A method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on bifunctional modified macroporous resin

Abstract

The present invention provides a method for catalyzing the synthesis of plant sterol ester compounds by amino and C18 alkyl bifunctional modified macroporous resin immobilized lipase. Use cheap and readily available stearic acid as a modifier to modify the macroporous resin (LXTE-700s) rich in amino functional groups on the surface with long-chain alkyl. The bifunctional modified macroporous resin immobilized enzyme prepared by this method has the characteristics of high catalytic activity, convenient separation and good reusability. In application, the carrier uses its Schiff base reaction with Candida plicata lipase and the hydrophobic interaction between C18 alkyl and Candida plicata lipase to jointly realize the immobilization of Candida plicata lipase, solving the problem that the enzyme is easy to leak from the carrier in diatomite immobilization technology. The fixed lipase is a catalyst, which catalyzes the esterification of plant sterols and fatty acids in a solvent-free system to synthesize plant sterol esters.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and specifically relates to a method for synthesizing phytosterol esters by catalyzing esterification reaction of phytosterol and fatty acid by using bifunctional modified macroporous resin immobilized lipase under solvent-free system conditions. Background Art

[0002] Phytosterols, also known as plant sterols, belong to the class of cyclopentane polyolefin derivatives. They are an important natural active ingredient and a natural regulator of cholesterol levels in the human body. They are hailed by scientists as the "key to life". With the establishment of the concept system of big health and big health care, more and more people are focusing on improving health literacy and self-health management. Introducing functional substances with complex physiological activities such as plant sterols (esters) into people's daily diet can help people shift from overdrawing their health and fighting diseases to a new health model of caring for health and preventing diseases.

[0003] At present, phytosterol esters are mainly synthesized by direct esterification of phytosterols with fatty acids or transesterification of phytosterols with corresponding fatty acid esters by chemical or enzymatic methods. Chemical synthesis and biosynthesis are the two main methods. Chemical synthesis has the advantages of high conversion rate and short reaction time, but due to the need for high temperature reaction conditions, dehydrated sterol by-products that are difficult to separate will appear in the product. Although biocatalytic synthesis has the disadvantages of long reaction time and poor catalyst stability, due to its catalytic specificity, the required temperature is much lower and the by-products produced are much less.

[0004] Traditional chemical catalytic reaction conditions are relatively intense, using high temperatures and strong acids (H2SO4, H3PO4, p-toluenesulfonic acid) or strong bases (NaOEt, NaOH, KOH, NaOMe) as catalysts. For example, the esterification reaction of plant sterols and oleic acid was carried out at 170°C using concentrated sulfuric acid as a catalyst. The conversion rate exceeded 98% after 2 hours, but 18.8% of by-products were produced. Studies have reported transesterification reactions using NaOMe as a catalyst, which obtained a conversion rate of almost 99% after 4 hours but with 26.7% by-products. Although chemical catalysis can synthesize plant sterol esters very efficiently, it inevitably produces by-products that are difficult to separate. The formation of by-products can lead to reduced yields and increased complexity in downstream processes. In addition, the removal of chemical catalysts is difficult and usually incomplete, which will result in residual catalysts in the product. The presence of by-products and residual catalysts may affect product quality and safety, which is a major problem that needs to be faced during application in the food field.

[0005] On the contrary, the production of phytosterol esters by biocatalysis meets the needs of sustainable development due to its mild conditions, safety and efficiency. In addition, the enzymatic synthesis of phytosterol esters has high selectivity, can avoid the formation of dehydrated sterol byproducts, and has good product color. However, at present, the literature reports that the process of enzymatic synthesis of phytosterol esters generally has problems such as long reaction time, poor enzyme reusability, and low yield of phytosterol esters. Therefore, some methods and technologies have been proposed to solve these problems, among which enzyme immobilization technology is widely used. Covalent bonding is a mature enzyme immobilization technology that connects enzymes to support materials (such as porous silica, polyacrylamide, agarose, porous glass, etc.) through highly stable strong connections. Covalent bonding has many advantages, such as producing stable enzymes and obtaining sufficient enzyme recovery for reuse. Covalent bonding increases the stereospecificity of the enzyme and improves its stability. In addition, the mobility of the enzyme attached to the carrier may be subject to some restrictions, which may limit some conformational changes required in the catalytic process, thereby reducing the activity of the enzyme. Adsorption immobilization is a method that relies on the principle of physical adsorption and can reversibly immobilize enzymes. The materials used for adsorption include ion exchange resins, alumina, activated carbon, etc. This method is relatively economical and easy to implement, but the binding force between the carrier and the enzyme is weak (such as hydrogen bonds, salt chains, ionic bonds, hydrophobic bonds, etc.). The specific active groups on the carrier material are the guarantee of successful adsorption. These groups help to produce interactions between the enzyme and the carrier. The advantage of immobilizing enzymes by adsorption is that the required activation steps are minimal, the required reagents are few, and it is an inexpensive method that is easy to implement. In addition, there are immobilization techniques such as cross-linking, trapping, and encapsulation.

