WO2022227122A1 - 一种利用双MOFs固定化脂肪酶制备生物柴油的方法 - Google Patents

一种利用双MOFs固定化脂肪酶制备生物柴油的方法 Download PDF

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WO2022227122A1
WO2022227122A1 PCT/CN2021/093556 CN2021093556W WO2022227122A1 WO 2022227122 A1 WO2022227122 A1 WO 2022227122A1 CN 2021093556 W CN2021093556 W CN 2021093556W WO 2022227122 A1 WO2022227122 A1 WO 2022227122A1
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zif
anl
mofs
oil
immobilized lipase
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French (fr)
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杜伟
胡应立
戴玲妹
刘德华
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东莞深圳清华大学研究院创新中心
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/008—Supramolecular polymers
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10G—CRACKING HYDROCARBON OILS; PRODUCTION OF LIQUID HYDROCARBON MIXTURES, e.g. BY DESTRUCTIVE HYDROGENATION, OLIGOMERISATION, POLYMERISATION; RECOVERY OF HYDROCARBON OILS FROM OIL-SHALE, OIL-SAND, OR GASES; REFINING MIXTURES MAINLY CONSISTING OF HYDROCARBONS; REFORMING OF NAPHTHA; MINERAL WAXES
    • C10G3/00—Production of liquid hydrocarbon mixtures from oxygen-containing organic materials, e.g. fatty oils, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to the field of biochemical industry, in particular to a method for preparing biodiesel by utilizing double MOFs immobilized lipase.
  • MOFs metal organic frameworks
  • MOFs metal organic frameworks
  • the immobilization of enzymes by MOFs is generally achieved by pore adsorption. After the enzyme molecules are immobilized in the pores of MOFs, the molecular conformation is not easily changed due to the confinement effect of the pore structure of MOFs, thus greatly improving its stability.
  • the crystalline pore diameter of MOFs is usually less than 5 nm, and it is theoretically impossible to achieve pore adsorption to immobilize lipase.
  • lipase is often combined with hydration molecules to form enzymes - Hydration molecules, whose molecular size is often tens of nanometers, make this micro-mesoporous carrier immobilize lipase by pore adsorption.
  • enzymes - Hydration molecules whose molecular size is often tens of nanometers, make this micro-mesoporous carrier immobilize lipase by pore adsorption.
  • the enzyme-hydration molecule The size is difficult to meet the catalytic synthesis requirements of lipase for biodiesel.
  • the main trend is to require a macroporous structure (pore size>50nm) to catalyze the preparation of biodiesel from macromolecular long-chain lipid substrates while adsorbing lipase.
  • the present invention discloses a method for preparing biodiesel by using double MOFs immobilized lipase.
  • the metal-organic framework material MOFs adopted in the present invention include but are not limited to ZIF-8 (ZIF-8 is an organic metal framework material with a stable zeolite-like structure composed of Zn 2+ and 2-methylimidazole ligands), and the present invention selects ZIF-8 synthesizes ZIF-8 surface immobilized lipase, and synthesizes macroporous ZIF-8 immobilized lipase by pore adsorption method, which realizes high-value and resource utilization of oil and fat, and achieves higher enzyme activity recovery rate and Product yield.
  • ZIF-8 is an organic metal framework material with a stable zeolite-like structure composed of Zn 2+ and 2-methylimidazole ligands
  • the present invention selects ZIF-8 synthesizes ZIF-8 surface immobilized lipase, and synthesizes macroporous ZIF-8 immobilized lipase by pore adsorption method, which realizes high-value and resource utilization of oil and fat, and achieves higher enzyme
  • the present invention provides a method for preparing biodiesel by utilizing dual MOFs immobilized lipase, comprising the following steps:
  • Step 2 using the ZIF-8 to prepare ANL/ZIF-8;
  • Step 3 preparing a three-dimensional ordered polystyrene template
  • Step 4 using the three-dimensional ordered polystyrene template to prepare M-ZIF-8;
  • Step 5 using the M-ZIF-8 to prepare ANL@M-ZIF-8;
  • step 6 the oil, short-chain alcohol, the ANL/ZIF-8, and the ANL@M-ZIF-8 are mixed and reacted to obtain biodiesel.
  • step 1 further comprises the following steps:
  • Step 1-1 dissolve Zn(NO 3 ) 2 ⁇ 6H 2 O in methanol to prepare solution A;
  • Step 1-2 dissolving 2-methylimidazole in methanol to prepare solution B;
  • step 1-3 the solution A and the solution B are mixed, and centrifuged at room temperature after shaking or magnetic stirring for 24 hours; wherein, the centrifugal separation speed is 10000rpm;
  • Steps 1-4 taking the precipitate after centrifugation, washing with methanol for several times, soaking in methanol for 1 day for activation, centrifuging, washing with methanol, and vacuum drying to obtain the ZIF-8 in the form of white powder.
  • step 2 further comprises the following steps:
  • Step 2-1 quantitatively weighing the dried ZIF-8, adding ultrapure water and liquid lipase ANL, mixing, shaking, centrifuging, and taking the supernatant to measure the protein concentration; wherein, shaking conditions are: The shaking temperature is 45°C, the cyclotron frequency is 200rpm, and the shaking time is more than 4 hours;
  • step 2-2 the centrifuged precipitate is washed with water, centrifuged, and lyophilized by a freeze dryer for >12 hours to obtain the ANL/ZIF-8, which is the surface-immobilized lipase of ZIF-8.
