Preparation method of biomass diesel oil immobilized lipase

Abstract

The invention discloses a preparation method of biomass diesel oil immobilized lipase, and belongs to the technical field of lipase immobilization. The preparation method specifically comprises the following steps: (1) drying polystyrene resin, and adding methanol for swelling, suction filtration, washing and drying to obtain pretreated resin; (2) adding L-lysine, L-arginine, L-histidine or L-cysteine into the enzyme solution of the biomass diesel lipase, and stirring for dissolving, so as to obtain a pretreated enzyme solution; and (3) adding the pretreated enzyme liquid into the pretreated resin for adsorption, suction filtration, washing and drying. According to the biomass diesel oil immobilized lipase prepared by the invention, the content of free FFA in a biomass diesel oil reaction can be reduced to 0.5% or below, the cycle use frequency exceeds 14 times, the biomass diesel oil immobilized lipase has the characteristics of simple preparation process, excellent catalytic performance, greatly improved stability and the like, and the production cost is greatly reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lipase immobilization, and particularly relates to a preparation method of biomass diesel immobilized lipase. BACKGROUND

[0002] Fatty acid methyl ester (FAME) generated by the conversion of biodiesel is non-toxic, renewable and biodegradable, and has similar physicochemical properties to petrochemical diesel. At the same time, compared with traditional fuels, biodiesel has high energy density, good lubricating performance, safe transportation and storage, high oxygen content, more complete combustion, and low emissions of CO2, CO, sulfides and particulate matter, and is a high-quality green alternative fuel.

[0003] Renewable raw materials for biodiesel production are divided into three types: edible oil, non-edible oil and waste oil. Among them, edible oils such as soybean oil, rapeseed oil and palm oil, as raw materials for biodiesel production, have high production costs, compete with the grain industry, and are prone to cause imbalance between food supply and demand. Especially for East Asian countries with high population density, edible oil supply is tight, and it is not suitable to produce biodiesel with a large amount of edible oil as raw material. Non-edible oil, waste oil and microalgae oil are cheap and easy to obtain, and can solve the problem of edible oil-derived biodiesel production, and are currently the main sustainable alternative raw materials. Non-edible oil and waste oil have high content of free fatty acids (FFA), which can be saponified with alkali, and cannot be directly used for the production of biodiesel through the traditional alkali-catalyzed transesterification route. Lipase can catalyze esterification and transesterification reactions, and can use relatively cheap high-FFA and water-containing oils as raw materials for the production of biodiesel. Therefore, compared with traditional chemical catalytic processes, lipase-catalyzed preparation of biodiesel has the characteristics of low energy consumption, low post-treatment cost and more extensive raw materials, and shows better environmental and economic advantages. However, in most cases, the high cost of non-renewable catalysis limits the industrial application of enzyme-catalyzed processes.

[0004] Enzyme immobilization technology can effectively improve the catalytic performance and operational stability of enzymes and reduce their cost, and is a widely used technology. Enzyme immobilization involves fixing enzymes on a suitable carrier, and the enzyme is fixed and functional on the carrier. Moreover, due to the close combination of the enzyme and the carrier, the enzyme will not easily fall off and be released in the applied solvent under external force. After treatment, the immobilized enzyme has certain stability and can be repeatedly used. In addition, immobilized lipase is more conducive to the separation and purification of products than free lipase, and shows superior performance in terms of pH tolerance, substrate selectivity, thermal stability and recycling usability.

[0005] In order to realize the highest efficiency of enzyme immobilization, that is, after the minimum amount of enzyme liquid is adsorbed on the carrier, the immobilized enzyme reaches the highest level of catalytic performance and stability after drying treatment. At present, the commonly used enzyme and carrier combination methods on the market include physical adsorption and covalent cross-linking. Among them, the surface of the carrier needs to be modified to achieve reasonable hydrophobicity; on the one hand, the combination ability of the carrier and the enzyme is considered, and on the other hand, the influence of the hydrophobicity of the carrier on the structure of the lipase protein is considered, thereby affecting the selectivity and stability of the enzyme. The covalent cross-linking method needs to form a covalent bond between the enzyme and the carrier through a relatively mild chemical reaction, and the negative influence of this process on the molecular structure and activity of the enzyme should be minimized. The covalent combination is relatively stable, and the enzyme molecules are not easy to fall off. Industrial-grade enzyme immobilization usually needs to use column adsorption technology, and the carrier is placed in a column-shaped adsorption container. A low-shear pump (peristaltic pump or diaphragm pump) is used to provide power to inject the enzyme liquid into the column. Through the column head distributor, the enzyme protein is uniformly adsorbed and combined with the carrier. After the immobilization is completed, the bottom end of the adsorption column is opened, and the top end is connected to compressed air to transfer the carrier to the tray and spread it out. Put it into a drying box and vacuum dry for 18-24h. The obtained product is the immobilized enzyme.

[0006] However, the existing biomass diesel immobilized lipase product can only reduce the free FFA content in the biomass diesel reaction to about 0.6%, while the chemical catalysis can be reduced to below 0.5%. At the same time, the recycling number of the enzyme method is not more than 10 times.