[0006] Macroporous resins have rich porous structures and surface groups, and are easy to obtain at low cost, making them potential carriers for enzyme immobilization. Macroporous resins are a type of organic polymer with good adsorption properties. They have been widely used because of their three-dimensional spatial pore structure, large pore size and specific surface area, insolubility in organic solvents, and stability to oxygen, heat and chemical reagents. Summary of the invention

[0007] The technical problem solved by the present invention is: the present invention uses cheap and readily available stearic acid as a modifier to modify the macroporous resin (LXTE-700s) rich in amino functional groups on the surface with long-chain alkyl groups, and compares the immobilization efficiency and enzyme activity recovery rate of the modified macroporous resins with different alkyl modification degrees, and the optimal ratio is -NH2 / -C18 when the ratio is 3:1. Glutaraldehyde is used to activate the surface amino groups, and the immobilization of the Candida plicata lipase is achieved by the Schiff base reaction between the carrier and the Candida plicata lipase and the hydrophobic interaction between the C18 alkyl and the Candida plicata lipase, and the enzyme loading amount finally optimized is 138.21 mg·g-1. The yield of plant sterol esterification catalyzed by the bifunctional resin immobilized Candida plicata lipase can reach 95.7%, and can still reach more than 75% after 6 repeated uses. In addition, the bifunctional resin carrier can also immobilize the Javanese Mucor lipase and be used to catalyze the cyano-substituted indole reaction, with a yield of up to 83.4%.

[0008] In order to solve the above technical problems, the technical solution proposed by the present invention is: a method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on a bifunctional modified macroporous resin,

[0009] (1) The macroporous resin (LXTE-700s) was pretreated with ethanol and distilled water and stored in phosphate buffer solution;

[0010] (2) Dissolve stearic acid in tetrahydrofuran, add N,N'-carbonyldiimidazole (CDI) and stir at room temperature. Then, add amino resin and stir at room temperature to obtain amino and C 18 Alkyl bifunctional modified macroporous resin (AOMR): AOMR was dispersed in 5 mL phosphate buffer and glutaraldehyde was added for amino activation.

[0011] (3) Preparation of CRL@AOMR: The activated AOMR was dispersed in 5 mL of different concentrations (100-180 mg g -1 ) and Candida rugosa lipase solutions with different pH values ​​(7.0-8.0), and then stirred at 25-45°C for 6-12h.

[0012] (4) Adding substrates of phytosterols and fatty acids into a reactor, and adding the immobilized lipase prepared in step (3), and performing an esterification reaction under solvent-free conditions under heating and stirring conditions.

[0013] In step (2), the amount of stearic acid added as a modifier is 2.0 mmol·g-1 relative to the mass of the macroporous resin;

[0014] In step (2), 1% of glutaraldehyde is added to activate the macroporous resin.

[0015] Preferably, the specific preparation steps of the bifunctional macroporous resin immobilized lipase include:

[0016] (1) Disperse 2 g of macroporous resin (LXTE-700s) in 10 mL of ethanol and stir gently for 2 h. Then, add 10 mL of distilled water to the suspension and continue stirring for 30 min. Then, remove the solution by suction filtration and repeat washing with distilled water at least five times. Finally, store the pretreated macroporous resin in phosphate buffer solution (0.1 mol·L -1 , pH 7.0).

[0017] (2) Dissolve 569 mg (2.0 mmol) of stearic acid in tetrahydrofuran, stir and mix, add 324 mg (2.0 mmol) of N,N'-carbonyldiimidazole (CDI) at room temperature, and stir at room temperature for 2 h. Then, add 1 g of amino resin (Aminomacroporous resin, AMR), and stir at room temperature for 24 h to obtain amino and C 18 Amino and octadecyl co-bonded macroporous resins (AOMR). The modified resin obtained by the reaction was repeatedly washed with ethanol for at least 5 times, and then washed with phosphate buffer solution (0.1 mol·L -1 Finally, AOMR was stored in phosphate buffer solution (0.1 mol / L, pH 7.0).

[0018] 0.1 g of wet AOMR was dispersed in 5 mL of phosphate buffer (0.1 mol / L, pH 8.0). Then, 1% (relative to the mass of the macroporous resin) of glutaraldehyde was added to the system and stirred at 30°C for 4 h. Finally, the activated resin was vacuum dried at 60°C.

[0019] (3) Dissolve the Candida plicata lipase in a buffer solution and centrifuge at 4°C and 6000 rpm for 10 min to obtain a uniform Candida plicata lipase solution (180 mg / g). Disperse 0.1 g of activated AOMR in 5 mL of the Candida plicata lipase solution at pH 7.5 and stir at 35°C for 10 h. The immobilized enzyme product is named: CRL@AOMR.

[0020] Preferably, the lipase is Candida rugosa lipase.

[0021] Preferably, the fatty acid in the esterification reaction catalyzed by the immobilized lipase is one of oleic acid, linoleic acid, linolenic acid, lauric acid, or a mixed fatty acid.

[0022] Preferably, one or a mixed fatty acid of oleic acid, linoleic acid, linolenic acid, and lauric acid is used as the reaction medium.

[0023] Preferably, the molar ratio of phytosterols to fatty acids is 1:5-1:9.

[0024] Preferably, the immobilized lipase is added in the reaction at 6.0-9.0 U·g -1 (relative to the mass of pine sterol); the reaction temperature of the reaction is 40-55°C; the reaction time of the reaction is 24-60 hours.

[0025] Preferably, the reaction medium is oleic acid, the molar ratio of phytosterols to fatty acids is 1:6; the amount of immobilized lipase added in the reaction is 8.0 U·g-1, the reaction temperature is 50°C, the reaction time is 48 hours, and the esterification rate measured in the gas phase is 95.7%.