  • step 3 further comprises the following steps:
  • Step 3-1 take a sufficient amount of styrene and wash with 10wt% NaOH solution and deionized water for several times in turn to remove the polymerization inhibitor in the styrene;
  • step 3-2 the solution of styrene and polyvinylpyrrolidone in step 3-1 was bubbled with nitrogen for 15 minutes, and K 2 S 2 O 8 was added under the condition of mechanical stirring at 450 rpm and heating at 75° C. The solution was reacted for 24 hours, and after the reaction was completed, it was cooled, filtered with suction, washed with deionized water and ethanol in sequence, and dried at 60° C. to obtain the three-dimensional ordered polystyrene template in the form of a filter cake.
  • step 4 further comprises the following steps:
  • Step 4-1 take Zn(NO 3 ) 2 .6H 2 O, 2-methylimidazole and methanol according to the proportion, and prepare a sufficient amount of ZIF-8 precursor methanol solution;
  • Step 4-2 take the three-dimensional ordered polystyrene template obtained in step 3 and soak it in the ZIF-8 precursor methanol solution for 1 hour, vacuumize to remove air bubbles for 10 minutes, and dry at 50°C for 12 hours, Obtain filter cake A;
  • Step 4-3 immerse the filter cake A in a mixed solution of CH 3 OH/NH 3 ⁇ H 2 O with a volume ratio of 1:1 at room temperature, vacuumize to remove air bubbles for 10 minutes, and place the reaction at room temperature and pressure for 10 minutes. 24 hours, during this process, the filter cake A is gradually broken into small pieces due to the increasing growth pressure of ZIF-8, and after the reaction is completed, suction filtration, wash with sufficient water and ethanol successively, and fully wash away the residual ammonia water. , dried at 50°C to obtain filter cake B;
  • step 4-4 the filter cake B was immersed in a sufficient amount of tetrahydrofuran for 24 hours, etched for several times and dried at 100 ° C to obtain the M-ZIF-8 in the form of white powder, which is macroporous ZIF-8 ;
  • the pore diameter of the M-ZIF-8 is greater than 100 nm, the solid is separated by centrifugation after each etching operation, and the etching operation is repeated more than 5 times to ensure that the polystyrene template has been fully etched.
  • step 5 further comprises the following steps:
  • Step 5-1 weigh the M-ZIF-8, add ultrapure water and ultrasonically disperse it to uniform dispersion, add liquid lipase ANL, mix, shake, and centrifuge (the solid is actually suspended above the solution after centrifugation), take The protein concentration of the supernatant was determined by the BCA method; wherein, the shaking conditions were: shaking temperature 45°C, cyclotron frequency 200rpm, shaking time >4 hours;
  • step 5-2 the centrifuged precipitate was washed with water, centrifuged, and lyophilized by a freeze dryer for >12 hours to obtain the ANL@M-ZIF-8, which is the macroporous ZIF-8 immobilized lipase.
  • the amount of the short-chain alcohol is 4.5 to 6 times the oil molar ratio, and the short-chain alcohol includes methanol, ethanol, Propanol, butanol, etc.
  • the dosage of ANL/ZIF-8 is 600-1000 standard enzyme activity units of oil quality
  • the dosage of ANL@M-ZIF-8 is 600-1000 standard enzyme activity units of oil quality .
  • step 6 is carried out in a one-stage or multi-stage enzyme reactor, the reaction temperature is controlled at 40-55 ° C, and the reaction is carried out for 8-20 hours, wherein the short chain is carried out.
  • the alcohol is added at a uniform speed within 2 hours; preferably, the one-stage or multi-stage enzyme reactor is coupled with an online dehydration device to remove the moisture in the reaction system online, wherein online dehydration refers to the use of membrane, molecular sieve or air stripping .
  • the membranes used in online dehydration are organic membranes, inorganic membranes or ceramic membranes, etc.; the molecular sieves used in online dehydration are or Molecular sieve, etc.; under normal circumstances, the reaction conversion rate of the ANL/ZIF-8 is over 74%, and the reaction conversion rate of the ANL@M-ZIF-8 is over 82%.
  • the liquid lipase in the present invention includes lipase derived from yeast, mold, bacteria or other microorganisms; the liquid lipase is a single lipase or a combination of multiple lipases, such as but not limited to Antarctic Candida At least one of yeast (Candida antarctica), Thermomyces lanuginosus, Aspergillus niger, Aspergillus oryzae, Rhizomucor miehei and Rhizopus oryzae, etc. A sort of.
  • the oil described in the present invention is a renewable biological oil, including vegetable oil, animal oil, waste edible oil, acidified oil, oil refining waste and microbial oil, etc.; wherein, the vegetable oil is castor oil, palm oil, rapeseed oil, etc. Oil, soybean oil, peanut oil, corn oil, cottonseed oil, rice bran oil, jatropha oil, Wenguan fruit oil or jatropha oil, etc.; the animal fat is fish oil, tallow, lard or suet oil, etc.; the The microbial oil is yeast oil or microalgae oil, etc.; the waste edible oil is slop oil or waste oil, etc.; the oil refining leftovers are acidified oil and the like.
  • the vegetable oil is castor oil, palm oil, rapeseed oil, etc. Oil, soybean oil, peanut oil, corn oil, cottonseed oil, rice bran oil, jatropha oil, Wenguan fruit oil or jatropha oil, etc.
  • the animal fat is fish oil, t
  • the present invention simultaneously uses ANL/ZIF-8 and ANL@M-ZIF-8 as dual MOFs to prepare biodiesel, which not only solves the problem that the pore size of the single MOFs mesoporous structure is insufficient and inhibits the catalytic synthesis effect, but also The physicochemical properties of the mesoporous structure of the two MOFs were integrated to form a multi-layered pore adsorption structure, which greatly improved the yield of biodiesel; among them, ZIF-8 surface-immobilized lipase was used as a conventional microporous surface-immobilized fat.