[0007] Therefore, how to simultaneously improve the selectivity and stability of the biomass diesel immobilized lipase, so that its catalytic performance reaches and exceeds the chemical method, and increases the number of recycling times is a problem that those skilled in the art need to solve. SUMMARY

[0008] Therefore, the purpose of the present application is to provide a preparation method of biomass diesel immobilized lipase to solve the problems in the prior art.

[0009] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0010] A preparation method of biomass diesel immobilized lipase, specifically comprising the following steps:

[0011] (1) drying polystyrene resin, adding methanol for swelling, filtering, washing, and drying to obtain pretreated resin;

[0012] (2) adding L-lysine, L-arginine, L-histidine or L-cysteine to the enzyme liquid of biomass diesel lipase, stirring and dissolving to obtain pretreated enzyme liquid;

[0013] (3) adding the pretreated enzyme solution into the pretreated resin to adsorb, filtering, washing, and drying, to obtain the immobilized lipase for the production of biomass diesel.

[0014] Further, in the step (1), the polystyrene resin is a polystyrene resin with a surface modification type C4. Further, the polystyrene resin with the surface modification type C4 is a spherical particle with a particle size of 400-800 μm.

[0015] Further, in the step (1), the polystyrene resin (dry weight) and the methanol are used in a ratio of 1 g:(2-6) mL, preferably 1 g:(3-5) mL, and more preferably 1 g:(3.5-4) mL.

[0016] Further, in the step (1), the swelling time is 0.5 h.

[0017] Further, in the step (1), since the synthetic polystyrene resin has a high water content, it cannot be accurately weighed for loading, so the polystyrene resin is first dried in a drying oven, the water content is determined according to GB 5757-2008 "Ion Exchange Resin Water Content Determination Method", and the dry weight is calculated and weighed.

[0018] Further, in the step (2), the concentration of L-lysine, L-arginine, L-histidine or L-cysteine in the pretreated enzyme solution is 0.1-0.7 M, preferably 0.2-0.5 M, and more preferably 0.3-0.4 M.

[0019] The above further beneficial effects are that L-histidine can prevent the aggregation of proteins in the enzyme solution, increase the solubility of proteins, and reduce the formation of aggregates during enzyme protein adsorption, thereby preventing the active center of the enzyme protein from being covered. The adsorption efficiency of the enzyme protein on the macroporous resin is improved, and L-histidine has strong buffering capacity. The enzyme catalyzes the biomass diesel reaction, and the oil contains a large amount of FFA, which accelerates the deactivation of the enzyme. L-histidine can inhibit the negative impact of free FFA on protein activity.

[0020] Further, in the step (3), the pretreated resin (dry weight) and the pretreated enzyme solution are used in a ratio of 1 g:(1.5-5) mL, preferably 1 g:(1.5-4) mL, and more preferably 1 g:(1.5-2.5) mL.

[0021] Further, in the step (3), the adsorption temperature is 25-45°C, preferably 30-40°C, and more preferably 35°C; and the adsorption time is 3-7 h, preferably 4-6 h, and more preferably 5 h.

[0022] Further, in the step (3), the drying method is low-temperature vacuum drying; the drying temperature is 35-55℃, preferably 35-45℃, more preferably 40℃; and the drying time is 12-20h, preferably 12-15h, more preferably 14h.

[0023] The above further beneficial effects are that, by low-temperature vacuum drying, the volatile components such as water and organic solvents in the resin are removed.

[0024] According to the above technical solution, compared with the prior art, the present application has the following beneficial effects:

[0025] 1. The immobilized lipase has significant catalytic activity. In the catalysis of biomass diesel reaction, it has significant catalytic activity on free fatty acid (FFA) and methanol, can efficiently promote the esterification reaction of FFA and methanol to generate fatty acid methyl ester, and improves the synthesis efficiency of biomass diesel. Under certain reaction conditions, its catalytic efficiency is significantly improved compared with free lipase, and it shows excellent ability to accelerate reaction progress.

[0026] 2. The immobilized lipase has excellent stability. In the complex reaction system, it can still maintain the integrity of its molecular structure and the stability of its catalytic activity in the face of the strong polarity of methanol and the temperature fluctuations during the reaction. Compared with free enzyme, the immobilized lipase has significantly enhanced tolerance to environmental factors such as temperature and pH, can maintain high catalytic performance in a wide temperature and pH range, reduces enzyme inactivation caused by environmental changes, and ensures the continuous and stable progress of biomass diesel reaction.

[0027] 3. The immobilized lipase has high selectivity. In the reaction of catalyzing FFA and methanol to generate fatty acid methyl ester, it can accurately recognize FFA and methanol as substrates, preferentially catalyze the esterification reaction between them, and has very low catalytic activity on other impurities or side reactions that may exist in the system, effectively reducing the generation of by-products, improving the purity of fatty acid methyl ester and the quality of biomass diesel.

[0028] 4. The immobilized lipase has outstanding repeated use performance. After completing a biomass diesel catalytic reaction, the immobilized enzyme can be recovered and reused in the next reaction through simple separation operation. After repeated use for many times, its catalytic activity can still remain at a high level, greatly reducing the production cost and improving the economic efficiency and sustainability of the biomass diesel production process.