[0026] Preferably, the method comprises the following steps:

[0027] (1) 2 g of macroporous resin (LXTE-700s) was dispersed in 10 mL of ethanol and gently stirred for 2 h. Subsequently, 10 mL of distilled water was added to the suspension and stirred for 30 min. The solution was then removed by suction filtration and washed repeatedly with distilled water at least five times. Finally, the pretreated macroporous resin was stored in a phosphate buffer solution (0.1 mol·L-1, pH 7.0).

[0028] (2) Dissolve 569 mg (2.0 mmol) of stearic acid in tetrahydrofuran, stir and mix, add 324 mg (2.0 mmol) of N,N'-carbonyldiimidazole (CDI) at room temperature, and stir at room temperature for 2 h. Then, add 1 g of amino resin (Aminomacroporous resin, AMR), stir at room temperature for 24 h, and obtain amino and C18 alkyl bifunctional group modified macroporous resin (Amino and octadecyl co-bonded macroporous resins, AOMR). The modified resin obtained by the reaction is repeatedly washed with ethanol for at least 5 times, and then washed with phosphate buffer solution (0.1 mol·L-1, pH 7.0) for at least 5 times. Finally, AOMR is stored in phosphate buffer solution (0.1 mol / L, pH 7.0).

[0029] 0.1 g of wet AOMR was dispersed in 5 mL of phosphate buffer (0.1 mol / L, pH 8.0). Then, 1% (relative to the mass of the macroporous resin) of glutaraldehyde was added to the system and stirred at 30°C for 4 h. Finally, the activated resin was vacuum dried at 60°C.

[0030] (3) Dissolve the Candida plicata lipase in a buffer solution and centrifuge at 4°C and 6000 rpm for 10 min to obtain a uniform Candida plicata lipase solution (180 mg / g). Disperse 0.1 g of activated AOMR in 5 mL of the Candida plicata lipase solution at pH 7.5 and stir at 35°C for 10 h. The immobilized enzyme product is named: CRL@AOMR.

[0031] (4) 10 mmol of pine sterol (a plant sterol obtained from a byproduct of the pulping industry using pine as raw material) and 60 mmol of oleic acid were added to the reaction flask, heated to 50°C and mixed evenly, then 8.0 U g-1 CRL@AOMR was added, stirred at 50°C, and the reaction progress was detected by TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v). After 48 hours of reaction, the esterification rate measured by the gas phase was 95.7%. The immobilized enzyme was filtered, separated from the reaction system, and washed three times with 50 mL of hexane. After drying, the obtained CRL@AOMR can be directly used for the next batch of new esterification reactions. After 6 consecutive cycles of reaction, the recovered immobilized lipase still maintained more than 75% of the initial activity.

[0032] The beneficial effects of the present invention are:

[0033] (1) Stearic acid was used as a modifier to modify the macroporous resin (LXTE-700s) with rich amino functional groups on its surface. 18 Modification of the alkyl group to give amino and C 18 Alkyl bifunctional modified macroporous resin (AOMR). The surface amino groups of the support are activated by glutaraldehyde, and the Schiff base reaction and C 18 The hydrophobic interaction between the alkyl group and the enzyme achieves the immobilization of Candida plicata lipase, solving the problem of enzyme leakage from the carrier in diatomaceous earth immobilization technology.

[0034] The hydrophobic interaction and multi-site covalent connection are combined to construct a C18 alkyl and amino bifunctional modified macroporous resin. The carrier has the characteristics of low cost, low toxicity, good biocompatibility, easy and rapid separation and recovery, and is an excellent carrier for lipase immobilization. The amino and C18 alkyl bifunctional modified macroporous resin immobilized enzyme prepared by this method has the characteristics of high catalytic activity, convenient separation and good reusability, and can be used to catalyze the esterification reaction of plant sterols and fatty acids to prepare plant sterol esters. The present invention provides a new method for synthesizing plant sterol esters in a green and efficient manner.

[0035] (2) The present invention uses a solvent-free system for esterification reaction, with the substrate fatty acid as the reaction medium. On the one hand, it can reduce the use of organic solvents and avoid organic solvent residues in the product; on the other hand, it can increase volume productivity, reduce the complexity of downstream processes, and avoid the generation of hazardous liquid waste that needs to be treated or recycled.

[0036] (3) The amino group and C 18 The enzyme immobilized on the alkyl bifunctional modified macroporous resin has good thermal stability, organic solvent tolerance, storage stability, and can be reused. After 6 reuses, the amino and C 18 The enzyme immobilized on the alkyl bifunctional group modified macroporous resin can still maintain good activity, retaining more than 75% of the initial activity.

[0037] (4) In previous studies, it was found that Candida plicata lipase (Candida plicata lipase) has good catalytic performance for the esterification reaction of plant sterols and oleic acid. On this basis, we tried to use amino macroporous resin (LXTE-700s) as a template, construct amide bonds through stearic acid activated by carbonyldiimidazole, and introduce C18 alkyl long chains. Glutaraldehyde was used to activate the remaining amino groups on the surface, and the immobilization of Candida plicata lipase was achieved by utilizing the Schiff base reaction with Candida plicata lipase and the hydrophobic interaction between C18 alkyl and enzyme. The carrier stabilizes the active conformation of the enzyme molecule and increases the stability of the enzyme when catalyzing the esterification reaction. The immobilized enzyme was applied to the esterification reaction of plant sterols and fatty acids, and satisfactory results were obtained. The amount of lipase added was small, and the enzyme addition amount was 8.0U·g -1 (relative to the mass of phytosterols), the method has the characteristics of mild reaction conditions, short reaction time, complete reaction in 48 hours, high yield of phytosterol esters up to 95.7%, no dehydrated sterol byproducts, high catalytic activity of immobilized enzyme, good reusability, etc. After the enzyme has been cyclically reacted for 6 consecutive times, the enzyme activity can still maintain 76.2% of the initial activity. That is, the reaction conditions of Example 3 are the best conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention is further described below in conjunction with the accompanying drawings.