  • Enzyme which can meet the basic reaction transformation requirements, macroporous ZIF-8 immobilized lipase immobilized lipase inside the pores of macroporous MOFs by pore adsorption method, which greatly improves the specific surface area and shows better performance than microporous surface immobilization.
  • Bio-lipase has higher catalytic efficiency, enzyme activity recovery rate, specific enzyme activity, and in the subsequent process of catalyzing oil to prepare biodiesel, it also shows higher short-chain alcohol tolerance. It has important development and application prospects in bioenergy and bio-based chemicals;
  • the present invention adopts a mild method to synthesize ANL@M-ZIF-8, and controls the ratio of raw materials and preparation conditions at the same time, which is conducive to the effective adsorption and immobilization of lipase in the macroporous mesoporous structure, and realizes the stability and stability of lipase. circular usability;
  • Fig. 1 is the main flow schematic diagram of the technological method in the present invention.
  • Embodiment 1 provides a method for preparing biodiesel by using dual MOFs immobilized lipase, which includes the following steps:
  • step 1 1.68g Zn(NO 3 ) 2 ⁇ 6H 2 O (5.65mmol) was dissolved in 80mL methanol to prepare solution A; 3.70g 2-methylimidazole (45mmol) was dissolved in 80mL methanol to prepare a solution B; Mix the solution A and solution B, and magnetically stir at room temperature for 24 hours, then centrifuge at 10,000 rpm; take the centrifuged precipitate, wash it several times with methanol, soak it in methanol for 1 day, and then activate it by centrifugation , washed with methanol, and dried in vacuo to obtain the ZIF-8 in white powder form;
  • Step 2 Quantitatively weigh 30 mg of the dried ZIF-8 using an analytical balance, add 980 ⁇ L of ultrapure water and 20 ⁇ L of liquid lipase ANL, mix well, shake at 45° C. at a frequency of 200 rpm for 5 hours, centrifuge, and take the solution. The protein concentration of the supernatant was measured; the centrifuged precipitate was washed with water, centrifuged, and lyophilized by a freeze dryer for 14 hours to obtain the ANL/ZIF-8;
  • Step 3 add enough styrene to the separatory funnel, wash with 10wt% NaOH solution and deionized water for several times in turn to remove the polymerization inhibitor in the styrene; add 65 mL of styrene and 500 mL of polyvinylpyrrolidone solution (K-30, 2.50g) was added to a round-bottom three-necked flask, bubbled with nitrogen for 15 minutes, heated at 75°C for 30 minutes under continuous mechanical stirring at 450rpm, and then rapidly added 50mL of K 2 S 2 O 8 (1.00g) solution initiates the reaction, continues mechanical stirring at 450rpm at 75°C for 24 hours, cools after the reaction is completed, and stacks two conventional filter papers on the Buchner funnel, pump suction filtration, after suction filtration for 24 hours, Wash the formed filter cake with deionized water and ethanol in turn, and place it in a 60°C oven to dry overnight to obtain the three-dimensional ordered polystyrene template in the
  • Step 4 take 8.15g Zn(NO 3 ) 2 ⁇ 6H 2 O, 6.75g 2-methylimidazole and 45mL methanol to prepare a sufficient amount of ZIF-8 precursor methanol solution; the three-dimensional ordered polystyrene
  • the template was immersed in the methanol solution of the ZIF-8 precursor for 1 hour, and after vacuuming to remove air bubbles for 10 minutes, the fully infiltrated filter cake was placed in an oven and dried at 50 ° C for 12 hours to obtain filter cake A; Filter cake A was immersed in a mixed solution of CH 3 OH/NH 3 ⁇ H 2 O with a volume ratio of 1:1 at room temperature, vacuumed to remove air bubbles for 10 minutes, and then left to react at room temperature and pressure for 24 hours.
  • the filter cake A is gradually broken into small pieces due to the increasing growth pressure of ZIF-8.
  • Step 5 weigh 60 mg of the M-ZIF-8, add 800 ⁇ L of ultrapure water and ultrasonically disperse it to a uniform dispersion, add 200 ⁇ L of liquid lipase ANL, mix well and shake at 45° C. at a frequency of 200 rpm for 5 hours and then centrifuge ( After the centrifugation, the solid was actually suspended above the solution), and the supernatant was taken to measure the protein concentration by the BCA method; the precipitate after the centrifugation was washed with water, centrifuged, and lyophilized by a freeze dryer for 14 hours to obtain the ANL@M-ZIF-8;
  • Step 6 place 200 g of rapeseed oil and methanol based on the oil and fat molar ratio of 4.5:1 in a one-stage or multi-stage enzyme reactor, and add ANL/ZIF-8 and ANL based on 600 standard enzyme activity units of oil and fat quality respectively.
  • @M-ZIF-8 the reaction temperature was controlled at 40 °C, and the reaction was carried out for 8 hours to obtain biodiesel. It was determined that the reaction conversion rate of ANL/ZIF-8 was 80%, and the reaction conversion rate of ANL@M-ZIF-8 was 80%. 92%; wherein, the liquid lipase ANL in steps 2 and 5 is derived from Candida antarctica, and methanol is added at a constant speed within 2 hours.
  • Example 2 This example provides a method for preparing biodiesel by using dual MOFs immobilized lipase, wherein steps 1 to 5 are the same as steps 1 to 5 in Example 1, and the differences are:
  • Step 6 put 500g soybean oil and ethanol based on the oil and fat molar ratio of 6:1 in a one-stage or multi-stage enzyme reactor, and add ANL/ZIF-8 and ANL@ 600 standard enzyme activity units based on oil quality respectively.
