X is a C1~C6 alkylene.

[0016] The additive is a water-soluble salt of nickel.

[0017] This invention innovatively modifies the magnetic carrier using Formula 1 beforehand, and then pre-cures it with phospholipase in the first stage, followed by aging with a nickel-based auxiliaries, and finally curing it with histidine-modified lipase in the second stage. Research in this invention shows that the combination of the aforementioned one-pot, two-stage curing method and means can achieve synergistic effects, enabling the co-curing of phospholipase and lipase. This avoids problems such as site competition and active site shielding inherent in co-curing, thereby achieving synergistic effects between phospholipase and lipase within the same carrier, enhancing the conversion rate and selectivity of diglycerides.

[0018] In this invention, the magnetic carrier can be any magnetic component, such as a silicon dioxide@iron oxide composite material. The magnetic carrier can be prepared using known methods.

[0019] In this invention, the magnetic carrier is pre-modified using Formula 1 and then combined with subsequent processes. This not only facilitates the efficient curing of phospholipase, but also avoids problems such as activity competition and peeling that exist in the subsequent lipase curing process.

[0020] In this invention, in Formula 1, R1~R3 ​​are C1~C3 alkoxy groups; X is a C2~C4 alkylene group;

[0021] The weight ratio of the magnetic carrier and the modifier of Formula 1 is 1:0.15~0.35, and can be further 1:0.2~0.3.

[0022] The modification temperature is 80℃~120℃, and can be further modified to 90~110℃.

[0023] The modification time is 6h~30h, and can be further extended to 15~25h.

[0024] The solvent used in the modification process includes at least one of anhydrous toluene and anhydrous xylene (e.g., ortho / meta / para-xylene).

[0025] In this invention, the modified carrier and lipase solution can be mixed for the first stage of solidification treatment.

[0026] In this invention, the phospholipase includes at least one of phospholipase A1, phospholipase A2, phospholipase C, and phospholipase D.

[0027] In this invention, the curing temperature of the first stage is 1℃~25℃, and can be further 4±2℃.

[0028] The first stage of curing takes 3 to 5 hours.

[0029] In this invention, the pH of the first curing stage can be 8±0.5.

[0030] The lipase loading in the phospholipase-cured material is 8000~12000U / g.

[0031] In this invention, after the first stage of curing, the aforementioned additives are directly added to the solution system and aged. This allows nickel to be infiltrated into the phospholipase-cured material based on concentration difference, and then chelated with lipase for the second stage of curing.

[0032] In this invention, the additives include at least one of nickel nitrate hexahydrate and nickel chloride hexahydrate.

[0033] The weight ratio of phospholipase curing material and additives is 1:0.05~0.3.

[0034] The aging process is carried out at temperatures ranging from 20°C to 40°C.

[0035] The aging process takes 1 to 4 hours.

[0036] In this invention, the lipase is at least one of the following: Thermomyces lanuginosus lipase, Thermomyces lanuginosus lipase variant, Candida antarctica lipase A (CALA), Candida antarctica lipase B (CALB), Hyphozyma lipase, Rhizomucor miehei lipase, P. alcaligenes lipase, P. pseudoalcaligenes lipase, and P. cepacia lipase.

[0037] In this invention, the curing temperature of the second stage is 20℃~30℃.

[0038] The curing time for the second stage is 2 to 10 hours; it can be further extended to 6 to 8 hours.

[0039] In this invention, the pH of the second curing stage can be 7.5 ± 0.5.

[0040] The lipase loading in the phospholipase-lipase complex immobilized enzyme is 10,000~60,000 U / g; it can be further increased to 15,000~50,000 U / g.

[0041] In this invention, the enzyme activity ratio of phospholipase and lipase in the phospholipase-lipase complex immobilized enzyme is 1:1~5.

[0042] The present invention also provides a phospholipase-lipase composite immobilized enzyme prepared by the above preparation method.