[0039] Figure 1 Schematic diagram of the bifunctional modified macroporous resin immobilized lipase of the present invention

[0040] Figure 2 Optimize results for glutaraldehyde concentration.

[0041] Figure 3 To optimize the molar ratio, enzyme addition amount, temperature and time of the esterification reaction of plant sterol esters.

[0042] Figure 4This is the reusability result of pNPP hydrolysis by bifunctional resin-immobilized enzyme.

[0043] Figure 5 The results show the reusability of bifunctional resin-immobilized enzyme in the esterification reaction of plant sterol esters. DETAILED DESCRIPTION

[0044] The present invention is further described below with reference to the following examples. The examples listed are for illustration only and indicate that the spirit and scope of the present invention are not limited to the details and modifications therein.

[0045] Embodiment 1:

[0046] (1) Pretreatment of aminated macroporous resin: 2 g of macroporous resin (LXTE-700s) was dispersed in 10 mL of ethanol and gently stirred for 2 h. Subsequently, 10 mL of distilled water was added to the suspension and stirred for 30 min. The solution was then removed by suction filtration and washed repeatedly with distilled water at least five times. Finally, the pretreated macroporous resin was stored in a phosphate buffer solution (0.1 mol·L-1, pH 7.0).

[0047] (2) Preparation of Amino and C18 alkyl difunctional modified macroporous resin (AOMR) Solvent screening: 341 mg (1.2 mmol) of stearic acid was dissolved in dichloromethane, dichloroethane, tetrahydrofuran, carbon tetrachloride and acetonitrile respectively, stirred and mixed, and 195 mg (1.2 mmol) of N, N'-carbonyldiimidazole was added at room temperature, and stirred at room temperature for 2 h. Subsequently, 1 g of amino resin (Amino macroporous resin, AMR) was added, and stirred at room temperature for 24 h to obtain AOMR. The modified resin obtained by the reaction was repeatedly washed with ethanol for at least 5 times, and then washed with phosphate buffer solution (0.1 mol·L-1, pH 7.0) for at least 5 times.

[0048] The functional group density of the prepared AOMR was measured. When dichloromethane and dichloroethane were used, the long reaction time would cause the resin to swell, and even if it was stirred at a lower speed, a large amount of resin would still break. When carbon tetrachloride was used as a solvent, the modification efficiency was low because the solubility of stearic acid in carbon tetrachloride was low. When tetrahydrofuran was used as a solvent, a reaction effect comparable to that of acetonitrile was obtained, but tetrahydrofuran was selected as the reaction solvent in view of its low toxicity.

[0049] Table 1. Functional group density of AOMR synthesized in different solvents

[0050]

[0051] (3) Optimization of the amount of modifier added in the preparation of AOMR: 0.4-2.4mmol·g-1 (relative to the mass of the resin) of stearic acid was dissolved in tetrahydrofuran, stirred and mixed, and N,N'-carbonyldiimidazole in the same amount as stearic acid was added at room temperature, and stirred at room temperature for 2h. Subsequently, 1g of amino resin (Amino macroporous resin, AMR) was added and stirred at room temperature for 24h to obtain amino and C18 alkyl co-bonded macroporous resin (AOMR) with different degrees of modification. The modified resin obtained by the reaction was repeatedly washed with ethanol at least 5 times, and then washed with phosphate buffer solution (0.1mol·L-1, pH 7.0) at least 5 times. The amino group concentration on the surface of AOMR was determined by titration, the functional group modification ratio was calculated, and the immobilization effect was compared. The enzyme addition amount of the immobilization reaction was 180mg·g-1, the temperature was 30℃, and the reaction was carried out at pH 7.0 for 6h.

[0052] The results showed that the addition amount of stearic acid modifier was 2.0 mmol·g -1 When -NH2 / -C 18 The ratio is 3:1, at which the enzyme loading capacity and enzyme activity recovery rate are optimal.

[0053] Table 2. Screening of modifier addition amount

[0054]

[0055] (4) 0.1 g of wet AOMR was dispersed in 5 mL of phosphate buffer (0.1 mol / L, pH 8.0). Then, 1%-10% (relative to the mass of the macroporous resin) of glutaraldehyde was added to the system and stirred at 30°C for 4 h. Finally, the activated resin was vacuum dried at 60°C. The results showed that the increase in glutaraldehyde concentration can indeed improve the immobilization efficiency of AOMR ( Figure 2 ). With the increase of glutaraldehyde concentration, the activity of immobilized enzyme showed a rapid downward trend.

[0056] Therefore, 1% glutaraldehyde was selected to activate the amino groups of the macroporous resin.

