A lipase-sepiolite composite microcapsule catalyst for biodiesel and a preparation method thereof
By preparing a lipase@sepiolite composite microcapsule catalyst, the problem of enzyme inactivation in enzymatic biodiesel production was solved, thereby improving enzyme stability and activity, reducing costs, and increasing conversion rate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI ZHONGQI ENVIRONMENTAL PROTECTION TECHNOLOGY CO LTD
- Filing Date
- 2024-01-11
- Publication Date
- 2026-06-09
AI Technical Summary
In existing enzymatic methods for biodiesel production, the enzymes have short catalytic lifespans, and immobilized enzymes are prone to inactivation and difficult to separate, which limits the development of enzymatic biodiesel production.
Using a lipase@sepiolite composite microcapsule catalyst, PB011 lipase was embedded in a Pickering emulsion with siloxane-modified sepiolite as the capsule wall to form a biomimetic microcapsule, thereby controlling the chemical reaction of the enzyme to take place in a tiny area.
It improves enzyme stability and activity, enhances reaction efficiency, reduces the cost of enzymatic synthesis of biodiesel, and ensures high conversion rate.
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Figure CN117839771B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biodiesel preparation technology, specifically relating to a lipase@sepiolite composite microcapsule catalyst for biodiesel and its preparation method. Background Technology
[0002] Biodiesel is a clean and safe new fuel produced from renewable resources such as vegetable or animal oils, which can replace petrochemical diesel. Its main components are a mixture of a series of long-chain fatty acid esters. Currently, biodiesel production mainly involves chemical and biological methods. The biological method is simple to operate, but the resulting product may undergo varying degrees of sintering during use. Chemical methods mainly include thermal cracking and transesterification. Enzymatic biodiesel production refers to the process of using lipases as catalysts to transesterify alcohols with vegetable oils to produce fatty acid esters. However, because many enzymes are highly dependent on water, and the alcohol system in the biodiesel transesterification process also affects enzyme activity, the catalytic life of the enzymes is relatively short, limiting the development of enzymatic biodiesel production. Summary of the Invention
[0003] To address the shortcomings of existing technologies, the present invention aims to provide a lipase@sepiolite composite microcapsule catalyst for biodiesel and its preparation method. Siloxane-modified sepiolite (APTES-SP) is used as a stabilizer for the oil-water interface in Pickering emulsions (Pickering emulsions refer to emulsions obtained using ultrafine solid particles as emulsifiers), and simultaneously serves as the capsule wall of the constructed microcapsules, embedding PB011 lipase within them.
[0004] To achieve the above objectives, the solution of the present invention is:
[0005] A method for preparing a lipase@sepiolite composite microcapsule catalyst, comprising the following steps:
[0006] (1) Add sepiolite to hydrochloric acid and seal for water bath reaction. Wash until pH 5, dry, sieve, then add calcium and distilled water and mix evenly. When a slurry is formed, dry it and wash repeatedly to obtain alkaline calcium-based sepiolite.
[0007] (2) Disperse alkaline calcium sepiolite in a mixture of anhydrous ethanol and water, add a modifier, stir to react, wash, and dry to obtain amphiphilic sepiolite.
[0008] (3) Prepare an amphiphilic sepiolite solution in toluene solution and disperse it by ultrasonication. Prepare a lipase solution in phosphate buffer solution and add it to the amphiphilic sepiolite solution. Homogenize to obtain lipase@sepiolite composite microcapsule catalyst.
[0009] Sepiolite is a monazite silicate mineral with an orthorhombic crystal system and space group Pna2(1). Its unit cell parameters are generally... The basic unit of the sepiolite crystal structure is the silicon-oxygen tetrahedron, with the chemical formula SiO4. In the sepiolite crystal structure, these silicon-oxygen tetrahedra interconnect to form a series of infinitely extending chain-like structures. These chain-like structures are arranged in space to form a special network structure. Each vertex of the silicon-oxygen tetrahedron is connected to three other tetrahedra, forming a network structure. The chemical formula of sepiolite is (Ca,Na)2-3Al3(AlSi3O 10 (OH)2, in which Ca and Na ions are located in the gaps of the network structure composed of silicon-oxygen tetrahedra, while Al ions are located outside the network structure.