  • M-ZIF-8 the reaction temperature was controlled at 50 °C, and the reaction was carried out for 10 hours to obtain biodiesel. It was determined that the reaction conversion rate of ANL/ZIF-8 was 84%, and the reaction conversion rate of ANL@M-ZIF-8 was 90%. %; wherein, the liquid lipase ANL in the steps 2 and 5 is derived from Aspergillus oryzae, and ethanol is added at a constant speed within 2 hours.
  • Example 3 This example provides a method for preparing biodiesel by using dual MOFs immobilized lipase, wherein steps 1 to 5 are the same as steps 1 to 5 in Example 1, and the differences are:
  • step 6 200 g of acidified oil and propanol with a molar ratio of 6:1 based on oil and fat are placed in a one-stage or multi-stage enzyme reactor, and ANL/ZIF-8 and ANL based on 1000 standard enzyme activity units of oil and fat mass are added respectively.
  • M-ZIF-8 the reaction temperature was controlled at 55 °C, and the reaction was performed for 12 hours to obtain biodiesel. It was determined that the reaction conversion rate of ANL/ZIF-8 was 78%, and the reaction conversion rate of ANL@M-ZIF-8 was 78%. 85%; wherein, the liquid lipase ANL in the steps 2 and 5 is derived from Rhizomucor miehei, and the propanol is added at a constant speed within 2 hours.
  • Example 4 This example provides a method for preparing biodiesel by using dual MOFs immobilized lipase, wherein steps 1-5 are the same as steps 1-5 in Example 1, and the differences are:
  • step 6 400 g of acidified oil and butanol with a molar ratio of 6:1 based on oil and fat are placed in a one-stage or multi-stage enzyme reactor, and ANL/ZIF-8 and ANL based on 800 standard enzyme activity units based on oil quality are added respectively.
  • @M-ZIF-8 through the online dehydration shown in Figure 1, the reaction temperature was controlled at 55 °C, and the reaction was carried out for 20 hours to obtain biodiesel.
  • reaction conversion rate of ANL/ZIF-8 was 74%, ANL@M - The reaction conversion rate of ZIF-8 is 82%; wherein, the liquid lipase ANL in the steps 2 and 5 is derived from Candida antarctica, and the butanol is added at a uniform rate within 2 hours.
  • the invention simultaneously uses ANL/ZIF-8 and ANL@M-ZIF-8 as double MOFs to prepare biodiesel, which not only solves the problem that the pore size of the single MOFs mesoporous structure is insufficient and inhibits the catalytic synthesis effect, but also integrates two MOFs.
  • the physical and chemical properties of the mesoporous structure of various MOFs form a multi-layered pore adsorption structure, which greatly improves the yield of biodiesel; among them, ZIF-8 surface-immobilized lipase, as a conventional microporous surface-immobilized lipase, can To meet the basic reaction transformation requirements, the macroporous ZIF-8 immobilized lipase immobilized the lipase inside the pores of the macroporous MOFs through the pore adsorption method, which greatly improved the specific surface area and showed better performance than the microporous surface immobilized lipase. Higher catalytic efficiency, recovery rate of enzymatic activity, specific enzymatic activity, and higher tolerance to short-chain alcohols in the subsequent process of catalyzing oil to prepare biodiesel. It has important development and application prospects in bio-based chemicals;
  • the present invention adopts a mild method to synthesize ANL@M-ZIF-8, and at the same time controls the ratio of raw materials and preparation conditions, which is conducive to the effective adsorption and immobilization of lipase in the macroporous mesoporous structure, and realizes the stability and circulation of lipase. practicality;
  • M-ZIF-8 is selected to synthesize, its macroporous mesoporous structure has a large specific area and high porosity, which is conducive to the immobilization and protection of lipase, preventing the loss or loss of enzymatic activity of lipase during the preparation process, and facilitating the product.
  • the separation of lipase and lipase facilitates the reuse of lipase.