[0029] 5、The immobilized carrier plays a key role in the performance of the lipase. The carrier has specific physical and chemical properties, such as high specific surface area, suitable pore size distribution and good hydrophobicity, which can provide a stable immobilization environment for the lipase. On the one hand, the specific interaction (such as covalent bond combination, adsorption, etc.) between the carrier and the lipase realizes the firm fixation of the enzyme, preventing the leakage of the enzyme during the reaction; on the other hand, the microstructure of the carrier is beneficial to the mass transfer of the substrate and the product, promoting the smooth progress of the catalytic reaction, thereby synergistically improving the overall performance of the immobilized lipase in the biomass diesel reaction.

[0030] 6、Through a large number of studies on the reaction of lipase catalyzing the synthesis of biomass diesel from kitchen waste oil, a series of enzyme catalysis kitchen waste oil synthesis biomass diesel reaction characteristics are found and summarized. According to the obtained conclusion, a series of resins are screened. Through specific immobilization research on various resins, a preparation method of biomass diesel immobilized lipase is obtained. The biomass diesel immobilized lipase prepared by the application can reduce the content of free FFA in the biomass diesel reaction to below 0.5%, and the recycling number is more than 14 times, which has the characteristics of simple preparation process, excellent catalytic performance, greatly improved stability and greatly reduced production cost.

[0031] 7、The application provides a simple and effective method for immobilizing lipase, which provides a product of lipase immobilized on a carrier with excellent catalytic performance and a product with better stability in the use process of terminal application.

[0032] 8、The immobilization type of the immobilized lipase is physical adsorption, without introducing crosslinking agent, and the negative influence on the enzyme molecular structure is minimized. The production process is simple, and compared with the immobilized enzyme product obtained by the known immobilization method, the immobilized lipase has higher enzyme activity, the content of FFA in the product is reduced to the optimal level at present, the quality of biomass diesel is improved, and the stability is better in the use process of terminal application, the pH tolerance is stronger, and the recycling number is also increased compared with other products.

[0033] 9、The immobilization process of the application is simple, the amount of enzyme protein consumed in the immobilization process is less, the catalytic performance and stability are greatly improved, and the application has great advantages in economy and product competitiveness. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is a catalytic performance comparison chart of the immobilized lipase of example 1 loaded with different types of polystyrene resins;

[0035] Figure 2 It is a catalytic performance comparison chart of the immobilized lipase of example 2 added with different amounts of L-lysine;

[0036] Figure 3 The catalytic performance comparison chart of the immobilized lipase after adding different amounts of L-arginine to Example 3;

[0037] Figure 4 The catalytic performance comparison chart of the immobilized lipase after adding different amounts of L-histidine to Example 4;

[0038] Figure 5 The catalytic performance comparison chart of the immobilized lipase after adding different amounts of L-cysteine to Example 5;

[0039] Figure 6 The catalytic performance comparison chart of the immobilized lipase after loading different amounts of enzyme liquid to Example 6;

[0040] Figure 7 The cycle number test result chart of the immobilized lipase of Example 7;

[0041] Figure 8 The optical microscope appearance integrity comparison chart of the immobilized lipase of Example 7 before and after the strength test;

[0042] Figure 9 The particle size distribution curve comparison chart of the immobilized lipase of Example 7 before and after the strength test;

[0043] Figure 10 The cycle number test result chart of the commercially available immobilized lipase A of Comparative Example 1;

[0044] Figure 11 The cycle number test result chart of the commercially available immobilized lipase B of Comparative Example 2;

[0045] Figure 12 The catalytic performance and cycle number test result chart of the commercially available immobilized lipase C of Comparative Example 3. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0047] Example 1

[0048] The preparation method of the biomass diesel immobilized lipase specifically includes the following steps:

[0049] (1) Polystyrene resin (301) with surface modification type C4 was dried in a 121℃ oven for 2h, 2g was weighed, swelled in 8mL methanol for 0.5h, suction filtered, washed with deionized water, and dried to obtain pretreated resin;

[0050] (2) 30mL of enzyme solution of biomass diesel lipase was taken, 3.834g of L-lysine (0.7M) was added, and stirred and dissolved to obtain pretreated enzyme solution;

[0051] (3) 8mL of enzyme solution was added to 2g of pretreated resin, mixed uniformly at 30℃, and then stirred for 3h for adsorption, the enzyme solution was removed by suction filtration, washed with deionized water, and dried in a 55℃ vacuum drying oven for 12h to obtain biomass diesel immobilized lipase.

[0052] Example 2

[0053] The preparation method of biomass diesel immobilized lipase specifically includes the following steps:

[0054] (1) Polystyrene resin (301) with surface modification type C4 was dried in a 121℃ oven for 2h, 2g was weighed, swelled in 8mL methanol for 0.5h, suction filtered, washed with deionized water, and dried to obtain pretreated resin;

[0055] (2) 8mL of enzyme solution of biomass diesel lipase was taken, 0.584g of L-lysine (0.4M) was added, and stirred and dissolved to obtain pretreated enzyme solution;

[0056] (3) 8mL of enzyme solution was added to 2g of pretreated resin, mixed uniformly at 30℃, and then stirred for 3h for adsorption, the enzyme solution was removed by suction filtration, washed with deionized water, and dried in a 55℃ vacuum drying oven for 12h to obtain biomass diesel immobilized lipase.