[0010] The sepiolite crystal structure exhibits a high degree of symmetry, with multiple symmetry elements appearing in the unit cell. Most importantly, it displays tetrahedral symmetry, with all four connecting dihedral angles greater than 90 degrees. If the silicon-oxygen tetrahedron is considered the fundamental unit, the symmetry of the sepiolite crystal structure manifests as a 48-point group symmetry.
[0011] Furthermore, in step (1), the temperature of the water bath reaction is 75-95℃ and the time is 3-5h.
[0012] Furthermore, in step (1), the drying temperature is 101-110℃.
[0013] Furthermore, in step (1), the drying reaction temperature is 45-65℃ and the time is 15-20h.
[0014] Furthermore, in step (2), the volume ratio of anhydrous ethanol to water in the mixture of anhydrous ethanol and water is 72:25.
[0015] Furthermore, in step (2), the modifier is 3-aminopropyltriethoxysilane.
[0016] Furthermore, in step (2), the stirring temperature is 70-90℃, the stirring speed is 400-600rpm, and the stirring time is 9-11h.
[0017] Furthermore, in step (2), the drying temperature is 50-70℃.
[0018] Furthermore, in step (3), the lipase is PB011 lipase.
[0019] PB011 lipase is simple to extract and inexpensive, costing more than ten times less than LpozymeTLIM, Novozym435, and Candida sp 99-12 lipases, which is beneficial for the industrialization of enzymatic biodiesel synthesis. Furthermore, PB011 lipase exhibits strong catalytic activity towards oleic acid and linoleic acid glycerides in oils. Therefore, using PB011 lipase to catalyze biodiesel synthesis is of great significance, as it can reduce the price of lipases used in the industrialization of enzymatic biodiesel synthesis, thus lowering costs while ensuring a high conversion rate of fatty acid methyl esters.
[0020] Enzyme catalysis takes place in organic or organic-aqueous mixed media. Enzyme immobilization is beneficial for improving enzyme stability and promoting enzyme recycling. Among various enzyme immobilization methods, enzyme encapsulation is a simple, mild, and universal method. It can create an aqueous microenvironment and utilize the "interfacial activation" mechanism to enhance enzyme activity in organic media.
[0021] Furthermore, in step (3), the homogenization time is 8-12 seconds.
[0022] A lipase@sepiolite composite microcapsule catalyst, which is obtained by the above preparation method.
[0023] Application of a lipase@sepiolite composite microcapsule catalyst as described above in biodiesel production.
[0024] Due to the adoption of the above solution, the beneficial effects of the present invention are:
[0025] Compared with conventional enzyme immobilization technology, biomimetic microcapsule enzyme immobilization technology can overcome the shortcomings of immobilized enzymes, such as easy inactivation and difficulty in separation. In biomimetic microcapsules, the enzyme chemical reaction is controlled in a small area, and the reactants / intermediates have a higher concentration in this confined microenvironment, thereby significantly improving the reaction efficiency. The sieving membrane of the biomimetic microcapsule has a selective effect on the guest molecules entering the capsule, making the reaction controllable. The special structure of the microcapsule is conducive to maintaining the stability and activity of the enzyme. Attached Figure Description
[0026] Figure 1 This is a schematic diagram of the structure of sepiolite. Detailed Implementation
[0027] The technical solution of the present invention will be further described in detail below with reference to several embodiments. These embodiments are implemented on the premise of the technical solution of the invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.
[0028] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.
[0029] Example 1:
[0030] (1) Preparation of amphiphilic sepiolite (APTES-VM)
[0031] Take 300 mL of 12% hydrochloric acid, stir and add 100 g of sepiolite, cover to prevent hydrochloric acid evaporation, react in a constant temperature water bath at 80℃ for 4 hours, filter and wash, rinse until pH is about 5 or chloride ions are absent, dry at 105℃, pulverize and pass through a 200-mesh sieve, take 10 g and put it into a 10 cm diameter flat-bottomed watch glass, add 3.5 g of Ca and 20 mL of distilled water and mix well. When a slurry material forms on the watch glass, remove the cover and place it in a 50℃ electric constant temperature drying oven for 18 hours. Take a sample every 2 hours, wash repeatedly to remove unreacted calcium hydroxide to obtain alkaline calcium-based sepiolite (Ca-SP).
