CN101200740A - A method for preparing vitamin A fatty acid ester catalyzed by lipase - Google Patents

Abstract

The invention relates to a method for producing vitamin A fatty acid esters. To be specific, the invention provides a method which uses fatty enzyme catalysis vitamin A and fatty acid to synthesize the vitamin A fatty acid, in particular a method that uses the immobilized microorganism lipase catalytic reaction to synthesize the vitamin A fatty acid. The method disclosed in the invention has the advantages of little lipase used amount, short reaction time, high vitamin A conversion, little fatty acid used amount, long service life of the lipase, etc. Therefore, the production cost is lowered, which is beneficial to the industrialized production.

Description

A method for preparing vitamin A fatty acid ester catalyzed by lipase

technical field

The invention relates to a method for producing vitamin A fatty acid ester. More specifically, the present invention provides a method for synthesizing vitamin A fatty acid esters by using lipase to catalyze the reaction of vitamin A and fatty acid, especially in the non-aqueous phase system, using immobilized microbial lipase to catalyze the reaction to synthesize vitamin A fatty acid esters Methods.

Background technique

Vitamin A is one of the essential nutrients for the human body. It is an indispensable micronutrient in the growth and development of children. It participates in various physiological processes of the body and can maintain and promote the growth, development, reproduction and stability of the cell membrane of the human body. The formation has obvious effect. Because of its anti-inflammatory, anti-oxidation, immune regulation, anti-skin aging, anti-cancer and other effects, it has been widely used in food, medicine, nutritional health products, cosmetics, and feed additives. However, vitamin A is very unstable and is easily oxidized in air, light, and high temperature. And vitamin A is irritating to the skin. To reduce its instability and irritation, vitamin A can be converted to vitamin A esters. But for a long time, industrial vitamin A fatty acid esters have been produced by chemical methods, using catalysts such as sodium methoxide, Grignard reagent, etc. to carry out a series of catalytic reactions under high temperature and high pressure to complete. Not only the total conversion rate is not high, but also there are many shortcomings that are difficult to overcome. Such as: catalyst toxicity, high temperature and high pressure reaction, high energy consumption, many side reactions, deep color of product, difficulty in separation and purification, etc., and the acid corrodes the equipment seriously, which is not conducive to production. With the development of biotechnology, especially the research of enzyme engineering, a new method has been provided for biocatalyst synthesis of fatty acid esters. Compared with traditional chemical methods, enzymatic synthesis has the characteristics of mild reaction conditions, strong specificity, less side reactions, environmental friendliness, and high-quality products. Especially in the non-aqueous phase, the lipase-catalyzed reaction has broadened the application range and field of the enzyme, and has become a hot spot in enzymatic research in recent years.

"Inada, Yuji" in "Preparation of Vitamin A Esters" (JP 62248495, 1986) discloses a method for catalyzing the synthesis of vitamin A esters with O-methoxypolyethylene glycol-modified Pseudomonas fluorescens lipase . The method is characterized in that lipase modified by O-methoxy polyethylene glycol is used as a catalyst, and in a water-saturated benzene solvent, the reaction temperature is 25-30 ° C, and vitamin A acetate and C5-C20 fatty acids are used. Valeric acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, etc., synthesize vitamin A ester under the condition of molar ratio of 1:10, and the yield of vitamin A ester reaches 80-85%. The preparation of the catalyst in the method is complicated and the cost is high; moreover, the organic solvent benzene is used as the reaction medium, and the benzene is toxic and pollutes the environment. Moreover, using too much fatty acid will increase the difficulty of subsequent separation and purification, which will inevitably lead to high product costs.

"Tan Tianwei, Liu Tao, Yin Chunhua" disclosed a method for catalyzing the synthesis of vitamin A fatty acid esters with immobilized lipase (Chinese patent application 200310116834.0, 2003). The ester method is that the substrate vitamin A acetate and C10-C18 fatty acid or fatty acid ester are mixed with an organic solvent and an immobilized lipase in a molar ratio of 1:1-1:7 to immobilize the lipase The dosage is 0.2-5 times the quality of vitamin A acetate, react for 9-50 hours under the condition of 20-50°C, take out the immobilized lipase, and the reaction solution is separated and crystallized to obtain vitamin A fatty acid ester; organic solvent It is a C6-C10 saturated alkane with a water content of 0-1%. The immobilized lipase uses diatomaceous earth, resin or fabric film as a carrier, and there is yeast lipase on the carrier. The yield of vitamin A ester is about 80%. Although the method has low catalyst cost and simplifies the separation and purification steps, the yield of the vitamin A ester is not high, and the service life of the immobilized enzyme is low, which adds difficulties to industrial production.