[0057] Example 3

[0058] The preparation method of biomass diesel immobilized lipase specifically includes the following steps:

[0059] (1) Polystyrene resin (301) with surface modification type C4 was dried in a 121℃ oven for 2h, 2g was weighed, swelled in 8mL methanol for 0.5h, suction filtered, washed with deionized water, and dried to obtain pretreated resin;

[0060] (2) 8mL of enzyme solution of biomass diesel lipase was taken, 0.584g of L-lysine (0.4M) was added, and stirred and dissolved to obtain pretreated enzyme solution;

[0061] (3) Add 8 mL of enzyme solution to 2 g of pretreated resin, mix at 30 °C and stir for 3 h for adsorption, filter to remove enzyme solution, wash with deionized water, and dry in a vacuum drying oven at 55 °C for 12 h to obtain biomass diesel immobilized lipase.

[0062] Example 4

[0063] The preparation method of biomass diesel immobilized lipase specifically includes the following steps:

[0064] (1) Place the polystyrene resin (301) with surface modification type C4 in an oven at 121℃ and dry for 2 hours. Weigh 2g, add 8mL of methanol to swell for 0.5 hours, filter, wash with deionized water, and dry to obtain the pretreated resin.

[0065] (2) Take 8 mL of biomass diesel lipase enzyme solution, add 0.372 g L-histidine (0.3 M), stir to dissolve, and obtain the pretreated enzyme solution;

[0066] (3) Add 8 mL of enzyme solution to 2 g of pretreated resin, mix at 30 °C and stir for 3 h for adsorption, filter to remove enzyme solution, wash with deionized water, and dry in a vacuum drying oven at 55 °C for 12 h to obtain biomass diesel immobilized lipase.

[0067] Example 5

[0068] The preparation method of biomass diesel immobilized lipase specifically includes the following steps:

[0069] (1) Place the polystyrene resin (301) with surface modification type C4 in an oven at 121℃ and dry for 2 hours. Weigh 2g, add 8mL of methanol to swell for 0.5 hours, filter, wash with deionized water, and dry to obtain the pretreated resin.

[0070] (2) Take 8 mL of biomass diesel lipase enzyme solution, add 0.485 g L-cysteine ​​(0.5 M), stir to dissolve, and obtain the pretreated enzyme solution;

[0071] (3) Add 8 mL of enzyme solution to 2 g of pretreated resin, mix at 30 °C and stir for 3 h for adsorption, filter to remove enzyme solution, wash with deionized water, and dry in a vacuum drying oven at 55 °C for 12 h to obtain biomass diesel immobilized lipase.

[0072] Example 6

[0073] The preparation method of biomass diesel immobilized lipase specifically includes the following steps:

[0074] (1) Polystyrene resin (301) with surface modification type C4 was dried in a 121 °C oven for 2 h, 2 g was weighed, swelled in 8 mL of methanol for 0.5 h, suction filtered, washed with deionized water, and dried to obtain the pretreated resin;

[0075] (2) 4 mL of enzyme solution of biomass diesel lipase was taken, 0.209 g of L-arginine (0.3 M) was added, and stirred and dissolved to obtain the pretreated enzyme solution;

[0076] (3) 4 mL of enzyme solution was added to 2 g of the pretreated resin, mixed at 30 °C, and stirred for 3 h for adsorption, the enzyme solution was removed by suction filtration, washed with deionized water, and dried in a 55 °C vacuum drying oven for 12 h to obtain the biomass diesel immobilized lipase.

[0077] Example 7

[0078] The method for preparing the biomass diesel immobilized lipase specifically comprises the following steps:

[0079] (1) Polystyrene resin (301) with surface modification type C4 was dried in a 121 °C oven for 2 h, 8 g was weighed, swelled in 32 mL of methanol for 0.5 h, suction filtered, washed with deionized water, and dried to obtain the pretreated resin;

[0080] (2) 16 mL of enzyme solution of biomass diesel lipase was taken, 0.836 g of L-arginine (0.3 M) was added, and stirred and dissolved to obtain the pretreated enzyme solution;

[0081] (3) 16 mL of enzyme solution was added to 8 g of the pretreated resin, mixed at 30 °C, and stirred for 3 h for adsorption, the enzyme solution was removed by suction filtration, washed with deionized water, and dried in a 55 °C vacuum drying oven for 12 h to obtain the biomass diesel immobilized lipase.

[0082] Comparative Example 1

[0083] A commercially available biomass diesel immobilized lipase A.

[0084] Comparative Example 2

[0085] A commercially available biomass diesel immobilized lipase B.

[0086] Comparative Example 3

[0087] A biomass diesel immobilized lipase C.

[0088] Performance test

[0089] The immobilized lipase has certain requirements for the strength of the resin, and the strength performance test method of the resin is as follows: the water bath is heated to 40℃, 50-100g of sigma 70% methyl oleate is weighed into a glass bottle, and 1% immobilized lipase is added. Put it into the water bath for incubation, connect the stirring paddle and cantilever stirring table, rotate at 50rpm, and stir for 7 days.