[0057] Embodiment 2:

[0058] (1) Preparation of CRL@AOMR: Dissolve the Candida plicata lipase in a buffer solution and centrifuge at 4°C and 6000 rpm for 10 min to obtain a uniform Candida plicata lipase solution. Disperse 0.1 g of activated AOMR (prepared under the optimal conditions in Example 1) in 5 mL of Candida plicata lipase solution of different concentrations and different pH values ​​(6.0-8.0), and then stir at 25-45°C for 4-12 h. The immobilized enzyme product is named: CRL@AOMR. The immobilization efficiency of lipase on a hydrophobic carrier is affected by many factors, including enzyme concentration (enzyme addition amount), pH value, temperature and reaction time. This study fully investigated the effects of these factors on the enzyme loading and optimized the immobilization conditions. (Table 3)

[0059] (2) Optimization of enzyme addition conditions of CRL@AOMR: Dissolve the Candida plicata lipase in a buffer solution with an enzyme addition amount of 100-180 mg·g-1, and centrifuge at 4°C and 6000 rpm for 10 min to obtain a uniform Candida plicata lipase solution. Disperse 0.1 g of activated AOMR in 5 mL of Candida plicata lipase solution with different concentrations and pH 7, and then stir at 25°C for 6 h.

[0060] The experiment showed that the enzyme loading increased with the increase of Candida plicata lipase concentration. When the enzyme addition amount was 100-140 mg·g-1, the increase of immobilization efficiency was relatively large, and when it was 140-180 mg·g-1, the increase tended to be gentle. Therefore, 180 mg·g-1 was determined to be the optimal enzyme addition amount.

[0061] (3) Optimization of temperature conditions for CRL@AOMR: Dissolve the Candida plicata lipase in a buffer solution with an enzyme addition amount of 180 mg g-1 and centrifuge at 4°C and 6000 rpm for 10 min to obtain a uniform Candida plicata lipase solution. Disperse 0.1 g of activated AOMR in 5 mL of Candida plicata lipase solutions of different concentrations and pH 7, and then stir at 20-40° for 6 h.

[0062] Experiments show that the higher the temperature, the higher the immobilization efficiency, which can be explained by the collision theory between enzyme molecules and carriers. However, too high a temperature will also destroy the tertiary structure of the free enzyme, and the immobilized inactivated enzyme molecules will lead to a decrease in the activity of the final expressed enzyme. Considering the enzyme immobilization efficiency and enzyme activity comprehensively, 35°C was selected as the optimal immobilization temperature.

[0063] (4) Optimization of pH conditions of CRL@AOMR: Dissolve the Candida plicata lipase in a buffer solution with an enzyme addition amount of 180 mg g-1 and centrifuge at 4 °C and 6000 rpm for 10 min to obtain a uniform Candida plicata lipase solution. 0.1 g of activated AOMR was dispersed in 5 mL of Candida plicata lipase solution with different concentrations and pH 6.0-8.0, and then stirred at 35 °C for 6 h.

[0064] The experiments showed that when the pH value was 7.5, the immobilization efficiency was at a higher level, which indicated that weakly alkaline conditions were more suitable for the immobilization of AOMR.

[0065] (5) Optimization of time conditions for CRL@AOMR: Dissolve the Candida plicata lipase in a buffer solution with an enzyme addition amount of 180 mg·g-1, and centrifuge at 4°C and 6000 rpm for 10 min to obtain a uniform Candida plicata lipase solution. Disperse 0.1 g of activated AOMR in 5 mL of Candida plicata lipase solution with different concentrations and pH 7.5, and then stir at 35° for 4-12 h. The experiment showed that with the extension of reaction time, the enzyme loading of CRL@AOMR gradually increased, but after 10 h, the carrier was saturated and the enzyme loading no longer increased. Therefore, 10 h was selected as the final reaction time.

[0066] In this study, Candida plicata lipase was successfully immobilized on AOMR in a phosphate buffer with a pH value of 7.5, with an enzyme addition amount of 180 mg·g-1 and a reaction time of 10 hours at 35°C. The loading amount of immobilized Candida plicata lipase was 138.2 mg·g-1, which was higher than other hydrophobic carriers reported in the literature. This shows that the modified design of AOMR can effectively improve the immobilization efficiency and has strong application potential.

[0067] Table 3. Optimization of CRL@AOMR immobilization conditions

[0068]

[0069] (6) Study on thermal stability of CRL@AOMR: The enzyme activities of free Candida plicata lipase and CRL@AOMR at 50°C and 60°C were measured to study the thermal stability of CRL@AOMR. Candida plicata lipase and CRL@AOMR were placed in test tubes and kept in a water bath at 50°C and 60°C for 60 min. Part of the enzyme was taken out every 15 min to measure the enzyme activity. The enzyme activity at the initial moment was set as 100%, and the residual enzyme activity within 60 min was studied.

[0070] At 50°C, it showed high stability and the activity did not change significantly with temperature, indicating that the immobilized Candida plicata lipase can be effectively used in this reaction. At 60°C, the activity of free Candida plicata lipase (optimum temperature 40°C) dropped sharply, which may be related to the irreversible denaturation and inactivation of the secondary structure of Candida plicata lipase at high temperature. In contrast, CRL@AOMR can effectively resist the influence of high temperature, and its activity can be maintained at a level above 80% even after exposure to 60°C for 30 minutes. This result strongly confirms that CRL@AOMR has higher thermal stability than free enzymes within a certain temperature range.