The current biocatalytic method for producing vitamin A fatty acid ester has many disadvantages, such as high cost and low yield of vitamin A fatty acid ester. Therefore, it is necessary to provide a method for preparing vitamin A fatty acid esters with low cost and high yield.

Contents of the invention

An object of the present invention is to provide a new method for preparing vitamin A fatty acid ester.

The invention uses lipase, especially immobilized lipase, as a catalyst to catalyze the reaction of vitamin A and fatty acid to synthesize vitamin A fatty acid ester. By this method, the object of the present invention is achieved. Preferably, the method is carried out in a non-aqueous system.

Compared with the method in the prior art, in the method disclosed in the present invention, lipase consumption is few, and reaction time is short, and vitamin A conversion rate is high, fatty acid consumption is few, advantages such as lipase service life are long, thereby reduces The production cost is reduced, which is conducive to industrialized production.

Detailed ways

In the present invention, when describing the reaction of vitamin A fatty acid ester, "vitamin A" refers to retinol (retinol); and in the general description, vitamin A generally refers to the ability to directly or indirectly provide Any substance that is a source of retinol, including but not limited to retinol, retinal, retinoic acid, and various derivatives thereof, such as retinyl acetate, retinyl fatty acid esters, retinyl palmitate Esters etc.

In one aspect, the present invention provides a method for preparing vitamin A fatty acid ester by using lipase to catalyze the reaction of vitamin A and fatty acid.

In the present invention, lipase refers to an enzyme capable of catalyzing fatty acid esterification and its reverse reaction, hydrolysis of fatty acid ester. Lipase is present in many organisms. The lipase of the present invention may be derived from any organism that naturally expresses a lipase. Likewise, the lipase may also be produced recombinantly, and the recombinant lipase may be in mutant or non-mutant form. In one embodiment, the lipase may be modified, such as with polyethylene glycol. Therefore, in the present invention, any reference to "lipase" or lipase from a specific source includes the natural form, recombinant form, mutant form, variant form and various modified forms of the lipase. The lipase of the present invention can be obtained commercially or prepared according to known methods. The preparation methods of lipase are well known to those skilled in the art, including, for example, isolation from natural sources, chemical synthesis, gene recombination preparation, random mutagenesis, site-directed mutagenesis , and recombinant expression, as well as various protein modification techniques. Numerous prior art documents are available in this regard, including, for example, Maniatis and Sambrook et al., A Laboratory Guide to Molecular Cloning, 2nd Edition, 1989.

In one embodiment, the lipase is derived from microorganisms, including but not limited to bacteria and yeast. In one embodiment, the lipase is derived from bacteria, such as Pseudomonas, preferably Pseudomonas fluorescence. In one embodiment, the lipase is derived from yeast, especially from the genera Saccharomyces, Candida and/or Yarrowia, preferably from a yeast selected from the group consisting of: Candida lipolytica, Candida antarctica, Yarrowia lipolytica, especially derived from Candida lipolytica. As used herein, "source" means the original origin of expression of the lipase gene. For example, a lipase obtained by expressing a lipase gene from an organism A in an organism B is considered to be derived from the organism A in the present invention. For example, the lipase of the present invention may be derived from one or more of Candida lipolytica, Candida antarctica, Yarrow lipolytica, Pseudomonas fluorescens and the like. In one embodiment, the lipase of the invention is derived from Candida lipolytica. Lipases from these and many other organisms are known in the art and methods for their preparation can be found in many literatures and are within the purview of those skilled in the art.

In one embodiment, the lipase may be free lipase or immobilized lipase.

As the lipase in free form, either isolated lipase or unisolated lipase may be used. Isolated lipase refers to lipase from which some or all of the contaminants in the original environment have been removed, including but not limited to lipase purified to varying degrees. Unisolated lipase refers to lipase present in the initial environment, including but not limited to lipase-containing fermentation broth and fermentation broth supernatant.

In a preferred embodiment, said lipase is an immobilized lipase.