[0090] The test method of the catalytic performance of the immobilized lipase is as follows: mix methyl ester and oleic acid, control the FFA% content at 2%-5%, and the water content is <0.1%. If the water content is high, remove the water under vacuum. If necessary, add NaOH solution to adjust the pH of the raw material to >4. Weigh the raw material, add 2% immobilized lipase and 10% methanol according to the mass ratio. After taking the material, tightly cover the bottle cap to prevent gas leakage, and put the reactant into a shaking bed for reaction at 40℃ and 200-250rpm for 4h. After the reaction is completed, take the sample and centrifuge to remove the immobilized lipase remaining in the supernatant. Put the sample in a conical flask and heat to 121℃ for 0.5h to remove the unreacted methanol in the sample. Test the FFA% content in the sample according to the method in GB 5009.229-2016 "Determination of acid value in food safety national standard food".

[0091] The test method of the catalytic performance of the immobilized lipase is as follows: repeat the steps of the catalytic performance test method of the immobilized lipase, filter and remove the raw material in the immobilized lipase after each reaction, rinse with n-hexane, and re-enter the newly prepared reactant. Repeat the reaction and test until the catalytic performance of the immobilized lipase begins to decline.

[0092] 1. Example 1 Test of the catalytic performance of the immobilized lipase

[0093] Take the following three types of resins: polystyrene resin with surface modification type C4 (301), polystyrene resin with surface modification type C8 (302), and polystyrene resin with surface modification type C18 (303). The modification types of the three resins are different, and their surface hydrophobicity increases in turn. The selectivity and stability of enzyme protein adsorbed on the resins are different. Biomass diesel immobilized lipase is prepared according to the method of Example 1.

[0094] Weigh 15g of methyl palmitate, 0.57g of oleic acid, 0.3g of 1-monopalmitin, 0.3g of tripalmitin, and 2.1g of methanol. Weigh three portions in turn, seal and preheat at 40℃ for 15min, add 0.45g of immobilized lipase to each portion, react for 4h, centrifuge to remove the resin from each sample, weigh 1g of the sample for FFA% test, and test according to the method in GB 5009.229-2016.

[0095] The FFA% of the substrate before reaction is 3.1%, and the test results are as followsFigure 1 As shown in Table 1, the final immobilized lipase catalytic results of the three resins under the same loading conditions are: C4(FFA%): 0.85%, C8(FFA%): 1.01%, C18(FFA%): 1.85%. It can be seen that within a certain range, the higher the degree of surface modification hydrophobicity, the less conducive to the progress of biomass diesel reaction. The polystyrene resin modified by C4 is the most suitable for preparing immobilized lipase.

[0096] 2. Catalytic performance test of immobilized lipase in Example 2

[0097] 7 portions of biomass diesel lipase enzyme solution were measured, each portion was 8 mL, and L-lysine was added in sequence, 0.146 g (0.1 M), 0.292 g (0.2 M), 0.438 g (0.3 M), 0.584 g (0.4 M), 0.731 g (0.5 M), 0.877 g (0.6 M), 1.02 g (0.7 M), and stirred and dissolved. Biomass diesel immobilized lipase was prepared according to the method of Example 2.

[0098] 15 g of methyl palmitate, 0.57 g of oleic acid, 0.3 g of 1-monopalmitin, 0.3 g of tripalmitin, and 2.1 g of methanol were weighed. 7 portions were weighed in sequence, preheated at 40°C for 15 min, 0.45 g of immobilized lipase was added to each portion, reacted for 4 h, and then sampled, centrifuged to remove the resin, and 1 g of sample was weighed for FFA% test according to the method in GB 5009.229-2016.

[0099] The FFA% before the substrate was not reacted was 3.1%, and the test results are shown in Table 2. Figure 2 As shown in Table 2, the final immobilized lipase catalytic results of the seven loading conditions are: L-lysine 0.1M(FFA%): 0.90%, L-lysine 0.2M(FFA%): 0.71%, L-lysine 0.3M: 0.62%, L-lysine 0.4M(FFA%): 0.52%, L-lysine 0.5M(FFA%): 0.63%, L-lysine 0.6M(FFA%): 0.77%, L-lysine 0.7M(FFA%): 0.85%. It can be seen that within a certain range, the amount of L-lysine added in the enzyme solution has a significant effect on the catalytic performance of the immobilized lipase. The optimal amount of L-lysine is about 0.4M.

[0100] 3. Catalytic performance test of immobilized lipase in Example 3

[0101] Take 7 portions of enzyme liquid, each 8 mL, and add L-arginine 0.139 g (0.1 M), 0.279 g (0.2 M), 0.418 g (0.3 M), 0.557 g (0.4 M), 0.697 g (0.5 M), 0.836 g (0.6 M), and 0.976 g (0.7 M) into the enzyme liquid in sequence and stir to dissolve. Prepare the biomass diesel immobilized lipase according to the method of Example 3.