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Abstract

本发明公开一种利用双MOFs固定化脂肪酶制备生物柴油的方法，其包括以下步骤：制备ZIF-8；利用ZIF-8制备ANL/ZIF-8；制备三维有序聚苯乙烯模板；利用三维有序聚苯乙烯模板制备M-ZIF-8；利用M-ZIF-8制备ANL@M-ZIF-8；将油脂、短链醇、ANL/ZIF-8、及ANL@M-ZIF-8混合反应，制得生物柴油。本发明同时采用ANL/ZIF-8和ANL@M-ZIF-8作为双MOFs用以制备生物柴油，不但解决了采用单一MOFs介孔结构的孔径不足而抑制催化合成效果的问题，而且集成两种MOFs的介孔结构的物理化学性能，形成多层次的孔吸附结构，进而极大地提高生物柴油的得率。

Description

一种利用双MOFs固定化脂肪酶制备生物柴油的方法 技术领域
本发明涉及生物化工领域，具体涉及一种利用双MOFs固定化脂肪酶制备生物柴油的方法。
背景技术
金属有机骨架材料(MOFs)作为一种新兴材料，主要用于脂肪酶的固定化，对油脂高值化转化具有重要开发应用前景。MOFs对酶的固定化一般通过孔吸附法来实现，酶分子固定化在MOFs孔内后，受到MOFs孔结构的限域效应，分子构象不易发生变化，因而大大地提高其稳定性。但通常MOFs结晶性孔孔径<5nm，理论上无法实现孔吸附对脂肪酶进行固定化。一些学者提出通过软模板法合成含有介孔结构的多级孔MOF进而用于脂肪酶的固定化，但由于受其介孔结构(20～30nm)传质限制，固定化的脂肪酶仍然只能用来催化小分子底物月桂酸和苯甲醇的酯化反应，而无法催化动植物油脂中的甘油三酯底物进行生物柴油的制备。以三油酸甘油酯底物制备生物柴油为例，平均分子尺寸在3.5nm左右，而不同脂肪酶分子平均尺寸在5～10nm以上，在实际催化体系中，脂肪酶往往与水合分子结合形成酶-水合分子，其分子尺寸往往在数十纳米，使得这种微介孔载体通过孔吸附法固定化脂肪酶，综合考虑酶及底物分子尺寸、以及传质要求，此种酶-水合分子的尺寸难以满足脂肪酶对生物柴油的催化合成需求。现阶段主要趋向于需要大孔结构(孔径>50nm)才能在吸附脂肪酶的同时，还能催化大分子长链油脂底物制备生物柴油。
技术问题
如何制备及采用ANL/ZIF-8和ANL@M-ZIF-8作为双MOFs，以解决采用单一MOFs而引起抑制催化合成效果的问题，进而提高生物柴油的生产得率。
技术解决方案
为了克服上述技术问题，本发明公开了一种利用双MOFs固定化脂肪酶制备生物柴油的方法。
本发明为实现上述目的所采用的技术方案是：
本发明采用的金属有机骨架材料MOFs包括但不限于ZIF-8(ZIF-8是由Zn 2+和2-甲基咪唑配体组成的稳定的类沸石结构的有机金属骨架材料)，本发明选用ZIF-8合成ZIF-8表面固定化脂肪酶、及通过孔吸附法合成大孔ZIF-8固定化脂肪酶，实现对油脂的高值化、资源化利用，达到更高的酶活回收率和产品得率。
为此，本发明提供一种利用双MOFs固定化脂肪酶制备生物柴油的方法，其包括以下步骤：
步骤1，制备ZIF-8；
步骤2，利用所述ZIF-8制备ANL/ZIF-8；
步骤3，制备三维有序聚苯乙烯模板；
步骤4，利用所述三维有序聚苯乙烯模板制备M-ZIF-8；
步骤5，利用所述M-ZIF-8制备ANL@M-ZIF-8；
步骤6，将油脂、短链醇、所述ANL/ZIF-8、及所述ANL@M-ZIF-8混合反应，制得生物柴油。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其中所述步骤1进一步包括以下步骤：
步骤1-1，将Zn(NO 3) 2·6H 2O溶于甲醇中，制得溶液A；
步骤1-2，将2-甲基咪唑溶于甲醇中，制得溶液B；
步骤1-3，将所述溶液A和溶液B混合，并于室温下震摇或磁力搅拌24h后离心分离；其中，离心分离转速为10000rpm；
步骤1-4，取离心后的沉淀，利用甲醇洗涤数次后浸泡于甲醇中活化1天后，经过离心、甲醇洗涤、真空干燥，得到白色粉末状的所述ZIF-8。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其中所述步骤2进一步包括以下步骤：
步骤2-1，定量称取已干燥的所述ZIF-8，加入超纯水和液体脂肪酶ANL，混匀、震摇、离心，取上清液测定蛋白浓度；其中，震摇条件为：震摇温度45℃，回旋频率200rpm，震荡时间>4小时；
步骤2-2，取离心后的沉淀水洗、离心，利用冻干机冻干>12小时，得到所述ANL/ZIF-8，即为ZIF-8表面固定化脂肪酶。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其中所述步骤3进一步包括以下步骤：
步骤3-1，取足量苯乙烯，依次用10wt％NaOH溶液和去离子水分别洗涤数次，以去除苯乙烯中的阻聚剂；
步骤3-2，将步骤3-1的苯乙烯和聚乙烯吡咯烷酮溶液，用氮气鼓泡15分钟后，在持续转速450rpm机械搅拌和于75℃下加热的条件下，加入K 2S 2O 8溶液引发反应24小时，待反应完成后冷却、抽滤、用去离子水和乙醇依次洗涤、60℃干燥，得到滤饼状的所述三维有序聚苯乙烯模板。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其中所述步骤4进一步包括以下步骤：
步骤4-1，按照比例取Zn(NO 3) 2·6H 2O、2-甲基咪唑和甲醇，配制成足量的ZIF-8前体甲醇溶液；
步骤4-2，取步骤3制得的所述三维有序聚苯乙烯模板浸泡于所述ZIF-8前体甲醇溶液中1小时，抽真空除气泡10分钟、于50℃下干燥12小时，得到滤饼A；