Lipase can be immobilized using methods known in the art. For example, any one of the following two methods can be used to prepare the immobilized lipase of the present invention: (A) mix the carrier and co-immobilizer in the range of mass volume ratio 1:1-1:3, and dry at room temperature to obtain Activated carrier; co-fixative is PEG6000, coconut oil, Tween 80, gelatin, lecithin and magnesium sulfate mixture, and its mass ratio is gelatin: lecithin: PEG6000: Tween 80: magnesium sulfate: coconut oil=5: 1: 1:2:1:1, the magnesium salt is magnesium chloride or magnesium sulfate; the aqueous solution of lipase is used to soak the activated carrier and the activated carrier at 1000-30000 units/g, mix, and dry at room temperature to obtain an enzyme activity of about 8000IU/g or (B) soak the carrier with lipase fermentation broth or fermentation broth supernatant, mix, and dry at room temperature to obtain an immobilized lipase with an enzyme activity of 8000 IU/g. The enzymatic activity of lipase in the present invention is defined as: under the condition of 40 DEG C, the enzyme amount of hydrolyzing olive oil in a phosphate buffer solution of pH 8.0 and releasing 1 μmol of fatty acid per minute is an enzyme activity unit. When preparing the immobilized lipase in the present invention, the level of about 8000 IU/g carrier can be prepared, and of course other enzyme activity levels can also be used to prepare the immobilized lipase. This is common knowledge of those skilled in the art.

In addition, the preparation of the immobilized enzyme of the present invention can be referred to the applicant's co-pending Chinese invention patent application No. 200510112638.5. In addition, the preparation of fermented liquid of crude enzyme, immobilized enzyme, modified enzyme and enzyme used in the present invention is also taught in the following documents: Tan TW, Zhang M, Wang BW, Ying CH, Deng L.Screening of high lipaseproducing Candida sp. and production of lipase by fermentation. Process Biochem 2003, 39(4): 459-65; Mingrui Yu, Shaowei Qin, Tianwei Tan, Purification and characterization of the extracellular lipase Lip2from Yarrowia0 lipolytica 2Biolytica, 384-391; KailiNie, Feng Xie, Fang Wang, Tianwei Tan, Lipase catalyzed methanolysis to produce biodiesel: Optimization of the biodiesel production. Journal of Molecular Catalysis B: Enzymatic 43(2006) 142-147; The publication number is CN1456674; Mingrui Yu, StefanLange, Sven Richter, Tianwei Tan, Rolf D.Schmid High～levelexpression of extracellular lipase Lip2 from Yarrowia lipolytica inPichia pastoris and its purification and characterization. ProteinExpression-5(3250-5) The contents of the above documents are all incorporated herein by reference in their entirety.

In addition, those skilled in the art will understand that the fermentation, transformation and immobilization of enzymes are not limited to the methods provided in the above documents, and those of ordinary skill can easily determine the production and preparation of suitable enzymes based on general knowledge in the art. method.

In the immobilization of the lipase of the present invention, any suitable carrier can be used. The support can be, for example, solid particles such as silica gel, diatomaceous earth or molecular sieves, preferably silica gel. Alternatively, the carrier can be a film-like fabric or a non-woven fabric, such as a natural fiber fabric such as cotton or a chemical fiber fabric such as a polyester fabric, especially a film-like fabric, preferably cotton, nylon, silk, polyester or cellulose. The fabric has the characteristics of large surface area, strong adsorption, low price, good stability and reusability.

The used carrier diatomaceous earth and resin are also known and commonly used carriers, and the fabric membrane is a commercially available fabric. The fermented liquid used in the present invention can be, for example, yeast-based lipase fermented liquid, either commercially available or self-prepared. For example: using Candida lipolytica for fermentation, the fermentation conditions can be temperature 26°C, pH natural, stirring speed 300rpm, ventilation volume 1VVM, medium composition can be: organic nitrogen source, organic carbon source, inorganic salt, etc. .

In one embodiment, the immobilized lipase of the fabric membrane of the present invention can be used in a stirred tank reactor, and the immobilized lipase is fixed on a cylindrical grid, and the grid is fixed on a rotating shaft and serves as a stirring paddle for the axial direction. rotate. Alternatively, immobilized lipases may also be used in other ways known in the art.

In one embodiment, the immobilized lipase on the fabric membrane of the present invention can be used in a fixed bed reactor, and the immobilized lipase is fixed on a cylindrical grid, and the cylindrical grid is fixed in the reactor. Alternatively, immobilized lipases may also be used in other ways known in the art.

In the method of the present invention, the fatty acid may be selected from: fatty acids, and mixtures thereof. The fatty acid can be selected from C4-C30 fatty acid, C5-C26 fatty acid, C6-C24 fatty acid, C6-C20 fatty acid, C8-C18 fatty acid, C8-C16 fatty acid, C10-C18 fatty acid, C10-C16 fatty acid, C12-C16 fatty acid , and mixtures thereof. For example, it may be lauric acid, myristic acid, palmitic acid, stearic acid, etc., or a mixture thereof. In the present invention, the fatty acid may be saturated or unsaturated fatty acid, preferably saturated fatty acid.