[0071] Embodiment 3:

[0072] (1) CRL@AOMR process Refer to Example 2. According to the optimal conditions, the Candida plicata lipase was successfully immobilized on AOMR in a phosphate buffer with a pH of 7.5, with an enzyme addition amount of 180 mg·g-1 and a reaction time of 10 hours at 35°C. The loading amount of the immobilized Candida plicata lipase was 138.2 mg·g-1. In this study, CRL@AOMR was used as a catalyst to optimize the process parameters for the direct esterification reaction of pine sterol with oleic acid ( Figure 3 ). In the reaction tube, 1.0 mmol of pine sterol and 5.0-9.0 mmol of oleic acid were added respectively, and 5.0-9.0 U·g -1 (relative to the mass of pine sterol) CRL@AOMR is used as a catalyst, the reaction temperature is controlled at 35-55°C, and the reaction time is controlled at 12-60h.

[0073] (2) Optimization of substrate molar ratio conditions: 1.0 mmol of pine sterol and 5.0-9.0 mmol of oleic acid were added to the reaction tube at 6 U·g -1 (relative to the mass of pine sterol) CRL@AOMR was used as catalyst, the reaction temperature was controlled at 50°C, and the reaction time was 36h.

[0074] like Figure 3 As shown in the figure, when the molar ratio is 1:5 to 1:6, the esterification yield ranges from 74.6% to 83.7%, which is mainly due to the small amount of oleic acid and the insufficient solubility of pine sterol in the reaction medium. However, it is worth noting that too much oleic acid will also have a negative impact on the esterification efficiency. The reason for this situation is that as the reaction continues, the relative concentration of pine sterol in the oleic acid medium gradually decreases, resulting in a decrease in the effective collision between the sterol substrate and the active center of the enzyme molecule. Instead, oleic acid occupies a large amount of the active center of the Candida plicata lipase. When the molar ratio of pine sterol to oleic acid is 1:6, the esterification yield reaches a high level.

[0075] (3) Optimization of enzyme addition conditions: 1.0 mmol of pine sterol and 6.0 mmol of oleic acid were added to the reaction tube at a concentration of 5.0-9.0 U·g -1 (relative to the mass of pine sterol) CRL@AOMR was used as catalyst, the reaction temperature was controlled at 50°C, and the reaction time was 36h.

[0076] like Figure 3 As shown, when the enzyme addition amount is 5.0-8.0U·g -1 When the enzyme addition amount exceeds 8.0U·g -1 The yield of the esterification reaction was basically stable when the cost of the biocatalyst was taken into consideration. Finally, 8.0 U·g -1 The optimal enzyme addition amount.

[0077] (4) Optimization of temperature conditions: 1.0 mmol of pine sterol and 6.0 mmol of oleic acid were added to the reaction tube at 8.0 U·g -1 (relative to the mass of pine sterol) CRL@AOMR was used as catalyst, the reaction temperature was controlled at 35-55°C, and the reaction time was 36h.

[0078] like Figure 3 As shown in the figure, the reason for the low yield at 35°C is the insufficient solubility of pine sterol and the high mass transfer resistance caused by the high viscosity of oleic acid at low temperatures, which makes it more difficult for the substrate to diffuse to the active site of CRL@AOMR. In addition, since high temperature will cause the destruction of the structure of Candida rugosa lipase, temperatures exceeding 55°C will also have a negative effect on the esterification yield. After evaluating the catalytic efficiency of the immobilized enzyme and the performance of the esterification reaction, it was finally concluded that 50°C was the most suitable reaction temperature.

[0079] (5) Reaction time optimization: 1.0 mmol of pine sterol and 6.0 mmol of oleic acid were added to the reaction tube at 8.0 U·g -1 (relative to the mass of pine sterol) CRL@AOMR was used as the catalyst, the reaction temperature was controlled at 50°C, and the reaction time was 12-60h. Figure 3 As shown, after 48 hours of reaction, the esterification yield can reach 95.7%, and extending the reaction time has no significant effect on the esterification yield.

[0080] (6) The conditions for the synthesis of pine sterol oleate catalyzed by CRL@AOMR were finally optimized as follows: 1.0 mmol of pine sterol and 6.0 mmol of oleic acid were added to the reaction tube, respectively, 8 U·g-1 (relative to the mass of pine sterol) of CRL@AOMR was used as the catalyst, the reaction temperature was controlled at 50°C, and the reaction time was 48 h.

[0081] Embodiment 4:

[0082] (1) In order to demonstrate the substrate range of CRL@AOMR, the esterification reaction of pine sterol with oleic acid, linoleic acid, linolenic acid, lauric acid, capric acid and mixed fatty acids was studied under the optimized reaction conditions of Example 3. The mixed fatty acids were composed of oleic acid, linoleic acid and linolenic acid mixed in equal proportions.

[0083] (2) In the reaction tube, 1.0 mmol of pine sterol and 6.0 mmol of oleic acid, linoleic acid, linolenic acid, lauric acid, capric acid and mixed fatty acids were added respectively, and 8 U·g-1 (relative to the mass of pine sterol) of CRL@AOMR was used as the catalyst. The reaction temperature was controlled at 50 °C and the reaction time was 48 h.

[0084] (3) The results show that when long-chain unsaturated fatty acids are used as substrates (oleic acid, linoleic acid, linolenic acid, including mixed acids of the three in equal proportions), ideal yields are obtained. Due to the high melting point of long-chain saturated fatty acids, the experiment only explored the reaction of medium- and long-chain saturated fatty acids (lauric acid, capric acid). Although the reaction effect when saturated fatty acids are used as substrates is relatively low, it can still reach more than 90%. The results show that CRL@AOMR has good substrate applicability and can be applied to the preparation of different types of sterol esters.