步骤4-3，将所述滤饼A在室温下浸入体积比为1:1的CH 3OH/NH 3·H 2O的混合溶液中，抽真空除气泡10分钟后常温常压下放置反应24小时，在此过程中所述滤饼A因ZIF-8的生长压力渐大而逐步破碎成小块，待反应完成后抽滤，用足量水和乙醇依次洗涤，充分洗去残余的氨水，于50℃下干燥，得到滤饼B；
步骤4-4，将所述滤饼B浸入足量四氢呋喃中24小时，多次刻蚀并于100℃下干燥，得到白色粉末状的所述M-ZIF-8，即为大孔ZIF-8；其中，所述M-ZIF-8的孔径大于100nm，每次刻蚀操作后离心分离出固体，重复刻蚀操作大于5次，以确保聚苯乙烯模板已被充分刻蚀。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其中所述步骤5进一步包括以下步骤：
步骤5-1，称取所述M-ZIF-8，加入超纯水超声分散至均匀分散，加入液体脂肪酶ANL，混匀、震摇、离心(离心后固体实际悬浮于溶液上方)，取上清液采用BCA法测定蛋白浓度；其中，震摇条件为：震摇温度45℃，回旋频率200rpm，震荡时间>4小时；
步骤5-2，取离心后的沉淀水洗、离心，利用冻干机冻干>12小时，得到所述ANL@M-ZIF-8，即为大孔ZIF-8固定化脂肪酶。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其中在所述步骤6中，所述短链醇的用量为油脂摩尔比的4.5～6倍，所述短链醇包括甲醇、乙醇、丙醇、丁醇等，所述ANL/ZIF-8用量为油脂质量的600～1000个标准酶活单位，所述ANL@M-ZIF-8用量为油脂质量的600～1000个标准酶活单位。
上述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其中所述步骤6于一级或多级酶反应器中进行，反应温度控制在40～55℃，反应8～20小时，其中短链醇在2个小时内匀速加入；优选地，一级或多级酶反应器与在线脱水设备相偶联，以在线除去反应体系中的水分，其中，在线脱水是指利用膜、分子筛或气提。在线脱水所用的膜为有机膜、无机膜或陶瓷膜等；在线脱水所用的分子筛为
Figure PCTCN2021093556-appb-000001
或
Figure PCTCN2021093556-appb-000002
分子筛等；通常情况下，所述ANL/ZIF-8的反应转化率为74％以上，所述ANL@M-ZIF-8的反应转化率为82％以上。
本发明中所述液体脂肪酶包括来源于酵母、霉菌、细菌或其它微生物的脂肪酶；所述液体脂肪酶为单种脂肪酶或多种脂肪酶的组合，例如但不限于来源于南极假丝酵母(Candida antarctica)、嗜热丝孢菌(Thermomyces lanuginosus)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、米黑根毛霉(Rhizomucor miehei)和米根霉(Rhizopus oryzae)等中的至少一种。
本发明中所述油脂为可再生生物油脂，包括植物油脂、动物油脂、废食用油、酸化油、油脂精练下脚料和微生物油脂等；其中，所述植物油脂为蓖麻油、棕榈油、菜籽油、大豆油、花生油、玉米油、棉子油、米糠油、麻风树油、文冠果油或小桐子油等；所述动物油脂为鱼油、牛油、猪油或羊油等；所述微生 物油脂为酵母油脂或微藻类油脂等；所述废食用油为潲水油或地沟油等；所述油脂精炼下脚料为酸化油等。
有益效果
(1)本发明同时采用ANL/ZIF-8和ANL@M-ZIF-8作为双MOFs用以制备生物柴油，不但解决了采用单一MOFs介孔结构的孔径不足而抑制催化合成效果的问题，而且集成了两种MOFs的介孔结构的物理化学性能，形成多层次的孔吸附结构，进而极大地提高生物柴油的得率；其中ZIF-8表面固定化脂肪酶作为常规的微孔表面固定化脂肪酶，可满足基本的反应转化需求，大孔ZIF-8固定化脂肪酶通过孔吸附法将脂肪酶固定化在大孔MOFs的孔内部，极大地提高比表面积，表现出优于微孔表面固定化脂肪酶更高的催化效率、酶活回收率、比酶活，在后续催化油脂制备生物柴油的过程中，也表现出更高的短链醇耐受性，在催化油脂高值化转化制备生物能源和生物基化学品中具有重要开发应用前景；
(2)本发明采用温和的方法合成ANL@M-ZIF-8，同时控制原材料的配比和制备条件，利于对脂肪酶有效吸附固定于大孔介孔结构内部，实现脂肪酶的稳定性和循环实用性；
(3)选择合成M-ZIF-8，其大孔介孔结构的比标面积大、孔隙率高，利于脂肪酶的固定和保护，防止脂肪酶在制备过程中流失或丧失酶活，方便产物和脂肪酶的分离，便于脂肪酶的重复利用。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1为本发明中工艺方法的主要流程示意图。
本发明的实施方式
下面通过具体实施例对本发明作进一步说明，以使本发明技术方案更易于理解、掌握，而非对本发明进行限制。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1：本实施例提供一种利用双MOFs固定化脂肪酶制备生物柴油的方法，其包括以下步骤：
步骤1，将1.68gZn(NO 3) 2·6H 2O(5.65mmol)溶于80mL甲醇中，制得溶液A；将3.70g 2-甲基咪唑(45mmol)溶于80mL甲醇中，制得溶液B；将所述溶液A和溶液B混合，并于室温下磁力搅拌24h后，以10000rpm的转速离心分离；取离心后的沉淀，利用甲醇洗涤数次后浸泡于甲醇中活化1天后，经过离心、甲醇洗涤、真空干燥，得到白色粉末状的所述ZIF-8；
