WO2022151024A1 - 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法 - Google Patents

液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法 Download PDF

Info

Publication number
WO2022151024A1
WO2022151024A1 PCT/CN2021/071430 CN2021071430W WO2022151024A1 WO 2022151024 A1 WO2022151024 A1 WO 2022151024A1 CN 2021071430 W CN2021071430 W CN 2021071430W WO 2022151024 A1 WO2022151024 A1 WO 2022151024A1
Authority
WO
WIPO (PCT)
Prior art keywords
liquid lipase
solution
sucrose
alginate
liquid
Prior art date
Application number
PCT/CN2021/071430
Other languages
English (en)
French (fr)
Inventor
张正颂
许传久
祁飞
戴永辉
Original Assignee
安徽金禾实业股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 安徽金禾实业股份有限公司 filed Critical 安徽金禾实业股份有限公司
Priority to CN202180000038.7A priority Critical patent/CN112888782A/zh
Priority to PCT/CN2021/071430 priority patent/WO2022151024A1/zh
Publication of WO2022151024A1 publication Critical patent/WO2022151024A1/zh

Links

Images

Classifications

    • 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
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • 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
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • 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)

Definitions

  • the invention belongs to the technical field of fine chemicals, and particularly relates to a method for immobilizing liquid lipase and a method for preparing sucrose-6-acetate.
  • Sucralose is a fresh sweetener.
  • Sucralose is a new type of sweetener jointly developed by British Taylor & Lyle and University of London, and applied for a patent in 1976. It is the only functional sweetener that uses sucrose as raw material. flavoring agent.
  • Sucralose has a diluting effect on sour and salty tastes, has a masking effect on unpleasant tastes such as astringency, bitterness, and wine taste, and has a synergistic effect on spicy and milky tastes, and has a wide range of applications. Therefore, this product is the most ideal strong sweetener today, which can be consumed by children, teenagers, youth, middle-aged, elderly and patients with various diseases without any nutritional doubts.
  • sucrose-6-acetate is an important intermediate in the synthesis of sucralose
  • the methods for synthesizing sucralose-6-acetate are mainly traditional chemical methods, such as acetic anhydride esterification, organometallic synthesis, orthoesters
  • the chemical synthesis reaction conditions of sucrose ester are generally harsh, the color of the synthesized product is relatively dark, and due to its poor selectivity, it is easy to produce by-products.
  • the present application is proposed to provide a method for immobilizing a liquid lipase and a method for preparing sucrose-6-acetate which overcome the above problems or at least partially solve the above problems.
  • a method for immobilizing a liquid lipase which performs the following steps in sequence:
  • Dissolving step adding alginate into deionized water to dissolve completely, and adding liquid lipase at a preset temperature to obtain a mixed solution;
  • the immobilization step mixing the mixed solution with the metal cross-linking agent solution and keeping it for a preset time to obtain a spherical gel to immobilize the liquid lipase; and,
  • Chelation step after washing the spherical gel, adding buffer solution and organic complexing agent solution to carry out chelation reaction, and washing with buffer solution, the immobilized liquid lipase is obtained.
  • sucrose-6-acetate comprising: mixing sucrose and fatty acid vinyl ester in a mixed organic solution, the immobilized liquid lipase obtained by the above method The step of carrying out dehydration esterification reaction under catalytic action to obtain sucrose-6-acetate solution.
  • the application utilizes alginate as a carrier to carry out secondary cross-linking with a metal cross-linking agent and an organic complexing agent to fix the liquid lipase, and the obtained gel has extremely high physical strength, which can be more stable in the reaction system. Exist, basically will not be broken and decomposed;
  • the immobilized enzyme can be used repeatedly for many times, which is much higher than the reuse rate of the immobilized enzyme reported in the prior art, and the industrial large-scale continuous esterification is used to produce sucrose-6-ethyl ester;
  • the preparation method of the present invention is simple and the raw materials required are widely sourced, which greatly reduces the cost of immobilizing liquid lipase, and further reduces the preparation cost of sucrose-6-ethyl ester.
  • Fig. 1 shows the schematic flow chart of the immobilization method of liquid lipase according to an embodiment of the present application
  • FIG. 2 shows a schematic structural diagram of a gel formed by sodium alginate and calcium lactate according to an embodiment of the present application.
  • the immobilized liquid lipase (hereinafter referred to as the immobilized enzyme) has low strength and poor reusability
  • an alginate as a carrier, which is combined with a metal crosslinking agent and a metal crosslinking agent.
  • the organic complexing agent undergoes secondary cross-linking to prepare a gel containing liquid lipase, which is extremely strong and has a high reusability rate.
  • Fig. 1 shows the immobilization method of liquid lipase according to an embodiment of the present application, including the following steps in sequence:
  • Dissolving step S110 adding alginate into deionized water to completely dissolve, and adding liquid lipase at a preset temperature to obtain a mixed solution.
  • the present application uses alginate as a carrier.
  • the alginate used as a carrier is a soluble salt, such as sodium alginate, excluding insoluble salts such as calcium alginate.
  • Sodium alginate (C 6 H 7 O 6 Na) n is mainly composed of the sodium salt of alginic acid, which is composed of ⁇ -D-mannuronic acid (M unit) and ⁇ -L-palladium.
  • M unit ⁇ -D-mannuronic acid
  • ⁇ -L-palladium A copolymer of luronic acid (G units) linked by ⁇ -1,4-glycosidic bonds and composed of GM, MM and GG fragments in varying proportions. Its molecular formula is formula (1), and its carboxyl group can be used as a cross-linking point for cross-linking with metal cross-linking agents and organic complexing agents to form irreversible gels.
  • the alginate is completely dissolved in deionized water.
  • a high-speed stirring device can be used for stirring and heating means to promote the dissolution.
  • the liquid lipase is dissolved in the obtained alginate solution at a preset temperature, and the liquid lipase can remain active at a certain temperature. If the temperature is too high or too low, it may cause fat.
  • the preset temperature can be determined according to the type of the selected liquid lipase, and the preset temperature is not the temperature at which the liquid lipase is inactivated.
  • Fixing step S120 Mix the mixed solution with the metal cross-linking agent solution, and keep it for a preset time to obtain a spherical gel, so as to fix the liquid lipase.