In one embodiment, the fatty acid may be a mixture of fatty acids or isolated fatty acids produced by hydrolysis of animal/vegetable oils and fats.

In a preferred embodiment, the fatty acid is palmitic acid.

In one aspect, the reaction of the present invention for preparing vitamin A fatty acid ester is carried out in a non-aqueous phase system. Such non-aqueous systems are known to those skilled in the art. For example, the non-aqueous phase system may be an organic solvent, or may also be an ionic liquid. The ionic liquid described herein refers to a substance that is composed of positively charged ions and negatively charged ions and is in a liquid state between -100°C and 200°C. Theoretically, there are no electrically neutral molecules in ionic liquids, but all anions and cations.

Enzyme catalysis in non-aqueous systems is superior to that in water. Organic solvents can maintain low water activity, which can reduce the thermodynamic and kinetic barriers of esterification and hydrolysis reactions. Moreover, organic substrates have higher solubility in organic solvents, which can increase the reaction rate. Therefore, the esterification reaction in a non-aqueous system can achieve high conversion and simplify the separation process.

In one embodiment, the organic solvent may be aromatic hydrocarbons and their derivatives, halogenated alkanes and their derivatives, linear and branched alkanes, cycloalkanes, etc., or mixtures thereof. For example, the organic solvent is benzene, toluene, xylene, methyl chloride, methylene chloride, chloroform, tetrachloromethane, ethyl chloride, ethylene dichloride, n-hexane, n-heptane, n-octane, n- Decane, cyclohexane, petroleum ether, etc., or a mixture thereof. In a preferred embodiment, the organic solvent is n-hexane or petroleum ether. Petroleum ether is a substance obtained by fractionation of petroleum. According to the fractionation boiling range, it can be divided into different types such as 30-60 and 60-90. Its main components are lower aliphatic alkanes such as pentane and hexane.

In some embodiments, the non-aqueous systems of the present invention may or may not contain water. For example, the water content of the non-aqueous phase system may be 0-10%, preferably 0-5%, more preferably 0-2%, most preferably 0-1%.

In a preferred embodiment, the organic solvent is petroleum ether with a water content of 0-1%. For example, it may be petroleum ether with a water content of 0, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%.

In one aspect, a water-absorbing agent can be added to the reaction system of the present invention to remove water, such as silica gel, molecular sieve, water-absorbing resin, and the like. The silica gel mentioned in this article refers to inorganic silica gel, which is mainly composed of silicon dioxide, has strong adsorption capacity, and is a commonly used chromatographic separation carrier. Silica gel can be divided into macroporous silica gel, coarse-porous silica gel, mesoporous silica gel, and fine-pore silica gel according to its pore size. Molecular sieve is a kind of aluminosilicate compound with cubic lattice. It is mainly composed of silicon and aluminum connected by oxygen bridges to form a hollow skeleton structure. These tiny pores have uniform diameters, which can adsorb molecules smaller than the diameter of the pores into the interior of the pores, while repelling molecules larger than the pores, so that molecules with different shapes and diameters, and molecules with different degrees of polarity can be absorbed. , Molecules with different boiling points and molecules with different degrees of saturation are separated, that is, they have the function of "sieving" molecules, so they are called molecular sieves. Molecular sieves have strong hygroscopicity, so they should avoid direct exposure to air during storage. Water-absorbent resin refers to various polymers with water-absorbing capacity, such as polyacrylic acid, cellulose and its derivatives.

In the preparation reaction of vitamin A fatty acid ester of the present invention, the molar ratio of vitamin A and fatty acid can be any available ratio, for example, it is preferably not more than 1:1 in consideration of raw material cost. In one embodiment, the molar ratio of vitamin A and fatty acid may be in the range of 1:1-1:5, for example in the range of 1:1-1:3. However, in the present invention, the molar ratio of vitamin A and fatty acid is not limited to this range, for example, the molar ratio may be greater than 1:1 or less than 1:5. In the method of the present invention, a 1:1 and even higher molar ratio of vitamin A and fatty acid can still give excellent vitamin A conversion. For example, in Example 2, a 1:1 molar ratio of vitamin A and palmitic acid resulted in a vitamin A conversion of 90%.