[0085] Table 4. Substrate suitability studies

[0086]

[0087] Embodiment 5:

[0088] In the reaction tube, 1.0 mmol of pine sterol and 6.0 mmol of oleic acid, linoleic acid, linolenic acid, lauric acid, capric acid and mixed fatty acids were added respectively, and 8 U·g-1 (relative to the mass of pine sterol) of CRL@AOMR was used as a catalyst. The reaction temperature was controlled at 50°C and the reaction time was 48 h. After the reaction, the immobilized enzyme was filtered, separated from the reaction system, and washed three times with 5 mL of hexane. After drying, the obtained CRL@AOMR can be directly used for the hydrolysis reaction of p-nitrophenyl palmitate (pNPP). When pNPP was used as a model substrate for testing, the hydrolysis yield could still reach more than 88% after 10 cycles of repeated use, which shows that the enzyme activity is rarely lost during the recycling of CRL@AOMR and has good operational stability ( Figure 4 ).

[0089] Embodiment 6:

[0090] (1) 2 g of macroporous resin (LXTE-700s) was dispersed in 10 mL of ethanol and gently stirred for 2 h. Subsequently, 10 mL of distilled water was added to the suspension and stirred for 30 min. The solution was then removed by suction filtration and washed repeatedly with distilled water at least five times. Finally, the pretreated macroporous resin was stored in a phosphate buffer solution (0.1 mol·L-1, pH 7.0).

[0091] (2) Dissolve 569 mg (2.0 mmol) of stearic acid in tetrahydrofuran, stir and mix, add 324 mg (2.0 mmol) of N,N'-carbonyldiimidazole (CDI) at room temperature, and stir at room temperature for 2 h. Then, add 1 g of amino resin (Aminomacroporous resin, AMR), stir at room temperature for 24 h, and obtain amino and C18 alkyl bifunctional group modified macroporous resin (Amino and octadecyl co-bonded macroporous resins, AOMR). The modified resin obtained by the reaction is repeatedly washed with ethanol for at least 5 times, and then washed with phosphate buffer solution (0.1 mol·L-1, pH 7.0) for at least 5 times. Finally, AOMR is stored in phosphate buffer solution (0.1 mol / L, pH 7.0).

[0092] 0.1 g of wet AOMR was dispersed in 5 mL of phosphate buffer (0.1 mol / L, pH 8.0). Then, 1% (relative to the mass of the macroporous resin) of glutaraldehyde was added to the system and stirred at 30°C for 4 h. Finally, the activated resin was vacuum dried at 60°C.

[0093] (3) Dissolve the Candida plicata lipase in a buffer solution and centrifuge at 4°C and 6000 rpm for 10 min to obtain a uniform Candida plicata lipase solution (180 mg / g). Disperse 0.1 g of activated AOMR in 5 mL of the Candida plicata lipase solution at pH 7.5 and stir at 35°C for 10 h. The immobilized enzyme product is named: CRL@AOMR.

[0094] (4) 10 mmol of pine sterol and 60 mmol of oleic acid were added to the reaction bottle, heated to 50 °C and mixed evenly, then 8.0 U g-1 CRL@AOMR was added and stirred at 50 °C. TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v) was used to detect the reaction progress. After 48 hours of reaction, the esterification rate measured by gas phase was 95.7%. The immobilized enzyme was filtered, separated from the reaction system, and washed with 50 mL of hexane three times. After drying, the obtained CRL@AOMR can be directly used for the next batch of new esterification reactions. After 6 consecutive cycles of reaction, the recovered immobilized lipase still maintained more than 75% of the initial activity. It surpassed the commercial macroporous resin ECR8806M. The reduction in esterification rate may be due to the detachment of part of the lipase from the carrier, or the change in the activity and structure of the enzyme due to the influence of the reaction conditions ( Figure 5 ).

Claims

1. A method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on a bifunctional modified macroporous resin, characterized in that: (1) The macroporous resin LXTE-700s was pretreated with ethanol and distilled water and stored in phosphate buffer solution; (2) Dissolve stearic acid in tetrahydrofuran, add N,N'-carbonyldiimidazole CDI at room temperature and stir; then add the macroporous resin LXTE-700s prepared in step (1) and stir at room temperature to obtain amino and C 18 Alkyl bifunctional group modified macroporous resin AOMR; AOMR is dispersed in phosphate buffer and glutaraldehyde is added for amino activation; (3) Preparation of CRL@AOMR: The activated AOMR was dispersed in different concentrations of 140-180 mg·g -1 and Candida rugosa lipase solutions with different pH values ​​of 7-8.0, and then stirred at 25-40°C for 6-12 h to obtain immobilized lipase; (4) adding substrates of phytosterols and fatty acids into a reactor, adding the immobilized lipase prepared in step (3), and carrying out an esterification reaction under solvent-free conditions under heating and stirring conditions; In step (2), the amount of stearic acid added as a modifier is 2.0 mmol·g relative to the mass of the macroporous resin. -1 ; In step (2), 1% of glutaraldehyde is added for activation relative to the mass of the macroporous resin.