  • the metal cross-linking agent is a soluble metal salt, which can only be used in a solution.
  • a compound containing divalent or trivalent metal ions as the metal cross-linking agent.
  • the viscosity of the solution is further increased, and the solubility of calcium alginate is lower than that of sodium alginate, forming a gel.
  • Adding an appropriate amount of calcium ions to the sodium alginate solution can affect the softness and hardness of the gel. Usually, the more calcium ions are added, the stronger the colloid.
  • the gel can be extruded using an extrusion machine, or an injection with a needle.
  • the chelating step S130 after washing the spherical gel, adding a buffer solution and an organic complexing agent solution to carry out a chelating reaction, and washing with a buffer solution, the immobilized liquid lipase is obtained.
  • the spherical gel obtained above is washed to wash away the unreacted metal cross-linking agent, and the washing can be carried out with buffer solutions such as deionized water or physiological saline. In order to speed up the washing process and promote the degree of washing, a vacuum filtration method .
  • a buffer solution and an organic complexing agent are added to carry out secondary cross-linking with the obtained spherical gel, that is, a chelating reaction is carried out.
  • the function of the buffer is to provide a reaction environment for the spherical gel and the organic complexing agent, so there is no limit to the amount of the buffer, just submerge the spherical gel.
  • the cross-linking groups available for cross-linking of the alginate that is, carboxyl groups
  • the metal cross-linking agent that is, carboxyl groups
  • the remaining uncross-linked carboxyl groups can be secondary with the organic complexing agent. cross-linked.
  • the present application uses alginate as a carrier to carry out secondary cross-linking with a metal cross-linking agent and an organic complexing agent to immobilize the liquid lipase, and the obtained gel has extremely high physical strength, which can be used in the reaction system. More stable existence, basically will not be broken and decomposed; and can be reused many times, much higher than the existing reported solid enzyme reuse rate, and more industrial large-scale continuous esterification is used to produce sucrose-6-acetate;
  • the invention has a simple preparation method and a wide range of required raw materials, which greatly reduces the immobilization cost of liquid lipase, and further reduces the preparation cost of sucrose-6-acetate.
  • the type of liquid lipase is not limited, and lipase is an enzyme with a variety of catalytic abilities, which can be used to catalyze hydrolysis, Alcoholysis, esterification, transesterification and reverse synthesis of esters, etc., such as enzymatic synthesis and transesterification in the organic phase.
  • the liquid lipase can be any one of Lipozyme TL IM, Lipozyme TL100L, Lipozyme RM IM.
  • the type of alginate in the above-mentioned immobilization method of liquid lipase, is not limited, and in other embodiments, the alginate is selected from sodium alginate.
  • the dosage of alginate, deionized water and liquid lipase is not limited, wherein, the minimum dosage of deionized water is sufficient to completely Dissolved alginate shall prevail; in other embodiments, the total mass and dosage ratio of alginate to deionized water and liquid lipase is 1:5 to 1:20; wherein, the mass of deionized water and liquid lipase is The dosage ratio is 0.1-10:1, and in some embodiments, the mass dosage ratio of deionized water and liquid lipase is 3:1.
  • the mass dosage of alginate is less than one-twentieth of the total mass dosage of deionized water and liquid lipase, the dosage is too low, resulting in that the formed gel cannot completely encapsulate the liquid lipase;
  • the mass amount of salt is more than one-fifth of the total mass amount of deionized water and liquid lipase, and if the amount is too much, the resulting gel will have too much polymer and too little liquid lipase, and will not use subsequent lipase. use.
  • the preset temperature in the dissolving step is not limited, and can be determined according to the type of the selected liquid lipase, so that the liquid lipase does not Deactivation shall prevail.
  • the preset temperature may be 10-37°C, and in still other embodiments, the preset temperature may be 28°C.
  • the type of metal cross-linking agent is not limited, and any metal-soluble metal compound that can make alginate form an irreversible gel can be used;
  • the metal crosslinking agent is inorganic salt of calcium, inorganic salt of zinc, inorganic salt of copper, inorganic salt of magnesium, inorganic salt of aluminum, inorganic salt of iron, zinc ion, copper ion, magnesium ion, The action mechanism of aluminum ion and iron ion is the same as that of calcium ion, which will not be repeated here; calcium or calcium lactate.
  • the amount and concentration of the metal cross-linking agent are not limited.
  • the mass dosage ratio of the acid salt is 1:1 ⁇ 1:20, in some other embodiments, the mass dosage ratio of the metal crosslinking agent and the mass dosage ratio of the alginate is 1:5, and the concentration of the metal crosslinking agent solution is 1wt% to 5wt%.
  • the mass dosage of the metal crosslinking agent is less than one-twentieth of the mass dosage of the alginate, the dosage is too small, the resulting gel strength is too low, and the gel cannot even be formed effectively; if the mass dosage of the metal crosslinking agent is greater than If the amount of alginate is twice the mass, if the amount is too much, the degree of cross-linking will be too deep, which is not conducive to the progress of secondary cross-linking.
  • the concentration of the metal cross-linking agent solution is less than 1 wt%, after the metal cross-linking agent solution is mixed with the sodium alginate solution, the volume of the mixed solution formed is too large, which makes it difficult for the metal ions to form a gel with the sodium alginate; If the concentration of the agent solution is greater than 5 wt%, the concentration of metal ions is too large, which will cause the local cross-linking of sodium alginate to be excessive, resulting in uneven gel strength.
  • the preset time in the immobilization step is not limited, and in other embodiments, the preset time is 0.5h-3h. If the preset time is less than 0.5h, the crosslinking time between sodium alginate and the metal crosslinking agent is too short to achieve the purpose of sufficient crosslinking; if the preset time is greater than 3h, the sodium alginate and the metal crosslinking agent The cross-linking time is too long.
  • the fixation temperature in the fixation step may be room temperature, and no special heating or cooling is required.
  • the function of the buffer in the present application is to provide a reaction environment for immobilization.
  • the present application does not limit the type of buffer.