[0032] 2.5 g of Ca-SP was dispersed in 100 mL of a mixture of anhydrous ethanol and water (75 / 25, v / v). After uniform ultrasonic dispersion, 3-aminopropyltriethoxysilane (APTES, 98%) was added. The mixture was stirred at 80 °C and 500 rpm for 10 h. The resulting product was washed three times with a mixture of ethanol and water and dried at 60 °C. The resulting product is amphiphilic sepiolite (APTES-VM).
[0033] (2) Preparation of lipase@sepiolite composite microcapsule catalyst
[0034] Using toluene as the oil phase and phosphate buffer as the aqueous phase, a lipase@sepiolite composite microcapsule (compound lipase@APTES-VM) with APTES-VM as the wall material and PB011 lipase as the core material was prepared using the Pickering emulsion method. Specifically, a 1 mg / mL APTES-VM solution was prepared in 2 mL of toluene solution and ultrasonically dispersed for 10 min to ensure uniform dispersion. 100 μL of a 4 mg / mL PB011 lipase solution was prepared in 0.1 M PBS buffer (pH 7.0) and added to the APTES-VM solution. The mixture was homogenized for 10 s to form a water-in-oil Pickering emulsion.
[0035] (3) Preparation of biodiesel
[0036] Soybean oil and ethanol (molar ratio 1:3) were added to the reactor. The ethanol was added in three portions. 3% lipase@sepiolite composite microcapsule catalyst (based on 100% of the soybean oil mass) was added. The enzyme-catalyzed reaction was carried out at 40°C for 70 hours. The lipase was recovered by filtration, and the filtrate was distilled to recover ethanol. The remaining portion was separated to filter out glycerol, and the remainder was biodiesel. The conversion rate was 97.2%. See Table 1.
[0037] Example 2:
[0038] (1) Preparation of amphiphilic sepiolite (APTES-VM)
[0039] Take 300 mL of 12% hydrochloric acid, stir and add 100 g of sepiolite. Cover to prevent hydrochloric acid evaporation, react in a 75℃ constant temperature water bath for 3 hours, filter and wash, rinse until pH is about 5 or chloride ions are absent, dry at 101℃, pulverize and pass through a 200-mesh sieve, take 10 g and put it into a 10 cm diameter flat-bottomed watch glass, add 3.5 g of Ca and 20 mL of distilled water and mix well. When a slurry material forms on the watch glass, remove the cover and place it in a 45℃ electric constant temperature drying oven for 15 hours. Take a sample every 2 hours, wash repeatedly to remove unreacted calcium hydroxide to obtain alkaline calcium-based sepiolite (Ca-SP).
[0040] 2.5 g of Ca-SP was dispersed in 100 mL of a mixture of anhydrous ethanol and water (75 / 25, v / v). After uniform ultrasonic dispersion, 3-aminopropyltriethoxysilane (APTES, 98%) was added. The mixture was stirred at 70 °C and 400 rpm for 9 h. The resulting product was washed three times with a mixture of ethanol and water and dried at 50 °C. The obtained product is amphiphilic sepiolite (APTES-VM).
[0041] (2) Preparation of lipase@sepiolite composite microcapsule catalyst
[0042] Using toluene as the oil phase and phosphate buffer as the aqueous phase, a lipase@sepiolite composite microcapsule (compound lipase@APTES-VM) with APTES-VM as the wall material and PB011 lipase as the core material was prepared using the Pickering emulsion method. Specifically, a 1 mg / mL APTES-VM solution was prepared in 2 mL of toluene and ultrasonically dispersed for 10 min to ensure uniform dispersion. 100 μL of a 4 mg / mL PB011 lipase solution was prepared in 0.1 M PBS buffer (pH 7.0) and added to the APTES-VM solution. The mixture was homogenized for 8 s to form a water-in-oil Pickering emulsion.