步骤2，利用分析天平定量称取30mg已干燥的所述ZIF-8，加入980μL超纯水和20μL液体脂肪酶ANL，混匀后于45℃、频率为200rpm下震摇5小时、离心，取上清液测定蛋白浓度；取离心后的沉淀水洗、离心，利用冻干机冻干14小时，得到所述ANL/ZIF-8；
步骤3，取足量苯乙烯加入至分液漏斗中，依次用10wt％NaOH溶液和去离子水分别洗涤数次，以去除苯乙烯中的阻聚剂；将65mL苯乙烯和500mL聚乙烯吡咯烷酮溶液(K-30，2.50g)添加至圆底三口烧瓶中，用氮气鼓泡15分钟后，在持续转速450rpm机械搅拌下，于75℃下加热30分钟，随后快速加入50mL的K 2S 2O 8(1.00g)溶液引发反应，于75℃下持续转速450rpm机械搅拌24小时，待反应完成后冷却，并于布氏漏斗上叠放两张常规滤纸后水泵抽滤，抽滤24小时后，用去离子水和乙醇依次洗涤形成的滤饼，并置于60℃烘箱中干燥过夜，得到滤饼状的所述三维有序聚苯乙烯模板；
步骤4，取8.15g Zn(NO 3) 2·6H 2O、6.75g 2-甲基咪唑和45mL甲醇，配制成足量的ZIF-8前体甲醇溶液；将所述三维有序聚苯乙烯模板浸泡于所述ZIF-8前体甲醇溶液中1小时，抽真空除气泡10分钟后，将充分浸润的滤饼放入烘箱中于50℃下干燥12小时，得到滤饼A；将所述滤饼A在室温下浸入体积比为1:1的CH 3OH/NH 3·H 2O的混合溶液中，抽真空除气泡10分钟后常温常压下放置反应24小时，在此过程中所述滤饼A因ZIF-8的生长压力渐大而逐步破碎成小块，待反应完成后抽滤，用足量水和乙醇依次洗涤，充分洗去残余的氨水，于50℃烘箱中干燥，得到滤饼B；将所述滤饼B浸入足量四氢呋喃中24小时，多次刻蚀并于100℃下干燥过夜，得到白色粉末状的所述M-ZIF-8；
步骤5，称取60mg所述M-ZIF-8，加入800μL超纯水超声分散至均匀分散，加入200μL液体脂肪酶ANL，混匀并于45℃、频率为200rpm下震摇5小时后离心(离心后固体实际悬浮于溶液上方)，取上清液采用BCA法测定蛋白浓度；取离心后的沉淀水洗、离心，利用冻干机冻干14小时，得到所述ANL@M-ZIF-8；
步骤6，将200g菜籽油和基于油脂摩尔比为4.5:1的甲醇置于一级或多级酶反应器中，分别加入基于油脂质量600个标准酶活单位的ANL/ZIF-8和ANL@M-ZIF-8，反应温度控制在40℃，反应8小时，获得生物柴油，经测定，ANL/ZIF-8的反应转化率为80％，ANL@M-ZIF-8的反应转化率为92％；其中，所述步骤2和步骤5中的液体脂肪酶ANL均来源于南极假丝酵母(Candida antarctica)，甲醇在2个小时内匀速加入。
实施例2：本实施例提供了一种利用双MOFs固定化脂肪酶制备生物柴油的方法，其中步骤1～5与实施例1中的步骤1～5相同，其不同之处在于：
步骤6，将500g大豆油和基于油脂摩尔比为6:1的乙醇置于一级或多级酶反应器中，分别加入基于油脂质量600个标准酶活单位的ANL/ZIF-8和ANL@M-ZIF-8，反应温度控制在50℃，反应10小时，获得生物柴油，经测定，ANL/ZIF-8的反应转化率为84％，ANL@M-ZIF-8的反应转化率为90％；其中，所述步骤2和步骤5中的液体脂肪酶ANL均来源于米曲霉(Aspergillus oryzae)，乙醇在2个小时内匀速加入。
实施例3：本实施例提供了一种利用双MOFs固定化脂肪酶制备生物柴油的方法，其中步骤1～5与实施例1中的步骤1～5相同，其不同之处在于：
步骤6，将200g酸化油和基于油脂摩尔比为6:1的丙醇置于一级或多级酶反应器中，分别加入基于油脂质量1000个标准酶活单位的ANL/ZIF-8和ANL@M-ZIF-8，反应温度控制在55℃，反应12小时，获得生物柴油，经测定，ANL/ZIF-8的反应转化率为78％，ANL@M-ZIF-8的反应转化率为85％；其中，所述步骤2和步骤5中的液体脂肪酶ANL均来源于米黑根毛霉(Rhizomucor miehei)，丙醇在2个小时内匀速加入。
实施例4：本实施例提供了一种利用双MOFs固定化脂肪酶制备生物柴油的方法，其中步骤1～5与实施例1中的步骤1～5相同，其不同之处在于：
步骤6，将400g酸化油和基于油脂摩尔比为6:1的丁醇置于一级或多级酶反应器中，分别加入基于油脂质量800个标准酶活单位的ANL/ZIF-8和ANL@M-ZIF-8，通过图1所示的在线脱水，反应温度控制在55℃，反应20小时，获得生物柴油，经测定，ANL/ZIF-8的反应转化率为74％，ANL@M-ZIF-8的反应转化率为82％；其中，所述步骤2和步骤5中的液体脂肪酶ANL均来源于南极假丝酵母(Candida antarctica)，丁醇在2个小时内匀速加入。
以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制。任何熟悉本领域的技术人员，在不脱离本发明技术方案范围情况下，都可利用上述揭示的技术手段和技术内容对本发明技术方案做出许多可能的变动和修饰，或修改为等同变化的等效实施例。故凡是未脱离本发明技术方案的内容，依据本发明之形状、构造及原理所作的等效变化，均应涵盖于本发明的保护范围。
工业实用性