  • the buffer is any one of physiological saline, phosphoric acid, citric acid, carbonic acid, acetic acid, barbituric acid, and tris, preferably physiological saline.
  • the type of organic complexing agent in the above-mentioned immobilization method of liquid lipase, is not limited, and a substance that can form a chemical bond with a carboxyl group is sufficient, and in other embodiments, it is formaldehyde , any one of C2-C8 linear or branched dialdehydes, preferably glutaraldehyde.
  • the mass and dosage ratio of alginate to organic complexing agent is 1:1 to 1:20; in other embodiments, seaweed
  • the mass and dosage ratio of the acid salt to the organic complexing agent is 1:3-1:5; the mass solubility of the organic complexing agent solution is 0.1wt%-2wt%.
  • the mass dosage of the organic complexing agent is less than one-twentieth of the mass dosage of the alginate, the dosage is too small, resulting in an excessively low gel strength; if the mass dosage of the organic complexing agent is greater than the alginate mass dosage If it is doubled, the dosage will be too much, which will cause a large workload in the subsequent washing process.
  • the concentration of the organic complexing agent solution is less than 0.1 wt%, after the organic complexing agent solution is mixed with the sodium alginate solution, the resulting mixed solution has a large volume, which makes it difficult for the organic complexing agent to form a gel with sodium alginate; If the concentration of the complexing agent solution is greater than 2 wt %, the concentration of the organic complexing agent is too large, which will cause excessive local cross-linking of sodium alginate and uneven gel strength.
  • the temperature and time of the chelation reaction are not limited.
  • the temperature is 1°C to 10°C, preferably 2°C to 4°C; and the time is 8h to 24h. If the temperature is lower than 1°C, the temperature is too low, and the chelation reaction is difficult to proceed; if the temperature is higher than 10°C, the temperature is too high, resulting in the chelation reaction being too fast and the degree of crosslinking being too deep.
  • a method for preparing sucrose-6-acetate comprising: preparing sucrose and fatty acid vinyl ester in a mixed organic solution by any of the above-mentioned methods
  • the immobilized liquid lipase is used as a catalyst for dehydration esterification to obtain a sucrose-6-acetate solution.
  • the immobilized enzyme prepared in the present application is used to catalyze the dehydration and esterification of sucrose and fatty acid vinyl esters in mixed organic solvents to prepare sucrose-6-acetate.
  • the immobilized enzyme has extremely high gel strength and can be recycled and reused many times.
  • sucrose-6-acetate is high, and the first use of immobilized enzyme catalyzes the esterification reaction to generate sucrose-6-acetate, and the yield can reach more than 70%, and in some embodiments, it can reach 74.2-78.5 %.
  • the above-mentioned preparation method of sucrose-6-acetate further comprises: recovering the immobilized liquid lipase and applying it to the dehydration esterification reaction of sucrose and fatty acid vinyl ester, repeating After 7 times of use, the yield can still reach 42.4-45.8%.
  • the gel strength of the immobilized enzyme is extremely high, it will not be broken in one reaction, so it can be recycled and reused repeatedly, at least 7 times. Since the gel of the immobilized enzyme will not be broken, the liquid lipase still remains Can maintain high enzymatic activity.
  • the type of fatty acid vinyl ester is not limited, and in other embodiments, the fatty acid vinyl ester is vinyl acetate, butyl Any of vinyl acetate, vinyl caprylate, vinyl caprate, vinyl palmitate, vinyl stearate, or vinyl oleate.
  • the type of mixed organic solvent is not limited.
  • the mixed organic solvent is composed of DMF and tert-butyl
  • the composition of alcohol and/or tert-amyl alcohol is 1:1-10 according to the volume ratio.
  • High performance liquid chromatography Agilent Poroshell 120EC-18 column, the mobile phase is acetonitrile: water (95; 5), the flow rate is 1.0ml/min, the injection volume is 20uL, the column temperature is 40°C, and the detection wavelength of the UV detector is set is 300nm.
  • the reaction ending concentration of sucrose is greater than the initial concentration of sucrose.
  • the undamaged immobilized enzyme 1 in the reaction system was filtered out by a filter press. It was observed with the naked eye that most of the spherical gels were not damaged. After the initial cleaning with normal saline, soak in normal saline at room temperature. place.
  • step d Use the immobilized enzyme 1 recovered in step d. (there will be a certain loss in each reaction process, and the recovered immobilized enzyme 1 will be used repeatedly each time without additional addition) as a catalyst to repeat steps a to d 7 times, and the mass of the immobilized enzyme 1 was weighed after each recovery, and the conversion rate of sucrose was determined by high performance liquid chromatography. The results are listed in Table 1.
  • the undamaged immobilized enzyme 2 in the reaction system is filtered out by a filter press, and after initial cleaning with physiological saline, soaked in physiological saline at room temperature and placed.
  • step d Use the immobilized enzyme 2 recovered in step d. (there will be a certain loss in each reaction process, and the recovered immobilized enzyme 2 will be used repeatedly each time without additional addition) as a catalyst to repeat steps a to d 7 times, and the mass of the immobilized enzyme 2 was weighed after each recovery, and the conversion rate of sucrose was determined by high performance liquid chromatography. The results are listed in Table 2.
  • the immobilized enzyme provided by the present application can be used repeatedly for at least 7 times, and the loss of the immobilized enzyme is very small each time; and the conversion rate of sucrose is very high, which can reach 74% in the first reaction Above, in the seventh reaction, the conversion rate of sucrose can still reach more than 40%.
  • the application utilizes alginate as a carrier to carry out secondary cross-linking with a metal cross-linking agent and an organic complexing agent to fix the liquid lipase, and the obtained gel has extremely high physical strength, which can be more stable in the reaction system. Exist, basically will not be broken and decomposed;
  • the immobilized enzyme can be used repeatedly for many times, which is much higher than the reuse rate of the immobilized enzyme reported in the prior art, and the industrial large-scale continuous esterification is used to produce sucrose-6-ethyl ester;
  • the preparation method of the present invention is simple and the raw materials required are widely sourced, which greatly reduces the cost of immobilizing the liquid lipase, and further reduces the preparation cost of sucrose-6-ethyl ester.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