[0102] Take 15 g of methyl palmitate, 0.57 g of oleic acid, 0.3 g of 1-monopalmitin, 0.3 g of tripalmitin, and 2.1 g of methanol. Take 7 portions in sequence, preheat at 40 °C for 15 min, add 0.45 g of immobilized lipase to each portion, react for 4 h, centrifuge to remove the resin from the sample, take 1 g of the sample for FFA% test, and test according to the method in GB 5009.229-2016.

[0103] The FFA% before the substrate is not reacted is 3.1%, and the test results are shown in Table 1. Figure 3 The final catalytic results of the immobilized lipase under the 7 loading conditions are as follows: L-arginine 0.1 M (FFA%): 1.05%, L-arginine 0.2 M (FFA%): 0.87%, L-arginine 0.3 M: 0.63%, L-arginine 0.4 M (FFA%): 0.71%, L-arginine 0.5 M (FFA%): 0.87%, L-arginine 0.6 M (FFA%): 1.10%, and L-arginine 0.7 M (FFA%): 1.25%. It can be seen that within a certain range, the amount of L-arginine added to the enzyme liquid has a significant effect on the catalytic performance of the immobilized lipase. The optimal amount of L-arginine is about 0.3 M.

[0104] 4. Catalytic performance test of the immobilized lipase in Example 4

[0105] Take 7 portions of enzyme liquid, each 8 mL, and add L-arginine 0.139 g (0.1 M), 0.279 g (0.2 M), 0.418 g (0.3 M), 0.557 g (0.4 M), 0.697 g (0.5 M), 0.836 g (0.6 M), and 0.976 g (0.7 M) into the enzyme liquid in sequence and stir to dissolve. Prepare the biomass diesel immobilized lipase according to the method of Example 3.

[0106] Take 15 g of methyl palmitate, 0.57 g of oleic acid, 0.3 g of 1-monopalmitin, 0.3 g of tripalmitin, and 2.1 g of methanol. Take 7 portions in sequence, preheat at 40 °C for 15 min, add 0.45 g of immobilized lipase to each portion, react for 4 h, centrifuge to remove the resin from the sample, take 1 g of the sample for FFA% test, and test according to the method in GB 5009.229-2016.

[0107] Before the substrate reacted, the FFA% was 3.1%, as shown in the test results. Figure 4 As shown, the catalytic performance of the immobilized lipase under the seven loading conditions was as follows: L-histidine 0.1M (FFA%): 0.90%, L-histidine 0.2M (FFA%): 0.61%, L-histidine 0.3M: 0.43%, L-histidine 0.4M (FFA%): 0.52%, L-histidine 0.5M (FFA%): 0.60%, L-histidine 0.6M (FFA%): 0.71%, and L-histidine 0.7M (FFA%): 0.85%. This demonstrates that within a certain range, the amount of L-histidine added to the enzyme solution significantly affects the catalytic performance of the immobilized lipase. The optimal amount of L-histidine is approximately 0.3M.

[0108] 5. Catalytic performance test of immobilized lipase in Example 5

[0109] Seven 8 mL portions of enzyme solution were measured, and L-cysteine ​​was added sequentially to each portion at concentrations of 0.097 g (0.1 M), 0.194 g (0.2 M), 0.291 g (0.3 M), 0.387 g (0.4 M), 0.485 g (0.5 M), 0.582 g (0.6 M), and 0.678 g (0.7 M), respectively. The solutions were stirred until dissolved. Biomass diesel-immobilized lipases were prepared according to the method described in Example 5.

[0110] Weigh out 15g of methyl palmitate, 0.57g of oleic acid, 0.3g of 1-monopalmitoylglycerol, 0.3g of trimalmitoylglycerol, and 2.1g of methanol. Take 7 portions of each portion, seal them, and preheat at 40℃ for 15 minutes. Add 0.45g of immobilized lipase to each portion and react for 4 hours. Centrifuge each portion to remove the resin, and weigh out 1g of the sample for FFA% testing according to the method in GB 5009.229-2016.

[0111] Before the substrate reacted, the FFA% was 3.1%, as shown in the test results. Figure 5 As shown, the catalytic results of the immobilized lipase under the seven loading conditions were as follows: L-cysteine ​​0.1M (FFA%): 1.01%, L-cysteine ​​0.2M (FFA%): 0.88%, L-cysteine ​​0.3M: 0.72%, L-cysteine ​​0.4M (FFA%): 0.63%, L-cysteine ​​0.5M (FFA%): 0.51%, L-cysteine ​​0.6M (FFA%): 0.66%, and L-cysteine ​​0.7M (FFA%): 0.79%. This indicates that within a certain range, the amount of L-lysine added to the enzyme solution significantly affects the catalytic performance of the immobilized lipase. The optimal amount of L-cysteine ​​is approximately 0.5M.

[0112] 6. Example 6 Catalytic performance test of immobilized lipase

[0113] Take 5 portions of enzyme solution, the proportion is 3 mL, 4 mL, 6 mL, 8 mL, 10 mL respectively. Add L-arginine 0.157 g (0.3 M), 0.209 g (0.3 M), 0.314 g (0.3 M), 0.418 g (0.3 M), 0.523 g (0.3 M) respectively in 5 portions of enzyme solution, and stir to dissolve. Prepare the biomass diesel immobilized lipase according to the method of Example 6.