[0043] The present invention also provides the application of the phospholipase-lipase complex solidified enzyme prepared by the above preparation method, using the lipase-lipase complex solidified enzyme as an enzyme catalyst and oil as a substrate to carry out an enzyme-catalyzed reaction to obtain diglycerides.

[0044] The oil is selected from at least one of the following: rice bran oil, palm oil, palm kernel oil, cottonseed oil, safflower seed oil, perilla seed oil, palm fruit oil, coconut oil, cocoa soybean oil, soybean oil, rapeseed oil, peanut oil, corn oil, sunflower seed oil, camellia oil, walnut oil, rice bran oil, wheat germ oil, tea seed oil, tallow tree seed oil, almond kernel oil, apricot kernel oil, tung oil seed oil, rubber seed oil, sesame oil, castor oil, evening primrose seed oil, and algae oil.

[0045] In this invention, the conditions for the enzyme-catalyzed reaction can be known. For example, the temperature of the enzyme-catalyzed reaction process can be 40~60℃, the pH can be 7~8, and the reaction time can be reasonably adjusted as needed, for example, it can be 1~20h, and further, it can be 4~12h.

[0046] Beneficial effects

[0047] This invention innovatively provides a method for successfully achieving simultaneous immobilization of phospholipase and lipase. Based on the control of the method described, the problems existing in simultaneous immobilization can be solved, and the synergistic effect of immobilized phospholipase and lipase in the preparation of diglyceride catalysis can be achieved.

[0048] By employing a composite immobilized enzyme system and optimized reaction conditions, the yield of diglycerides can be increased to 60%–70%, a significant improvement compared to traditional methods, greatly shortening the production cycle and increasing production efficiency. The immobilized composite enzyme exhibits excellent reusability, allowing for over 30 reuses with an activity retention rate exceeding 80% after each use, drastically reducing enzyme usage and lowering production costs. This invention avoids the use of strong acids and alkalis and the generation of large amounts of wastewater associated with traditional chemical methods, making the entire production process greener and more environmentally friendly, aligning with the concept of sustainable development. Furthermore, the reusability of the immobilized enzyme reduces enzyme waste, further minimizing environmental impact. Attached Figure Description

[0049] Figure 1 This is a diagram of the immobilized phospholipase-lipase complex enzyme prepared in Example 1. Detailed Implementation

[0050] The present invention provides an optional method for preparing a phospholipase-lipase complex immobilizing enzyme, the steps of which include, for example:

[0051] S1: Preparation of mesoporous silica-magnetic nanoparticle composite support:

[0052] First, mesoporous silica was synthesized by sol-gel method, then Fe3O4 magnetic nanoparticles were loaded on its surface by co-precipitation method, and then the surface of the carrier was modified by Formula 1 to obtain the modified carrier.

[0053] S2: Immobilized enzyme preparation:

[0054] Phospholipase solution was immobilized with a modified carrier in the first stage to obtain phospholipase-cured material. Then, an auxiliary agent was added to the system for aging treatment. Next, histidine-containing lipase was added and immobilized in the second stage to obtain phospholipase-lipase composite cured enzyme.

[0055] S3: Preparation of diglycerides:

[0056] An immobilized complex enzyme is added to a reaction vessel equipped with a stirrer, and an enzyme-catalyzed reaction is carried out using oil as a substrate.

[0057] S4: After the reaction is complete, the immobilized complex enzyme is separated from the reaction system using an external magnetic field for reuse. The separated product is purified by molecular distillation, with the distillation temperature controlled at 150-250℃ and the operating pressure at 0.01-5 Pa. The corresponding fraction is collected to obtain a high-purity diglyceride product.

[0058] This invention does not have special requirements for the magnetic carrier. For example, it can be prepared by the following steps: Tetraethyl orthosilicate, ethanol, ammonia and water are mixed in a volume ratio of 1:4:0.5:20 and stirred at 60°C for 24 h to obtain mesoporous silica; then FeCl2·4H2O and FeCl3·6H2O are dissolved in water in a molar ratio of 1:2, the above mesoporous silica is added, ammonia is added at 80°C under nitrogen protection to adjust the pH to 10, and the mixture is stirred for 1 h to obtain a composite carrier (magnetic carrier) loaded with Fe3O4.