一种液体脂肪酶的固定化方法，包括依次进行以下步骤：溶解步骤：将海藻酸盐加入去离子水中完全溶解，并在预设温度下，加入液体脂肪酶，得到混合溶液；固定步骤：将混合溶液与金属交联剂溶液混合，并保持预设时间，得到球状凝胶，以使液体脂肪酶固定；以及，螯合步骤：将球状凝胶洗涤后，加入缓冲液和有机络合剂溶液进行螯合反应后，采用缓冲液洗涤，即得到固定化的液体脂肪酶。得到的凝胶物理强度极高，能在反应体系中更加稳定的存在，基本不会破碎分解；能够重复使用多次，更适合利用工业大规模连续性酯化生产蔗糖-6-乙酸酯；制作方法简单、成本低。

Description

液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法 技术领域
本发明属于精细化工技术领域，具体涉及液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法。
发明背景
三氯蔗糖是新鲜甜味剂，三氯蔗糖是由英国泰莱公司与伦敦大学共同研制，并于1976年申请专利的一种新型甜味剂,它是唯一一个以蔗糖为原料的功能性甜味剂。三氯蔗糖对酸味和咸味有淡化效果，对涩味、苦味、酒味等不快的味道有掩盖效果，对辣味、奶味有增效作用，应用范围十分广泛。因此，该产品是当今最理想的强力甜味剂，可供儿童、少年、青年、中年、老年和各种疾病患者食用，没有任何营养学疑问。
而蔗糖-6-乙酸酯是合成三氯蔗糖的重要中间体，合成蔗糖-6-乙酸酯的方法主要是传统的化学法，如乙酸酐酯化法、有机金属合成法、原酸酯法等，蔗糖酯的化学合成反应条件一般比较苛刻，合成的产品颜色比较深，而且由于其选择性差，容易产生副产物。
现有技术中，也有关于酶法合成蔗糖-6-乙酯的报道，如中国专利CN102618601A等，但目前市面上能够买到的制备蔗糖-6-乙酯固体化的液体酶催化剂重复使用率很差，大约重复使用三次后，固体化的液体酶催化剂大部分都已破碎，造成液体酶基本失活，液体酶的损失严重，蔗糖-6-乙酯的合成成本高。
发明内容
鉴于上述问题，提出了本申请以便提供一种克服上述问题或者至少部分地解决上述问题的一种液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法。
根据本申请的一方面，提供一种液体脂肪酶的固定化方法，依次进行以下步骤：
溶解步骤：将海藻酸盐加入去离子水中完全溶解，并在预设温度下，加入液体脂肪酶，得到混合溶液；
固定步骤：将混合溶液与金属交联剂溶液混合，并保持预设时间，得到球状凝胶，以使液体脂肪酶固定；以及，
螯合步骤：将球状凝胶洗涤后，加入缓冲液和有机络合剂溶液进行螯合反应后，采用缓冲液洗涤，即得到固定化的液体脂肪酶。
根据本申请的另一方面，提供了一种蔗糖-6-乙酸酯的制备方法，包括：将蔗糖与脂肪酸乙烯酯在混合有机溶液中，在上述的方法制得的固定化的液体脂肪酶催化作用下进行脱水酯化反应，获得蔗糖-6-乙酸酯溶液的步骤。
综上所述，本申请的有益效果在于：
1.本申请利用海藻酸盐作为载体通过与金属交联剂和有机络合剂进行二次交联，以固定液体脂肪酶，得到的凝胶物理强度极高，能在反应体系中更加稳定的存在，基本不会破碎分解；
2.由于凝胶强度高，固化酶能够重复使用多次，远高于现有报道的固化酶重复利用率，更利用工业大规模连续性酯化生产蔗糖-6-乙酯；
3.本发明制作方法简单、所需原材料来源广泛的材料，大幅度的降低了液体脂肪酶固定化的成本，进一步地，降低了蔗糖-6-乙酯的制备成本。
上述说明仅是本申请技术方案的概述，为了能够更清楚了解本申请的技术手段，而可依照说明书的内容予以实施，并且为了让本申请的上述和其它目的、特征和优点能够更明显易懂，以下特举本申请的具体实施方式。
附图简要说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本申请的限制。而且在整个附图中，用相同的参考符号表示相同的部件。在附图中：
图1示出了根据本申请一个实施例的液体脂肪酶的固定化方法的流程示意图；
图2示出了根据本申请一个实施例的海藻酸钠与乳酸钙形成的凝胶的结构示意图。
实施本发明的方式
下面将参照附图更详细地描述本申请的示例性实施例。虽然附图中显示了本申请的示例性实施例，然而应当理解，可以以各种形式实现本申请而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本申请，并且能 够将本申请的范围完整的传达给本领域的技术人员。
本申请的构思在于，针对现有技术中，固定化的液体脂肪酶(下称固化酶)强度低，重复使用性差的现状，提供了一种采用海藻酸盐为载体，与金属交联剂和有机络合剂进行二次交联，制备含有液体脂肪酶的凝胶，该凝胶强度极高，重复使用率极高。
在本申请中，采用的原料和仪器均为市售产品。
图1示出了根据本申请一个实施例的液体脂肪酶的固定化方法，包括依次进行以下步骤：
溶解步骤S110：将海藻酸盐加入去离子水中完全溶解，并在预设温度下，加入液体脂肪酶，得到混合溶液。
本申请以海藻酸盐为载体，在本申请中，作为载体的海藻酸盐是可溶性的盐，如海藻酸钠，不包括像海藻酸钙等不溶性的盐。
下述以海藻酸钠为例，海藻酸钠(C 6H 7O 6Na) n主要由海藻酸的钠盐组成，由β-D-甘露糖醛酸(M单元)与α-L-古洛糖醛酸(G单元)依靠β-1,4-糖苷键连接并由不同比例的GM、MM和GG片段组成的共聚物。其分子式为式(1)，其羧基团可作为交联点，与金属交联剂和有机络合剂进行交联，以形成不可逆转的凝胶。
式(1)
Figure PCTCN2021071430-appb-000001
将海藻酸盐完全溶解于去离子水中，为了加速海藻酸盐的溶解可采用高速的搅拌装置进行搅拌以及加热手段以促进溶解。待海藻酸盐完全溶解后，在预设温度下，将液体脂肪酶溶解于得到的海藻酸盐溶液中，液体脂肪酶在一定温度下才能保持活性，如果温度过高或过低，可能造成脂肪酶的失活，因此预设温度可以根据选择的液体脂肪酶的种类进行确定，预设温度为不是液体脂肪酶失活的温度。
固定步骤S120：将混合溶液与金属交联剂溶液混合，并保持预设时间，得到球状凝胶，以使液体脂肪酶固定。
金属交联剂为可溶性金属盐，可只配制成溶液使用，在本申请中，优选使用含 有二价或三价金属离子的化合物作为金属交联剂，以乳酸钙和海藻酸钠为例，将海藻酸钠溶液加入到乳酸钙溶液中后，海藻酸钠会与乳酸钙中的钙离子发生离子交换形成海藻酸钙，而海藻酸钙中的钙离子与不同聚合物链上的羧酸根形成离子键，将不同的聚合物链连接在一起，也就是会发生交联反应，类似于在直链上加入更多的支链，使不同的聚合物链形成网状结构，其结构如图2所示，使溶液的黏稠度进一步增加，并且海藻酸钙的溶解度相对于海藻酸钠更低，形成凝胶。在海藻酸钠溶液中加入适量的钙离子，可影响凝胶的软硬程度，通常加入的钙离子越多，胶体越坚固。为了得到球状凝胶，可采用挤出机器，或者采用带有针头的注射剂将凝胶挤出。
以及，螯合步骤S130：将球状凝胶洗涤后，加入缓冲液和有机络合剂溶液进行螯合反应后，采用缓冲液洗涤，即得到固定化的液体脂肪酶。