[0114] Take 15 g of methyl palmitate, 0.57 g of oleic acid, 0.3 g of 1-monopalmitin, 0.3 g of tripalmitin, and 2.1 g of methanol. Take 4 portions in turn, seal and preheat at 40°C for 15 min, add 0.45 g of immobilized lipase to each portion, react for 4 h, centrifuge to remove the resin, take 1 g of sample for FFA% test, and test according to the method in GB 5009.229-2016.

[0115] The FFA% of the substrate before reaction is 3.1%, and the test results are shown in Figure 6 The final catalytic results of the immobilized lipase under the five loading conditions are: 1:1.5 (FFA%): 0.49%, 1:2 (FFA%): 0.38%, 1:3 (FFA%): 0.42%, 1:4 (FFA%): 0.47%, and 1:5 (FFA%): 0.51%. It can be seen that within a certain range, the amount of enzyme solution added has a certain effect on the catalytic performance of the immobilized lipase. Considering the cost and performance, the optimal amount of enzyme solution added is about 1:2.

[0116] 7. Example 7 Cycle number test of immobilized lipase

[0117] Take 15 g of methyl palmitate, 0.57 g of oleic acid, 0.3 g of 1-monopalmitin, 0.3 g of tripalmitin, and 2.1 g of methanol. Take 3 portions in turn, seal and preheat at 40°C for 15 min, add 0.45 g of immobilized lipase to each portion, react for 4 h, centrifuge to remove the resin, take 1 g of sample for FFA% test, and test according to the method in GB 5009.229-2016.

[0118] Repeat the above immobilized lipase performance test steps until the catalytic performance of the immobilized lipase begins to decline, and the test is completed.

[0119] The test results are shown in Figure 7 The first catalytic result (FFA%) of the immobilized lipase is 0.38%, and the performance begins to decline after 14 cycles, with a result (FFA%) of 0.48%.

[0120] 8. Strength test of immobilized lipase of Example 7

[0121] After determining the most suitable resin and loading method for loading (Example 7), a strength test was performed on the immobilized enzyme, with the standard being that there was no difference in the integrity of the immobilized enzyme before and after the test.

[0122] The water bath was heated to 40°C, 100 g of sigma 70% methyl oleate was weighed into a glass bottle, and 1 g of immobilized lipase was added. It was placed in the water bath for incubation, and a stirring paddle and cantilevered stirring platform were connected. The stirring speed was 50 rpm. Stirring was performed for 7 days. The particle size before and after stirring was observed, and the particle size distribution curve was measured.

[0123] The results of the observation under the optical microscope are shown in Figure 8 The particle size before and after stirring remained consistent.

[0124] The particle size distribution curve was measured by a laser particle size analyzer, and the results are shown in Figure 9 The particle size distribution remained consistent before and after the test, indicating that the strength of the immobilized lipase met the requirements.

[0125] 9. Catalytic performance and stability test of immobilized lipase of Comparative Example 1

[0126] A commercially available biomass diesel immobilized lipase A was used to perform a biomass diesel catalytic reaction to detect its catalytic performance and stability. It was compared with the self-made biomass diesel immobilized lipase.

[0127] 5 g of methyl palmitate, 0.19 g of oleic acid, 0.1 g of 1-monopalmitin, 0.1 g of tripalmitin, and 0.7 g of methanol were weighed. After being sealed and preheated at 40°C for 15 min, 0.15 g of immobilized lipase A was added, and the reaction was performed for 4 h. The sample was centrifuged to remove the resin, 1 g of the sample was weighed for FFA% (acid value) test, and the test was performed according to the method in GB 5009.229-2016.

[0128] The FFA% of the substrate before reaction was 3.1%, and the catalytic result (FFA%) of the immobilized lipase A was 0.65%. It can be seen that the self-made immobilized lipase is superior to the immobilized lipase A in performance.

[0129] 10. Cycle number test of immobilized lipase of Comparative Example 1

[0130] A commercially available biomass diesel immobilized lipase A was used to perform a biomass diesel catalytic reaction to detect its catalytic performance and stability. It was compared with the self-made biomass diesel immobilized lipase.

[0131] Take 5 g of methyl palmitate, 0.19 g of oleic acid, 0.1 g of 1-monopalmitin, 0.1 g of tripalmitin, and 0.7 g of methanol. Seal and preheat at 40°C for 15 min, then add 0.15 g of immobilized lipase A, and react for 4 h. Take a sample, centrifuge to remove the resin, take 1 g of the sample for FFA% test, and test according to the method in GB 5009.229-2016. Repeat the performance test steps of the immobilized lipase A in Comparative Example 1 until the catalytic performance of the immobilized lipase begins to decline, and the test is completed.

[0132] The test results are shown in Table 1. Figure 10 The first catalytic result (FFA%) of the immobilized lipase A is 0.65%. After 9 cycles, the performance begins to decline significantly, and the result (FFA%) is 0.81%.