[0059] In the following examples, Equation 1 is taken as Equation 1A:

[0060] Formula 1A.

[0061] The modification scheme for histidine lipase can be known or can be a known commercial product. For example, the technical implementation can be found in the literature "Research on the rational regulation of lipase-catalyzed asymmetric organic reactions".

[0062] Example 1

[0063] Step 1: Preparation of immobilized enzymes:

[0064] Step 1.1: The prepared magnetic support was modified using Formula 1A. The modification conditions were: the weight ratio of the magnetic support to the modifier of Formula 1 was 1:0.3, the solvent was anhydrous toluene, the temperature was 100℃, and the time was 12h to obtain the modified support. 1g of the modified support was taken and 5mL of PLA1 phospholipase (phospholipase A1) buffer solution (enzyme concentration 10mg / mL, enzyme activity 200U / mg, pH=8.0) was added. The mixture was stirred at 4℃ for 4 hours, and the immobilized phospholipase was obtained by centrifugation and washing.

[0065] Step 1.2:

[0066] Subsequently, nickel chloride hexahydrate was added to the system from step 1.1, with an immobilized phospholipase to nickel chloride hexahydrate weight ratio of 1:0.06. The mixture was stirred at 25°C for 2 hours, and then 5 mL of histidine-modified CALB lipase (Candida antarcticis lipase B) buffer solution (enzyme concentration 20 mg / mL, enzyme activity 300 U / mg, pH=7.5; phospholipase / lipase activity ratio 1:3) was added. The reaction was carried out at 25°C for 8 hours, followed by centrifugation and washing to obtain the immobilized phospholipase-lipase complex enzyme (immobilized complex enzyme, product image see...). Figure 1 ).

[0067] application:

[0068] Preparation of diglycerides:

[0069] The above-mentioned immobilized complex enzyme was added to a 250 mL reactor equipped with a stirrer. Soybean oil was used as the substrate, and the reaction was carried out for 6 hours at 45 °C, pH 7.0, with the complex enzyme accounting for 3% of the substrate weight, and a stirring speed of 200 r / min.

[0070] Product separation and purification: After the reaction, the immobilized enzyme was separated using a magnetic field. The product was purified using molecular distillation, with the distillation temperature controlled at 150℃ and the operating pressure at 0.05 Pa. The distillate was collected. Analysis showed that the diglyceride yield was 62% and the purity was 96%.

[0071] Example 2

[0072] Step 1: Preparation of immobilized enzymes:

[0073] Step 1.1: The prepared magnetic support was modified using Formula 1A. The modification conditions were: the weight ratio of the magnetic support to the modifier of Formula 1 was 1:0.2, the solvent was anhydrous toluene, the temperature was 110℃, and the time was 16h to obtain the modified support. 1g of the modified support was taken and 5mL of phospholipase (same type as in Example 1) buffer solution (enzyme concentration 10mg / mL, enzyme activity 200U / mg, pH=8.0) was added. The mixture was stirred at 4℃ for 3 hours, and the immobilized phospholipase was obtained by centrifugation and washing.

[0074] Step 1.2:

[0075] Subsequently, nickel chloride hexahydrate was added to the system from step 1.1, with the weight ratio of immobilized phospholipase to nickel chloride hexahydrate being 1:0.1. The mixture was stirred at 25°C for 3 hours, and then 5 mL of lipase (same type as in Example 1) buffer solution (enzyme concentration 10 mg / mL, enzyme activity 300 U / mg, pH=7.5; phospholipase / lipase activity ratio 1:1.5) was added. The mixture was reacted at 25°C for 8 hours, and then centrifuged and washed to obtain the immobilized phospholipase-lipase complex enzyme (immobilized complex enzyme).