将上述得到的球状凝胶进行洗涤以洗涤掉未反应的金属交联剂，洗涤可采用去离子水或生理盐水等缓冲液，为加快洗涤过程和促进洗涤程度，可采用减压抽滤的方式。
将球状凝胶洗涤后，再加入缓冲液和有机络合剂，与得到的球状凝胶进行二次交联，即进行螯合反应。在这里缓冲液的作用是为了球状凝胶与有机络合剂提供反应环境，因此对于缓冲液的用量不做限制，淹没球状凝胶即可。
在固定步骤中，海藻酸盐的可供交联的交联基团，即羧基，并未全部与金属交联剂进行交联，余下的未交联的羧基可与有机络合剂进行二次交联。通过两次交联过程，海藻酸盐的交联度被进一步的加深，形成的凝胶强度极强，不易破损。
综上所述，本申请利用海藻酸盐作为载体通过与金属交联剂和有机络合剂进行二次交联，以固定液体脂肪酶，得到的凝胶物理强度极高，能在反应体系中更加稳定的存在，基本不会破碎分解；且能够重复使用多次，远高于现有报道的固体酶重复利用率，更利用工业大规模连续性酯化生产蔗糖-6-乙酸酯；本发明制作方法简单、所需原材料来源广泛的材料，大幅度的降低了液体脂肪酶固定化的成本，进一步地，降低了蔗糖-6-乙酸酯的制备成本。
液体脂肪酶的种类
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，对液体脂肪酶的种类不做限制，脂肪酶是一类具有多种催化能力的酶，可以用来催化水解、醇解、酯化、转酯化及酯类的逆向合成反应等，如在有机相中可以酶促合成和酯交换。在本申请的另一些实施例中，液体脂肪酶可为Lipozyme TL IM、Lipozyme TL100L、 Lipozyme RM IM中的任意一种。
海藻酸盐的种类
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，对海藻酸盐的种类不做限制，在另一些实施例中，海藻酸盐选自海藻酸钠。
海藻酸盐、去离子水、液体脂肪酶的用量
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，对海藻酸盐、去离子水、液体脂肪酶的用量不做限制，其中，去离子水的最少用量以能够完全溶解海藻酸盐为准；在另一些实施例中，海藻酸盐与去离子水和液体脂肪酶的总质量用量比为1:5～1:20；其中，去离子水与液体脂肪酶的质量用量比为0.1～10:1，在一些实施例中，去离子水与液体脂肪酶的质量用量比为3:1。若海藻酸盐的质量用量少于去离子水和液体脂肪酶的总质量用量的二十分之一，则用量过低，造成形成的凝胶不能够完全包埋液体脂肪酶；若海藻酸盐的质量用量多于去离子水和液体脂肪酶的总质量用量的五分之一，则用量过多，则形成的凝胶内，聚合物过多，液体脂肪酶过少，不利用后续的利用。
脂肪酶加入温度
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，对溶解步骤中预设温度不做限制，可以根据选择的液体脂肪酶的种类来确定，以使液体脂肪酶不失活为准。在另一些实施例中，预设温度可以为10～37℃，在又一些实施例中，预设温度可以为28℃。
金属交联剂种类
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，对金属交联剂的种类不做限制，可使海藻酸盐形成不可逆凝胶的金属可溶性金属化合物均可；在另一些实施例中，金属交联剂为钙的无机盐、锌的无机盐、铜的无机盐、镁的无机盐、铝的无机盐、铁的无机盐，锌离子、铜离子、镁离子、铝离子以及铁离子做的作用机理与钙离子一样，在此不作赘述；在另一些实施例中，金属交联剂为钙的无机盐，在又一些实施例中，金属交联剂为氯化钙或乳酸钙。
金属交联剂用量
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，对金属交联剂的用量和浓度不做限制，在另一些实施例中，金属交联剂的质量用量与海藻酸盐的质量用量比为1：1～1:20，在又一些实施例中，金属交联剂的质量用量与海藻酸 盐的质量用量比为1:5，金属交联剂溶液的浓度为1wt％～5wt％。若金属交联剂的质量用量小于海藻酸盐质量用量的二十分之一，则用量过少，形成的凝胶强度过低，甚至不能有效形成凝胶；若金属交联剂的质量用量大于海藻酸盐质量用量的一倍，则用量过多，则交联程度过深，不利于二次交联的进行。若金属交联剂溶液的浓度小于1wt％，在金属交联剂溶液与海藻酸钠溶液混合后，形成的混合溶液体积过大，导致金属离子不易与海藻酸钠形成凝胶；若金属交联剂溶液的浓度大于5wt％，则金属离子的浓度过大，会造成海藻酸钠局部交联过度，形成的凝胶强度不均匀。
固定化时间和温度
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，对固定步骤中的预设时间不做限制，在另一些实施例中，该预设时间为0.5h～3h。如果该预设时间小于0.5h，则海藻酸钠与金属交联剂的交联时间过短，不能达到充分交联的目的；如果该预设时间大于3h，则海藻酸钠与金属交联剂的交联时间过长，在海藻酸钠与金属交联剂的用量都确定的情况下，在3h内已经能够交联完全，再延长时间也不能够加深交联程度，并且带不来其他有益效果。固定步骤中的固定温度为室温即可，无需特殊加热或冷却。
缓冲液种类
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，缓冲液在本申请的中的作用是为固定化提供反应环境，本申请对缓冲液的种类不做限制，在另一些实施例中，缓冲液为生理盐水、磷酸、柠檬酸、碳酸、醋酸、巴比妥酸、三羟甲基氨基甲烷中的任意一种，优选生理盐水。
有机络合剂的种类
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，对有机络合剂的种类不做限制，可以与羧基形成化学键的物质即可，在另一些实施例中为甲醛、C2～C8直链或支链二醛中的任意一种，优选戊二醛。
有机络合剂的用量及溶度
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，海藻酸盐与有机络合剂的质量用量比为1：1～1:20；在另一些实施例中，海藻酸盐与有机络合剂的质量用量比为1:3～1:5；有机络合剂溶液的质量溶度为0.1wt％～2wt％。若有机络合剂的质量用量小于海藻酸盐质量用量的二十分之一，则用量过少，导致形成的凝胶强度过低；若有机络合剂的质量用量大于海藻酸盐质量用量的一倍，则用量过 多，会造成后续洗涤工序工作量大。若有机络合剂溶液的浓度小于0.1wt％，在有机络合剂溶液与海藻酸钠溶液混合后，形成的混合溶液体积大，导致有机络合剂不易与海藻酸钠形成凝胶；若有机络合剂溶液的浓度大于2wt％，则有机络合剂的浓度过大，会造成海藻酸钠局部交联过度，形成的凝胶强度不均匀等问题。
螯合反应条件
在本申请的一些实施例中，在上述的液体脂肪酶的固定化方法中，对螯合步骤中，对螯合反应的温度和时间不做限制，在另一些实施例中，螯合反应在温度为1℃～10℃，优选2℃～4℃；时间为8h～24h条件下进行。若温度低于1℃，则温度过低，螯合反应难以进行；若温度高于10℃，则温度过高，则造成螯合反应过快且交联程度过深。