[0043] (3) Preparation of biodiesel
[0044] Soybean oil and ethanol (molar ratio 1:3) were added to the reactor. The ethanol was added in three portions. 3% lipase@sepiolite composite microcapsule catalyst (based on 100% of the soybean oil mass) was added. The enzyme-catalyzed reaction was carried out at 45°C for 72 hours. The lipase was recovered by filtration, the filtrate was distilled to recover ethanol, and the remaining portion was separated to filter out glycerol. The remainder was biodiesel, with a conversion rate of 95.8%. See Table 1.
[0045] Example 3:
[0046] (1) Preparation of amphiphilic sepiolite (APTES-VM)
[0047] Take 300 mL of 12% hydrochloric acid, stir and add 100 g of sepiolite. Cover to prevent hydrochloric acid evaporation, react in a 95℃ constant temperature water bath for 5 h, filter and wash, rinse until pH is about 5 or chloride ions are absent, dry at 110℃, pulverize and pass through a 200-mesh sieve, take 10 g and put it into a 10 cm diameter flat-bottomed watch glass, add 3.5 g of Ca and 20 mL of distilled water and mix well. When a slurry material forms on the watch glass, remove the cover and place it in a 65℃ electric constant temperature drying oven for 20 h. Take a sample every 2 h, wash repeatedly to remove unreacted calcium hydroxide to obtain alkaline calcium-based sepiolite (Ca-SP).
[0048] 2.5 g of Ca-SP was dispersed in 100 mL of a mixture of anhydrous ethanol and water (75 / 25, v / v). After uniform ultrasonic dispersion, 3-aminopropyltriethoxysilane (APTES, 98%) was added. The mixture was stirred at 90 °C and 600 rpm for 11 h. The resulting product was washed three times with a mixture of ethanol and water and dried at 70 °C. The obtained product is amphiphilic sepiolite (APTES-VM).
[0049] (2) Preparation of lipase@sepiolite composite microcapsule catalyst
[0050] Using toluene as the oil phase and phosphate buffer as the aqueous phase, a lipase@sepiolite composite microcapsule (compound lipase@APTES-VM) with APTES-VM as the wall material and PB011 lipase as the core material was prepared using the Pickering emulsion method. Specifically, a 1 mg / mL APTES-VM solution was prepared in 2 mL of toluene and ultrasonically dispersed for 10 min to ensure uniform dispersion. 100 μL of a 4 mg / mL PB011 lipase solution was prepared in 0.1 M PBS buffer (pH 7.0) and added to the APTES-VM solution. The mixture was homogenized for 12 s to form a water-in-oil Pickering emulsion.
[0051] (3) Preparation of biodiesel
[0052] Soybean oil and ethanol (molar ratio 1:3) were added to the reactor. The ethanol was added in three portions. 3% lipase@sepiolite composite microcapsule catalyst (based on 100% of the soybean oil mass) was added. The enzyme-catalyzed reaction was carried out at 60°C for 67 hours. The lipase was recovered by filtration, the filtrate was distilled to recover ethanol, and the remaining portion was separated to filter out glycerol. The remaining product was biodiesel, with a conversion rate of 96.4%. See Table 1.
[0053] Comparative Example 1:
[0054] (1) Preparation of amphiphilic sepiolite (APTES-VM)
[0055] Take 300 mL of 12% hydrochloric acid, stir and add 100 g of sepiolite, cover to prevent hydrochloric acid evaporation, react in a constant temperature water bath at 80℃ for 4 hours, filter and wash, rinse until pH is about 5 or chloride ions are absent, dry at 105℃, pulverize and pass through a 200-mesh sieve, take 10 g and put it into a 10 cm diameter flat-bottomed watch glass, add 3.5 g of Ca and 20 mL of distilled water and mix well. When a slurry material forms on the watch glass, remove the cover and place it in a 50℃ electric constant temperature drying oven for 18 hours. Take a sample every 2 hours, wash repeatedly to remove unreacted calcium hydroxide to obtain alkaline calcium-based sepiolite (Ca-SP).