[0076] application:

[0077] Diglyceride preparation: The above-mentioned immobilized complex enzyme was added to a 250 mL reaction vessel equipped with a stirrer, using rapeseed oil as the substrate and the immobilized complex enzyme as 5% of the substrate weight. The reaction was carried out at 50 °C, pH 7.5, and stirring speed of 300 r / min for 8 hours.

[0078] Product separation and purification: After the reaction, the immobilized enzyme was separated using a magnetic field. The product was purified using molecular distillation, with the distillation temperature controlled at 180℃ and the operating pressure at 1 Pa. The distillate was collected. Analysis showed that the diglyceride yield was 68% and the purity was 95.5%.

[0079] Example 3

[0080] Step 1: Preparation of immobilized enzymes:

[0081] Step 1.1: The prepared magnetic support was modified using Formula 1A. The modification conditions were: the weight ratio of the magnetic support to the modifier of Formula 1 was 1:0.2, the solvent was anhydrous toluene, the temperature was 90℃, and the time was 24h to obtain the modified support. 1g of the modified support was taken and 5mL of phospholipase (same type as in Example 1) buffer solution (enzyme concentration 12mg / mL, enzyme activity 200U / mg, pH=8.0) was added. The mixture was stirred at 4℃ for 3 hours, and the immobilized phospholipase was obtained by centrifugation and washing.

[0082] Step 1.2:

[0083] Subsequently, nickel chloride hexahydrate was added to the system from step 1.1, with the weight ratio of immobilized phospholipase to nickel chloride hexahydrate being 1:0.2. The mixture was stirred at 25°C for 4 hours, and then 5 mL of lipase (same type as in Example 1) buffer solution (enzyme concentration 32 mg / mL, enzyme activity 300 U / mg, pH=7.5; phospholipase / lipase activity ratio 1:4) was added. The mixture was reacted at 25°C for 6 hours, and then centrifuged and washed to obtain the immobilized phospholipase-lipase complex enzyme (immobilized complex enzyme).

[0084] Preparation of diglycerides: The above-mentioned immobilized complex enzyme was added to a 250 mL reaction vessel equipped with a stirrer, using camellia oil as the substrate and the immobilized complex enzyme as 2% of the substrate weight. The reaction was carried out at 48℃, pH 7.2 and stirring speed of 250 r / min for 7 hours.

[0085] Product separation and purification: After the reaction, the immobilized enzyme was separated using a magnetic field. The product was purified using molecular distillation, with the distillation temperature controlled at 200℃ and the operating pressure at 5 Pa. The distillate was collected. Analysis showed that the diglyceride yield was 65% and the purity was 94.3%.

[0086] Comparative Example 1

[0087] Compared with Example 1, the only difference is that step 1.1 was not performed, and the magnetic carrier was directly processed in step 1.2 to obtain immobilized lipase.

[0088] Diglyceride yield was 38%, purity was 92%.

[0089] Comparative Example 2

[0090] Compared with Example 1, the only difference is that step 1.2 was not performed, and the immobilized phospholipase obtained in step 1.1 was directly used as an enzyme catalyst.

[0091] Tests showed that the diglyceride yield was 35% and the purity was 90%.

[0092] Comparative Example 3

[0093] Compared with Example 1, the only difference is that no solidification treatment was performed. Instead, a phospholipase and lipase solution (with the same concentration as in Example 1) was prepared and mixed in a 1:3 ratio.

[0094] Tests showed that the diglyceride yield was 42% and the purity was 91%.

[0095] Comparative Example 4

[0096] Compared with Example 1, the only difference is that the immobilized enzyme of Comparative Example 1 and the immobilized enzyme prepared in Comparative Example 2 are mixed, while the enzyme activity ratio and other operations are the same as in Example 1.

[0097] Comparative Example 5

[0098] Compared with Example 1, the only difference is that the magnetic carrier is pretreated in step 1.2, followed by the modification of Formula 1A in step 1.1 and subsequent phospholipase immobilization treatment. All other operations and parameters are the same as in Example 1.