在本申请的又一些实施例中，还提供了一种蔗糖-6-乙酸酯的制备方法，该方法包括：将蔗糖与脂肪酸乙烯酯在混合有机溶液中，在上述任一的方法制得的固定化的液体脂肪酶作为催化剂的作用下进行脱水酯化反应，以获得蔗糖-6-乙酸酯溶液的步骤。采用本申请的制得的固化酶来催化蔗糖和脂肪酸乙烯酯在混合有机溶剂中进行脱水酯化反应制备蔗糖-6-乙酸酯，该固化酶凝胶强度极高，能够多次回收重复使用；且蔗糖-6-乙酸酯的产率高，在首次使用固化酶催化酯化反应生成蔗糖-6-乙酸酯，其产率能够达到70％以上，在一些实施例中达到74.2～78.5％。
在本申请的一些实施例中，上述的蔗糖-6-乙酸酯的制备方法还包括：将固定化的液体脂肪酶回收并将其用于蔗糖与脂肪酸乙烯酯的脱水酯化反应，在反复使用7次后，产率仍然能够达到42.4～45.8％。
由于固化酶的凝胶强度极高，在一次反应中基本不会发生破碎，因此能够反复回收并利用，至少能够反复使用7次以上，由于固化酶的凝胶不会破碎，因此液体脂肪酶仍然能够保持很高的酶活性。
脂肪酸乙烯酯的种类
在本申请的一些实施例中，在上述的蔗糖-6-乙酸酯的制备方法中，对脂肪酸乙烯酯的种类不做限制，在另一些实施例中为脂肪酸乙烯酯为乙酸乙烯酯、丁酸乙烯酯、辛酸乙烯酯、癸酸乙烯酯、棕榈酸乙烯酯、硬脂酸乙烯酯或油酸乙烯酯中的任一种。
混合有机溶剂
在本申请的一些实施例中，在上述的蔗糖-6-乙酸酯的制备方法中，对混合有 机溶剂的种类不做限制，在另一些实施例中，混合有机溶剂是由DMF与叔丁醇和/或叔戊醇按照体积比为1:1～10组成。
测试手段或条件
高效液相色谱：Agilent Poroshell 120EC-18柱，流动相为乙腈：水(95；5)，流速为1.0ml/min，进样体积为20uL,柱温为40℃，紫外检测器的检测波长设置为300nm。
蔗糖的转化率的计算方法：
由高效液相色谱检测出的数据计算得出，具体是，由蔗糖的反应结束浓度比蔗糖的初始浓度。
实施例1：固化酶1的制备
(1)取500kg海藻酸钠加4.4m 3离子水，60℃蒸汽加热，开启搅拌至完全溶解。
(2)降至室温后(30℃以下)，加入600kg脂肪酶液，搅拌均匀静置直到气泡完全消失。
(3)配制0.9wt％生理盐水5m 3，10m 3的3％的CaCl 2溶液。
(4)匀速泵入(2)中混合溶液，至CaCl 2溶液中进行固定，固定过程在2h左右。
(5)固定结束后，用0.9wt％生理盐水洗涤球状凝胶两次，再加入淹没过球状凝胶液位的生理盐水和0.5wt％戊二醛20L，通入冷气控制温度在2～4℃环境下交联5h。
(6)交联结束后，采用生理盐水洗涤，得到固化酶，并保存备用。
实施例2：采用固化酶1制备蔗糖-6-乙酯
a.配制混合有机溶液，DMF：叔戊醇体积比为1:4，总体积为6m 3，泵入4.8m 3的叔戊醇于反应釜中，再泵入1.2m 3DMF，开启搅拌。
b.投入蔗糖123kg(0.06mol/L)于反应釜中，开启搅拌完全溶解后，泵入乙酸乙烯酯310kg(0.6mol/L)于6m 3的反应釜中。
c.随后投入480kg实施例1中制备得到的固化酶1于反应釜中，在30℃保温反应，200rpm的搅拌速度反应9h。
d.反应结束后，经压滤机过滤过滤出反应体系中的没有破损的固化酶1，肉 眼观察到，绝大部分球状凝胶没有破损，使用生理盐水初次清洗后，于生理盐水中室温浸泡放置。
e.取反应后的母液，采用0.22um滤膜过滤至液相小瓶，并采用高效液相色谱HPLC定量分析蔗糖的转化率，结果列于表1。
f.使用步骤d.中回收的固化酶1(在每次反应过程中会有一定的损失，每次将回收后的固化酶1反复使用，不再额外添加)作为催化剂重复步骤a～d 7次，并在每次回收后称取固化酶1的质量，并采用高效液相色谱测定蔗糖的转化率，结果列于表1。
表1 采用固化酶1制备蔗糖-6-乙酯的结果
Figure PCTCN2021071430-appb-000002
实施例3：固化酶2的制备
(1)取400kg海藻酸钠加3.8m 3离子水，55℃蒸汽加热，开启搅拌至完全溶解。
(2)降至室温后(30℃以下)，加入500kg脂肪酶液，搅拌均匀静置直到气泡完全消失。
(3)配制0.9wt％生理盐水6m 3，12m 3的3％的CaCl 2溶液。
(4)匀速泵入(2)中混合溶液，至CaCl 2溶液中进行固定，固定过程在3h左右。
(5)固定结束后，用0.9wt％生理盐水洗涤球状凝胶两次，再加入淹没过球状凝胶液位的生理盐水和0.5wt％戊二醛50L，通入冷气控制温度在2～4℃环境下交联8h。
(6)交联结束后，采用生理盐水洗涤，得到固化酶2，并保存备用。
实施例4：采用固化酶2制备蔗糖-6-乙酯
a.配制混合有机溶液，DMF：叔戊醇体积比为1:4，总体积为6m 3，泵入4.8m 3的叔戊醇于反应釜中，再泵入1.2m 3DMF，开启搅拌。
b.投入蔗糖123kg(0.06mol/L)于反应釜中，开启搅拌完全溶解后，泵入乙酸乙烯酯310kg(0.6mol/L)于6m 3的反应釜中。
c.随后投入450kg实施例2中制备得到的固化酶2于反应釜中，在30℃保温反应，200rpm的搅拌速度反应10h。
d.反应结束后，经压滤机过滤过滤出反应体系中的没有破损的固化酶2，使用生理盐水初次清洗后，于生理盐水中室温浸泡放置。
e.取反应后的母液，采用0.22um滤膜过滤至液相小瓶，并采用HPLC定量分析蔗糖的转化率，结果列于表2。
f.使用步骤d.中回收的固化酶2(在每次反应过程中会有一定的损失，每次将回收后的固化酶2反复使用，不再额外添加)作为催化剂重复步骤a～d 7次，并在每次回收后称取固化酶2的质量，并采用高效液相色谱测定蔗糖的转化率，结果列于表2。
表2 采用固化酶2制备蔗糖-6-乙酯的结果
Figure PCTCN2021071430-appb-000003
通过表1和表2可以看出，本申请提供的固化酶能够反复使用至少7次，每一次固化酶的损失均很小；且蔗糖的转化率很高，在首次反应时，可达74％以上，在第7次反应时，蔗糖的转化率仍然可以达到40％以上。
综上所述，本申请的有益效果在于：
1.本申请利用海藻酸盐作为载体通过与金属交联剂和有机络合剂进行二次交联，以固定液体脂肪酶，得到的凝胶物理强度极高，能在反应体系中更加稳定的存在，基本不会破碎分解；
2.由于凝胶强度高，固化酶能够重复使用多次，远高于现有报道的固化酶重复利用率，更利用工业大规模连续性酯化生产蔗糖-6-乙酯；
3.本发明制作方法简单、所需原材料来源广泛的材料，大幅度的降低了液体脂肪酶固定化的成本，进一步地，降低了蔗糖-6-乙酯的制备成本。
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方法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。因此，凡是未脱离本发明技术方案的内容，依据本发明的技 术实质对以上实施例所做的任何简单修改、等同替换、等效变化及修饰，均仍属于本发明技术方案保护的范围内。