2. The method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on bifunctional modified macroporous resin according to claim 1, characterized in that: The specific preparation steps of the bifunctional group modified macroporous resin immobilized lipase include: (1) Disperse 2 g of macroporous resin LXTE-700s in 10 mL of ethanol and stir gently for 2 h. Then, add 10 mL of distilled water to the suspension and continue stirring for 30 min. Then, remove the solution by suction filtration and repeat washing with distilled water at least five times. Finally, store the pretreated macroporous resin in 0.1 mol·L phosphate buffer solution. -1 , pH 7.0; (2) Dissolve 569 mg of stearic acid in tetrahydrofuran, stir and mix, add 324 mg of N,N'-carbonyldiimidazole CDI at room temperature, and stir at room temperature for 2 h; then, add 1 g of amino resin (Aminomacroporous resin, AMR) pretreated in step (1), and stir at room temperature for 24 h to obtain amino and C 18 Amino and octadecyl co-bonded macroporous resins (AOMR); the modified resin obtained by the reaction was repeatedly washed with ethanol for at least 5 times, and then washed with 0.1 mol·L phosphate buffer solution -1 , pH 7.0 at least 5 times; finally, AOMR was stored in phosphate buffer solution 0.1 mol / L, pH 7.0; 0.1 g wet AOMR was dispersed in 5 mL phosphate buffer 0.1 mol / L, pH 8.0; then, 1% glutaraldehyde relative to the mass of the macroporous resin was added to the system and stirred at 30°C for 4 h; finally, the activated resin was vacuum dried at 60°C; (3) Dissolve the Candida puckerygium lipase in a buffer solution and centrifuge at 4°C, 6000 rpm for 10 min to obtain a uniform Candida puckerygium lipase solution of 180 mg / g. Disperse 0.1 g of activated AOMR in 5 mL of the Candida puckerygium lipase solution with a pH of 7.5 and stir at 35°C for 10 h. The immobilized enzyme product is named: CRL@AOMR.

3. The method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on bifunctional modified macroporous resin according to claim 1, characterized in that: The fatty acid for the esterification reaction catalyzed by the immobilized lipase is one of oleic acid, linoleic acid, linolenic acid, lauric acid, capric acid or a mixed fatty acid, wherein the mixed fatty acid is a mixture of oleic acid, linoleic acid and linolenic acid in equal molar ratios.

4. The method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on bifunctional modified macroporous resin according to claim 3, characterized in that: The reaction medium is one of oleic acid, linoleic acid, linolenic acid, lauric acid, capric acid or a mixed fatty acid.

5. The method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on bifunctional modified macroporous resin according to claim 1, characterized in that: The molar ratio of the phytosterol to the fatty acid is 1:5-1:

9.

6. The method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on bifunctional modified macroporous resin according to claim 1, characterized in that The immobilized lipase is added in the reaction at a concentration of 6.0-9.0 U·g relative to the mass of the phytosterols. -1 ; The reaction temperature of the reaction is 40-55°C; The reaction time of the reaction is 24-60 hours.

7. The method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on bifunctional modified macroporous resin according to claim 1, characterized in that: The reaction medium is oleic acid, the molar ratio of the phytosterol to the fatty acid is 1:6; the addition amount of the immobilized lipase in the reaction is 8.0 U·g -1 , the reaction temperature is 50°C, and the reaction time is 48 hours.

8. The method for synthesizing phytosterol ester compounds by catalyzing lipase immobilized on bifunctional modified macroporous resin according to claim 7, characterized in that: The following steps are involved: (1) Disperse 2 g of macroporous resin LXTE-700s in 10 mL of ethanol and stir gently for 2 h. Then, add 10 mL of distilled water to the suspension and continue stirring for 30 min. Then, remove the solution by suction filtration and repeat washing with distilled water at least five times. Finally, store the pretreated macroporous resin in 0.1 mol·L phosphate buffer solution. -1 , pH 7.0; (2) Dissolve 569 mg of stearic acid in tetrahydrofuran, stir and mix, add 324 mg of N,N'-carbonyldiimidazole (CDI) at room temperature, and stir at room temperature for 2 h; then, add 1 g of the amino resin (Aminomacroporous resin, AMR) pretreated in step (1), and stir at room temperature for 24 h to obtain amino and C18 alkyl bifunctional modified macroporous resins (Amino and octadecyl co-bonded macroporous resins, AOMR); wash the modified resin obtained by the reaction with ethanol repeatedly for at least 5 times, and then wash it with 0.1 mol·L phosphate buffer solution. -1 , pH 7.0 at least 5 times; finally, AOMR was stored in phosphate buffer solution 0.1 mol / L, pH 7.0; 0.1 g wet AOMR was dispersed in 5 mL phosphate buffer 0.1 mol / L, pH 8.0; then, 1% glutaraldehyde relative to the mass of the macroporous resin was added to the system and stirred at 30°C for 4 h; finally, the activated resin was vacuum dried at 60°C; (3) Dissolve the Candida plicata lipase in a buffer solution and centrifuge at 4°C and 6000 rpm for 10 min to obtain a uniform Candida plicata lipase solution of 180 mg / g; disperse 0.1 g of activated AOMR in 5 mL of the Candida plicata lipase solution with a pH of 7.5, and then stir at 35°C for 10 h; the immobilized enzyme product is named: CRL@AOMR; (4) Add 10 mmol of pine sterol and 60 mmol of oleic acid into the reaction flask, heat to 50 °C and mix well, then add 8.0 U·g -1 CRL@AOMR was stirred at 50°C and the reaction progress was detected by TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10: 1, v / v / v). After 48 hours of reaction, the esterification rate measured by gas phase was 95.7%. The immobilized enzyme was filtered to separate it from the reaction system and washed three times with 50 mL of hexane. After drying, the obtained CRL@AOMR could be directly used for the next batch of new esterification reactions. After 6 consecutive cycles of reaction, the recovered immobilized lipase still maintained more than 75% of its initial activity.

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