[0099] The tests for each case were the same as in Example 1; the test results are shown in Table 1:

[0100] Yield retention after 30 cycles: The yield is calculated as a percentage of the initial yield after 30 cycles of enzyme catalysis.

[0101] Purity retention rate after 30 cycles: The purity is calculated as a percentage of the initial purity after 30 cycles of enzyme catalysis; this is the yield retention rate.

[0102] ;

[0103] As can be seen from Example 1 and the comparative example, an innovative method is provided that can successfully achieve simultaneous immobilization of phospholipase and lipase. Based on the control of the method, the problems existing in simultaneous immobilization can be solved. Furthermore, based on the control of the preparation method, the synergistic effect of immobilized phospholipase and lipase in the preparation of diglyceride catalytic enzymes can be achieved.

Claims

1. A method for preparing a phospholipase-lipase composite immobilized enzyme, characterized in that, The magnetic carrier was modified using the modifier of Formula 1 to obtain the modified carrier; then the modified carrier and phospholipase were mixed and cured in the first stage to obtain the phospholipase-cured material. Add an auxiliary agent to the first stage of the solidification reaction system for aging treatment, then add a histidine-modified lipase and carry out the second stage of solidification to obtain the phospholipase-lipase complex solidified enzyme. Formula 1 In Formula 1, R1~R3 ​​are C1~C3 alkoxy groups; X is a C2~C4 alkylene group; The additive is a water-soluble salt of nickel.

2. The method for preparing the phospholipase-lipase composite immobilized enzyme as described in claim 1, characterized in that, The magnetic carrier is a silicon dioxide@iron tetroxide composite material.

3. The method for preparing the phospholipase-lipase composite immobilized enzyme as described in claim 1, characterized in that, The weight ratio of the magnetic carrier to the modifier of Formula 1 is 1:0.15~0.35; The modification temperature is 80℃~120℃; The modification time is 6h~30h.

4. The method for preparing the phospholipase-lipase composite immobilized enzyme as described in claim 1, characterized in that, Phospholipases include at least one of phospholipase A1, phospholipase A2, phospholipase C, and phospholipase D.

5. The method for preparing the phospholipase-lipase complex immobilized enzyme as described in claim 1 or 4, characterized in that, The curing temperature for the first stage is 1℃~25℃; The first stage of curing takes 3 to 5 hours. The phospholipase loading in the phospholipase-cured material is 8000~12000U / g.

6. The method for preparing the phospholipase-lipase composite immobilized enzyme as described in claim 1, characterized in that, The additives include at least one of nickel nitrate hexahydrate and nickel chloride hexahydrate; The weight ratio of phospholipase curing material to additives is 1:0.05~0.3; The aging process is carried out at temperatures ranging from 20°C to 40°C. The aging process takes 1 to 4 hours.

7. The method for preparing the phospholipase-lipase composite immobilized enzyme as described in claim 1, characterized in that, The lipase mentioned is at least one of the following: Thermophys salina lipase, Thermophys salina lipase variant, Candida antarcticis lipase A, Candida antarcticis lipase B, Hyphozyma lipase, Rhizopus miltiorrhiza lipase, Alcaligenes lipase, Pseudomonas alkaligenes lipase, Pseudomonas pseudoalkaligenes lipase, and Pseudomonas cepacia lipase.

8. The method for preparing the phospholipase-lipase complex immobilized enzyme as described in claim 1 or 7, characterized in that, The curing temperature for the second stage is 20℃~30℃; The second stage of curing takes 2 to 10 hours. The lipase loading in the phospholipase-lipase complex immobilized enzyme is 10,000~60,000 U / g.

9. A phospholipase-lipase complex immobilized enzyme prepared by the preparation method according to any one of claims 1 to 8.

10. The application of a phospholipase-lipase complex immobilized enzyme prepared by the method according to any one of claims 1 to 8, characterized in that, Using the aforementioned phospholipase-lipase complex immobilizing enzyme as an enzyme catalyst and oil as a substrate, an enzyme-catalyzed reaction was carried out to obtain diglycerides.

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