Claims (10)

  1. 一种液体脂肪酶的固定化方法，其特征在于，依次进行以下步骤：
    溶解步骤：将海藻酸盐加入去离子水中完全溶解，并在预设温度下，加入液体脂肪酶，得到混合溶液；
    固定步骤：将所述混合溶液与金属交联剂溶液混合，并保持预设时间，得到球状凝胶，以使所述液体脂肪酶固定；以及，
    螯合步骤：将所述球状凝胶洗涤后，加入缓冲液和有机络合剂溶液进行螯合反应后，采用缓冲液洗涤，即得到固定化的液体脂肪酶。
  2. 根据权利要求1所述的液体脂肪酶的固定化方法，其特征在于，在所述溶解步骤中，所述液体脂肪酶为Lipozyme TL IM、Lipozyme TL100L、Lipozyme RM IM中的任意一种；
    所述海藻酸盐为海藻酸钠；
    所述海藻酸盐与所述去离子水和所述液体脂肪酶的总质量用量比为1:5～1:20；其中，所述去离子水与所述液体脂肪酶的质量用量比为0.1～10:1，优选3:1；
    所述预设温度为10～37℃。
  3. 根据权利要求1所述的液体脂肪酶的固定化方法，其特征在于，所述金属交联剂为钙的无机盐、锌的无机盐、铜的无机盐、镁的无机盐、铝的无机盐、铁的无机盐，优选钙的无机盐，更优选氯化钙；
    所述金属交联剂的质量用量与所述海藻酸盐的质量用量比为1：1～1:20，优选1:5；
    所述金属交联剂溶液的浓度为1wt％～5wt％。
  4. 根据权利要求1所述的液体脂肪酶的固定化方法，其特征在于，在所述固定步骤中，所述预设时间为0.5h～3h。
  5. 根据权利要求1所述的液体脂肪酶的固定化方法，其特征在于，所述缓冲液为生理盐水、磷酸、柠檬酸、碳酸、醋酸、巴比妥酸、三羟甲基氨基甲烷中的任意一种，优选生理盐水；
    有机络合剂为甲醛、C2～C8直链或支链二醛中的任意一种，优选戊二醇。
  6. 根据权利要求5所述的液体脂肪酶的固定化方法，其特征在于，所述海藻酸盐与所述有机络合剂的质量用量比为1：1～1:20，优选1:3～1:5；所述有机络合剂溶液的质量溶度为0.1wt％～2wt％。
  7. 根据权利要求1所述的液体脂肪酶的固定化方法，其特征在于，在所述螯合步骤中，所述螯合反应在温度为1℃～10℃，优选2℃～4℃；时间为8h～24h条件下进行。
  8. 一种蔗糖-6-乙酸酯的制备方法，其特征在于，包括：将蔗糖与脂肪酸乙烯酯在混合有机溶液中，在权利要求1-7中任一项所述的液体脂肪酶的固定化方法制得的固定化的液体脂肪酶催化作用下进行脱水酯化反应，获得蔗糖-6-乙酸酯溶液的步骤。
  9. 如权利要求8所述的蔗糖-6-乙酸酯的制备方法，其特征在于，还包括：将所述固定化的液体脂肪酶回收并将其用于蔗糖与脂肪酸乙烯酯的脱水酯化反应。
  10. 如权利要求9所述的蔗糖-6-乙酸酯的制备方法，其特征在于，所述脂肪酸乙烯酯为乙酸乙烯酯、丁酸乙烯酯、辛酸乙烯酯、癸酸乙烯酯、棕榈酸乙烯酯、硬脂酸乙烯酯或油酸乙烯酯中的任一种；
    所述混合有机溶液是由DMF与叔丁醇和/或叔戊醇按照体积比为1:1～10组成。
PCT/CN2021/071430 2021-01-13 2021-01-13 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法 WO2022151024A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202180000038.7A CN112888782A (zh) 2021-01-13 2021-01-13 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法
PCT/CN2021/071430 WO2022151024A1 (zh) 2021-01-13 2021-01-13 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/071430 WO2022151024A1 (zh) 2021-01-13 2021-01-13 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法

Publications (1)

Publication Number Publication Date
WO2022151024A1 true WO2022151024A1 (zh) 2022-07-21