本发明同时采用ANL/ZIF-8和ANL@M-ZIF-8作为双MOFs用以制备生物柴油，不但解决了采用单一MOFs介孔结构的孔径不足而抑制催化合成效果的问题，而且集成了两种MOFs的介孔结构的物理化学性能，形成多层次的孔吸附结构，进而极大地提高生物柴油的得率；其中ZIF-8表面固定化脂肪酶作为常规的微孔表面固定化脂肪酶，可满足基本的反应转化需求，大孔ZIF-8固定化脂肪酶通过孔吸附法将脂肪酶固定化在大孔MOFs的孔内部，极大地提高比表面积，表现出优于微孔表面固定化脂肪酶更高的催化效率、酶活回收率、比酶活，在后续催化油脂制备生物柴油的过程中，也表现出更高的短链醇耐受性，在催化油脂高值化转化制备生物能源和生物基化学品中具有重要开发应用前景；
其次，本发明采用温和的方法合成ANL@M-ZIF-8，同时控制原材料的配比和制备条件，利于对脂肪酶有效吸附固定于大孔介孔结构内部，实现脂肪酶的稳定性和循环实用性；
再者，选择合成M-ZIF-8，其大孔介孔结构的比标面积大、孔隙率高，利于脂肪酶的固定和保护，防止脂肪酶在制备过程中流失或丧失酶活，方便产物和 脂肪酶的分离，便于脂肪酶的重复利用。

Claims (10)

  1. 一种利用双MOFs固定化脂肪酶制备生物柴油的方法，其特征在于，其包括以下步骤：
    步骤1，制备ZIF-8；
    步骤2，利用所述ZIF-8制备ANL/ZIF-8；
    步骤3，制备三维有序聚苯乙烯模板；
    步骤4，利用所述三维有序聚苯乙烯模板制备M-ZIF-8；
    步骤5，利用所述M-ZIF-8制备ANL@M-ZIF-8；
    步骤6，将油脂、短链醇、所述ANL/ZIF-8、及所述ANL@M-ZIF-8混合反应，制得生物柴油。
  2. 根据权利要求1所述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其特征在于，所述步骤1进一步包括以下步骤：
    步骤1-1，将Zn(NO 3) 2·6H 2O溶于甲醇中，制得溶液A；
    步骤1-2，将2-甲基咪唑溶于甲醇中，制得溶液B；
    步骤1-3，将所述溶液A和溶液B混合，并于室温下震摇或磁力搅拌24h后离心分离；
    步骤1-4，取离心后的沉淀，利用甲醇洗涤数次后浸泡于甲醇中活化1天后，经过离心、甲醇洗涤、真空干燥，得到所述ZIF-8。
  3. 根据权利要求2所述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其特征在于，所述步骤2进一步包括以下步骤：
    步骤2-1，定量称取已干燥的所述ZIF-8，加入超纯水和液体脂肪酶ANL，混匀、震摇、离心，取上清液测定蛋白浓度；
    步骤2-2，取离心后的沉淀水洗、离心，利用冻干机冻干，得到所述ANL/ZIF-8。
  4. 根据权利要求3所述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其特征在于，所述步骤3进一步包括以下步骤：
    步骤3-1，取足量苯乙烯，依次用10wt％NaOH溶液和去离子水分别洗涤数次，以去除苯乙烯中的阻聚剂；
    步骤3-2，将步骤3-1的苯乙烯和聚乙烯吡咯烷酮溶液，用氮气鼓泡后，在持续机械搅拌和于75℃下加热的条件下，加入K 2S 2O 8溶液引发反应，待反应完成后冷却、抽滤、洗涤、干燥，得到滤饼状的所述三维有序聚苯乙烯模板。
  5. 根据权利要求4所述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其特征在于，所述步骤4进一步包括以下步骤：
    步骤4-1，按照比例取Zn(NO 3) 2·6H 2O、2-甲基咪唑和甲醇，配制成足量的ZIF-8前体甲醇溶液；
    步骤4-2，取步骤3制得的所述三维有序聚苯乙烯模板浸泡于所述ZIF-8前体甲醇溶液中，抽真空除气泡、干燥，得到滤饼A；
    步骤4-3，将所述滤饼A在室温下浸入体积比为1:1的CH 3OH/NH 3·H 2O的混合溶液中，抽真空除气泡后常温常压下放置反应，待反应完成后抽滤，用足量水和乙醇洗涤、干燥，得到滤饼B；
    步骤4-4，将所述滤饼B浸入足量四氢呋喃中，多次刻蚀、干燥，得到所述M-ZIF-8。
  6. 根据权利要求5所述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其特征在于，所述步骤5进一步包括以下步骤：
    步骤5-1，称取所述M-ZIF-8，加入超纯水超声分散，加入液体脂肪酶ANL，混匀、震摇、离心，取上清液测定蛋白浓度；
    步骤5-2，取离心后的沉淀水洗、离心，利用冻干机冻干，得到所述ANL@M-ZIF-8。
  7. 根据权利要求6所述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其特征在于，在所述步骤6中，所述短链醇的用量为油脂摩尔比的4.5～6倍，所述ANL/ZIF-8用量为油脂质量的600～1000个标准酶活单位，所述ANL@M-ZIF-8用量为油脂质量的600～1000个标准酶活单位。
  8. 根据权利要求7所述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其特征在于，所述步骤6于一级或多级酶反应器中进行，反应温度控制在40～55℃，反应8～20小时，其中短链醇在2个小时内匀速加入。
  9. 根据权利要求8所述的利用双MOFs固定化脂肪酶制备生物柴油的方法， 其特征在于，所述液体脂肪酶包括来源于酵母、霉菌、细菌或其它微生物的脂肪酶。
  10. 根据权利要求9所述的利用双MOFs固定化脂肪酶制备生物柴油的方法，其特征在于，所述油脂为可再生生物油脂，包括植物油脂、动物油脂、废食用油、酸化油、油脂精练下脚料或微生物油脂。
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