[0133] 11. Number of cycles of the immobilized lipase in Comparative Example 2

[0134] A commercially available biomass diesel immobilized lipase B was used for biomass diesel catalytic reaction to detect the number of repeated use of its catalytic performance, and compared with the self-made biomass diesel immobilized lipase.

[0135] Take 5 g of methyl palmitate, 0.19 g of oleic acid, 0.1 g of 1-monopalmitin, 0.1 g of tripalmitin, and 0.7 g of methanol. Seal and preheat at 40°C for 15 min, then add 0.15 g of immobilized lipase A, and react for 4 h. Take a sample, centrifuge to remove the resin, take 1 g of the sample for FFA% test, and test according to the method in GB 5009.229-2016. Repeat the performance test steps of the immobilized lipase A in Comparative Example 1 until the catalytic performance of the immobilized lipase begins to decline, and the test is completed.

[0136] The test results are shown in Table 1. Figure 11 The first catalytic result (FFA%) of the immobilized lipase B is 0.75%. After 5 cycles, the performance begins to decline significantly, and the result (FFA%) is 1.18%.

[0137] 12. Number of cycles of the immobilized lipase in Comparative Example 3

[0138] A commercially available biomass diesel immobilized lipase C was used for biomass diesel catalytic reaction to detect the number of repeated use of its catalytic performance, and compared with the self-made biomass diesel immobilized lipase.

[0139] Take 5 g of methyl palmitate, 0.19 g of oleic acid, 0.1 g of 1-monopalmitin, 0.1 g of tripalmitin, and 0.7 g of methanol. Seal and preheat at 40°C for 15 min, add 0.15 g of immobilized lipase A, react for 4 h, centrifuge to remove the resin, take 1 g of the sample for FFA% (acid value) test according to the method in GB 5009.229-2016. Repeat the performance test steps of immobilized lipase A in Comparative Example 1 until the catalytic performance of the immobilized lipase begins to decline, and the test is completed.

[0140] The test results are shown in Table 1. Figure 12 The first catalytic results (FFA%) of the immobilized lipase are 0.70%, and the performance begins to decline significantly after 6 cycles, with a result (FFA%) of 0.92%.

[0141] The biomass diesel immobilized lipase in the above comparative examples is a mainstream product on the market, and its performance belongs to the first echelon. However, compared with the self-made immobilized lipase, there is a big difference in performance. Therefore, the self-made immobilized lipase technology in the present application is a major breakthrough in performance and economy.

[0142] There are many biomass diesel immobilized enzyme products on the market, such as Novozymes and Condea, etc. Their products have excellent catalytic performance, but in the biomass diesel catalytic reaction, FFA can only be reduced to about 0.6, and the number of cycles is less than 10. The immobilized enzyme prepared by the present application can reduce FFA to below 0.5 in the biomass diesel catalytic reaction, and the number of cycles reaches 14, greatly improving the quality of oil products and having great advantages in economy.

[0143] The immobilized lipase product obtained by the method of the present application has higher enzyme activity than known immobilized lipases and greatly improves the ability to inhibit enzyme activity decline. Another important aspect of the present application is that this immobilization method is easy to scale up by applying other larger standard equipment, so the equipment setting range given above can be adjusted to optimize larger equipment.

[0144] The above description of the disclosed embodiments enables those skilled in the art to carry out or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a biomass-diesel immobilized lipase, characterized by, Specifically comprising the following steps: (1) drying polystyrene resin, adding methanol for swelling, suction filtration, washing, drying, to obtain pretreated resin; (2) adding L-lysine, L-arginine, L-histidine or L-cysteine to the enzyme solution of the biomass diesel fat enzyme, stirring and dissolving to obtain pretreated enzyme solution; (3) adding the pretreated enzyme solution to the pretreated resin for adsorption, suction filtration, washing, drying, to obtain the biomass diesel immobilized fat enzyme.

2. The method for preparing biomass diesel-immobilized lipase according to claim 1, characterized in that, In step (1), the polystyrene resin is a polystyrene resin with C4 surface modification type, in the form of spherical particles, with a particle size of 400-800 μm.

3. The method for preparing biomass diesel-immobilized lipase according to claim 2, characterized in that, In step (1), the amount ratio of polystyrene resin to methanol is 1g:(2-6)mL.

4. The method for preparing biomass diesel-immobilized lipase according to claim 1, characterized in that, In step (1), the swelling time is 0.5h.

5. The method for preparing biomass diesel-immobilized lipase according to claim 1, characterized in that, In step (2), the concentration of L-lysine, L-arginine, L-histidine or L-cysteine in the pretreated enzyme solution is 0.1-0.7M.

6. The method for preparing biomass diesel-immobilized lipase according to claim 1, characterized in that, In step (3), the amount ratio of pretreated resin to pretreated enzyme solution is 1g:(1.5-5)mL.

7. The method for preparing biomass diesel-immobilized lipase according to claim 1, characterized in that, In step (3), the adsorption temperature is 25-45℃, and the time is 3-7h.

8. The method for preparing biomass diesel-immobilized lipase according to claim 1, characterized in that, In step (3), the drying method is low-temperature vacuum drying, the temperature is 35-55℃, and the time is 12-20h.

Images