[0056] 2.5 g of Ca-SP was dispersed in 100 mL of a mixture of anhydrous ethanol and water (75 / 25, v / v). After uniform ultrasonic dispersion, 3-aminopropyltriethoxysilane (APTES, 98%) was added. The mixture was stirred at 80 °C and 500 rpm for 10 h. The resulting product was washed three times with a mixture of ethanol and water and dried at 60 °C. The resulting product is amphiphilic sepiolite (APTES-VM).
[0057] (2) Preparation of sepiolite catalyst
[0058] Sepiolite with APTES-VM as the wall material was prepared using the Pickering emulsion method with toluene as the oil phase and phosphate buffer as the aqueous phase. The specific preparation method was as follows: A 1 mg / mL APTES-VM solution was prepared in 2 mL of toluene solution and ultrasonically dispersed for 10 min to ensure uniform dispersion. The solution was then homogenized for 10 s using a homogenizer to form a water-in-oil Pickering emulsion.
[0059] (3) Preparation of biodiesel
[0060] Soybean oil and ethanol (molar ratio 1:3) were added to the reactor in three portions. 3% sepiolite catalyst (based on 100% of the soybean oil mass) was added, and the enzyme-catalyzed reaction was carried out at 40°C for 70 hours. Lipase was recovered by filtration, the filtrate was distilled to recover ethanol, and the remaining portion was separated to filter out glycerol. The remaining product was biodiesel, with a conversion rate of 73.2%. See Table 1.
[0061] Table 1. Biodiesel yields of each example and comparative example.
[0062] serial number Biodiesel yield (%) Example 1 97.2 Example 2 95.8 Example 3 96.4 Comparative Example 1 73.2
[0063] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
Claims
1. A method for preparing a lipase@sepiolite composite microcapsule catalyst, characterized in that, It includes the following steps: (1) Add sepiolite to hydrochloric acid and seal for water bath reaction. Wash until pH 5, dry, sieve, then add calcium and distilled water and mix evenly until a slurry is formed. Then dry the material and wash repeatedly to obtain alkaline calcium-based sepiolite. (2) Disperse alkaline calcium sepiolite in a mixture of anhydrous ethanol and water, add a modifier, stir to react, wash, and dry to obtain amphiphilic sepiolite. (3) Prepare an amphiphilic sepiolite solution in toluene solution and disperse it by ultrasonication. Prepare a lipase solution in phosphate buffer solution and add it to the amphiphilic sepiolite solution. Homogenize to obtain lipase@sepiolite composite microcapsule catalyst. In step (1), the drying reaction is carried out at a temperature of 45-65°C for 15-20 hours. In step (2), the modifier is 3-aminopropyltriethoxysilane; In step (3), the lipase is PB011 lipase; In step (3), the homogenization time is 8-12 seconds.
2. The method for preparing a lipase@sepiolite composite microcapsule catalyst according to claim 1, characterized in that, In step (1), the temperature of the water bath reaction is 75-95℃ and the time is 3-5h.
3. The method for preparing a lipase@sepiolite composite microcapsule catalyst according to claim 1, characterized in that, In step (1), the drying temperature is 101-110℃.
4. The method for preparing a lipase@sepiolite composite microcapsule catalyst according to claim 1, characterized in that, In step (2), the volume ratio of anhydrous ethanol to water in the mixture of anhydrous ethanol and water is 72:
25.
5. The method for preparing a lipase@sepiolite composite microcapsule catalyst according to claim 1, characterized in that, In step (2), the stirring temperature is 70-90℃, the stirring speed is 400-600rpm, and the stirring time is 9-11h.
6. The method for preparing a lipase@sepiolite composite microcapsule catalyst according to claim 1, characterized in that, In step (2), the drying temperature is 50-70℃.
7. A lipase@sepiolite composite microcapsule catalyst, characterized in that, It is obtained by the preparation method described in any one of claims 1-6.
8. The application of the lipase@sepiolite composite microcapsule catalyst as described in claim 7 in the preparation of biodiesel.