Family

ID=76040140

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/071430 WO2022151024A1 (zh) 2021-01-13 2021-01-13 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法

Country Status (2)

Country Link
CN (1) CN112888782A (zh)
WO (1) WO2022151024A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022151024A1 (zh) * 2021-01-13 2022-07-21 安徽金禾实业股份有限公司 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104651345A (zh) * 2013-11-19 2015-05-27 金雪 一种固定化脂肪酶的制备方法
CN107858343A (zh) * 2017-11-27 2018-03-30 中国科学院南海海洋研究所 一种固定化脂肪酶及其制备方法
CN109575090A (zh) * 2018-12-10 2019-04-05 安徽金禾实业股份有限公司 一种蔗糖-6-乙酸酯的制备方法
CN111575327A (zh) * 2020-05-25 2020-08-25 安徽金禾实业股份有限公司 一种米黑根毛霉脂肪酶催化合成蔗糖-6-乙酸酯的方法
CN112888782A (zh) * 2021-01-13 2021-06-01 安徽金禾实业股份有限公司 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101268090A (zh) * 2005-09-22 2008-09-17 法马德医疗保险私人有限公司 利用全细胞生物催化生产蔗糖-6-乙酸酯的方法
CA2632659A1 (en) * 2005-12-09 2007-06-14 V.B. Medicare Pvt. Ltd. Enzymatic production of sucrose-6-ester, an intermediate for the manufacture of halo sugars
CN111763703B (zh) * 2020-07-02 2022-07-19 浙江工业大学 一种有机溶剂中酶法合成蔗糖-6-乙酯的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104651345A (zh) * 2013-11-19 2015-05-27 金雪 一种固定化脂肪酶的制备方法
CN107858343A (zh) * 2017-11-27 2018-03-30 中国科学院南海海洋研究所 一种固定化脂肪酶及其制备方法
CN109575090A (zh) * 2018-12-10 2019-04-05 安徽金禾实业股份有限公司 一种蔗糖-6-乙酸酯的制备方法
CN111575327A (zh) * 2020-05-25 2020-08-25 安徽金禾实业股份有限公司 一种米黑根毛霉脂肪酶催化合成蔗糖-6-乙酸酯的方法
CN112888782A (zh) * 2021-01-13 2021-06-01 安徽金禾实业股份有限公司 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LI QUNLIANG, ZHANG XIN, GUO XIAOBO, YAO PINGJIA, WEI YUANAN: "Enzymatic Synthesis of Sucrose-6-acetate by a Novel Immobilized Fructosyltransferase From Aspergillus sp. GX-0010", IRANIAN JOURNAL OF BIOTECHNOLOGY, vol. 16, no. 2, 15 May 2018 (2018-05-15), pages 114 - 119, XP055949963, DOI: 10.21859/ijb.1380 *
XUE YANG; PU ZHENG; YE NI; ZHIHAO SUN;: "Highly efficient biosynthesis of sucrose-6-acetate with cross-linked aggregates of Lipozyme TL 100 L", JOURNAL OF BIOTECHNOLOGY, ELSEVIER, AMSTERDAM NL, vol. 161, no. 1, 11 May 2012 (2012-05-11), Amsterdam NL , pages 27 - 33, XP028433160, ISSN: 0168-1656, DOI: 10.1016/j.jbiotec.2012.05.014 *
YANG XU'E, ET AL.: "Immobilization of Lipozyme TL 100 L and catalytic synthesis of sucrose-6-acetate", CHEMICAL INDUSTRY AND ENGINEERING PROGRESS, vol. 30, no. 11, 12 November 2011 (2011-11-12), pages 2517 - 2522, XP055949962, ISSN: 1000-6613, DOI: 10.16085/j.issn.1000-6613.2011.11.001 *
ZHONG XIANG; QIAN JUNQING; GUO HUI; HU YUANYUAN; LIU MIN: "Biosynthesis of sucrose-6-acetate catalyzed by surfactant-coatedCandida rugosalipase immobilized on sol-gel supports", BIOPROCESS AND BIOSYSTEMS ENGINEERING, SPRINGER, DE, vol. 37, no. 5, 14 September 2013 (2013-09-14), DE , pages 813 - 818, XP035315934, ISSN: 1615-7591, DOI: 10.1007/s00449-013-1053-9 *

Also Published As

Publication number Publication date
CN112888782A (zh) 2021-06-01

Similar Documents

Publication Publication Date Title
JP6153521B2 (ja) トウモロコシ繊維からのヘミセルロースの抽出方法
WO2022151024A1 (zh) 液体脂肪酶的固定化方法及蔗糖-6-乙酸酯的制备方法
CN100381405C (zh) 黄腐酸络合液肥及其生产方法
CN100422198C (zh) N-乙酰d-氨基葡萄糖的生产方法
CN108559763A (zh) 一种提高壳寡糖聚合度3-6糖含量的制备方法
CN108329205B (zh) 双（2-乙酰氧基苯甲酸）钙脲化合物的制备方法
CN111500661A (zh) 一种同时制备l-鼠李糖和异槲皮素的方法
CN102146423B (zh) 栀子苷元的制备方法
CN105712871B (zh) 一种纯化长链二元酸的方法
CN111363064B (zh) 一种高取代度和高分子量的羧甲基壳聚糖的制备方法
DE2023944A1 (de) Verfahren zur Herstellung von modi fiziertem Sacchandmaterial
US20070087997A1 (en) Methods for producing modified microcrystalline chitosan and uses therefor
CN113855588A (zh) 一种羊毛水解制备角蛋白溶液的方法
CN105385722A (zh) 一种预处理木质纤维生物质提高其产糖率的方法
CN110527003B (zh) 一种低温速溶琼脂糖的制备方法
CN115611759A (zh) 一种(r)-3-氨基丁酸螯合钙的制备方法
CN114507262B (zh) 一种聚唾液酸的水解工艺
CN110938020B (zh) 一种月桂酰精氨酸乙酯盐酸盐的制备工艺
CN111620967A (zh) 一种生产甲壳素高效去色素的生产工艺
CN105198940B (zh) 一种葡萄糖醛酸内酯制备工艺
JPH0725803B2 (ja) 水溶性低分子化キトサンの製造方法
CN102978297B (zh) 一种高浓度木糖水解液的制备方法
CN1249301A (zh) 乳酸钠生产工艺
CN114369075B (zh) 利用2-酮基-l-古龙酸的水溶液一步制备vc结晶的方法
CN111748105B (zh) 一种壳聚糖的溶解方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21918240

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21918240

Country of ref document: EP

Kind code of ref document: A1