In the preparation reaction of vitamin A fatty acid ester of the present invention, the amount of lipase can be any suitable amount. The amount of lipase is related to many factors, these factors include but not limited to the amount and concentration of vitamin A, the amount and concentration of fatty acids, the molar ratio of the two substrates, reaction system, reaction temperature, reaction time, lipase itself Specific activity, source of lipase, etc. In one embodiment, the amount of immobilized lipase is 0.2-5 times the mass of vitamin A. For example, it may be 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.8 times, 1 times, 2 times, 3 times, 4 times, 5 times and any ratio therebetween. In a specific embodiment, the amount of immobilized lipase is not limited thereto.

In the preparation reaction system of vitamin A fatty acid ester of the present invention, its reaction temperature can be any temperature that can carry out reaction. The optimum reaction temperature is related to specific reaction conditions, such as lipase source, lipase concentration, substrate type, organic solvent, reaction time, etc. For example, the reaction temperature may be 20-50°C, 25-45°C, 30-40°C, 30-37°C, etc. In a specific embodiment, the reaction temperature may be 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, etc., for example 30°C. During the reaction, the reaction temperature can be kept constant or changed according to certain rules. In general, the reaction temperature is kept constant during the reaction.

In the reaction system for the preparation of vitamin A fatty acid ester of the present invention, the reaction time can be any length of time that can carry out the reaction, which can be set according to actual needs. For the expected conversion rate and other desired effects, the specific reaction time is related to various reaction conditions, such as the source of lipase, the amount and concentration of lipase, the type and amount of substrate, organic solvent and so on. For example, the reaction time can be 1 minute to 1 week, 5 minutes to 2 days, or 10 minutes to 1 day, etc. In a specific embodiment, the reaction time can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours , 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days etc.

In one aspect, the present invention provides a method for preparing vitamin A fatty acid ester by using immobilized lipase to catalyze the reaction between vitamin A and fatty acid in a non-aqueous phase system.

In one embodiment, the substrate vitamin A and fatty acid are mixed with organic solvent and immobilized lipase in a molar ratio of 1:1-1:5, and the amount of immobilized lipase is 0.3% of vitamin A quality. -5 times, under the condition of 20-50°C, react for 1-24 hours, take out the immobilized lipase, and the reaction solution is separated and crystallized to obtain vitamin A fatty acid ester; the organic solvent is petroleum with a water content of 0-1%. Ether; immobilized lipase is a yeast lipase immobilized on a carrier, for example: Candida lipolytica lipase, Candida antarctica lipase, etc.

The product obtained by the invention has light color, high quality and low cost. A new method is provided for the industrial production of fatty acid esters.

Compared with the traditional chemical synthesis method, the method of the invention has mild reaction conditions, low energy consumption, and greatly reduces the production cost. Simultaneously, the invention uses a biocatalyst, which has strong reaction specificity and few by-products. The product has the characteristics of light color and high quality.

Compared with the synthetic method using vitamin A acetate as substrate, the present invention has the following advantages: 1, higher conversion rate is arranged; 2, the reaction time is shortened; 3, lipase such as immobilized enzyme can be reused, thereby its 4. The molar ratio of vitamin A and fatty acid can be smaller, reducing the amount of excess fatty acid.

Example

The present invention will be further described below in conjunction with some specific embodiments. It should be noted that these specific examples are only for illustration, and should not be construed as limiting the protection scope of the present invention. In addition, comparative examples are provided to further illustrate the advantages of the present invention over currently used methods.

The lipase used in the embodiments of the present invention refers to the applicant's Chinese invention patent application No. 200510112638.5 and Tan TW, Zhang M, Wang BW, Ying CH, Deng L. Screening of high lipase producing Candida sp. and production of lipase by fermentation. Prepared by the method in Process Biochem 2003; 39(4): 459-65.

The conversion rate of vitamin A was calculated according to the following method. First, the vitamin A palmitate generated in the reaction is analyzed by HPLC, and the chromatographic conditions are as follows: the chromatographic column is Alltech C18 (250 × 4.6mm, 4.5 μm); the mobile phase is 100% methanol; the detector is Shimadzu 10A UV detector ; Detection wavelength 327nm; Flow rate 1ml/min. Make a standard curve with the vitamin A palmitate standard substance, calculate the quality of generating vitamin A palmitate with the external standard method according to the standard curve, then calculate the conversion rate of vitamin A according to the following equation:

Conversion rate of vitamin A = (m/M)/(m ₀ /M ₀ )×100%

Wherein, m is the quality (g) that generates vitamin A palmitate; M is the molecular weight 524.9 (g/mol) of vitamin A palmitate; m ₀ is the quality (g) of substrate vitamin A; M ₀ is vitamin A The molecular weight is 328.4 (g/mol).

Example 1:

1. Immobilization of Lipase

Cotton cloth and co-fixative were mixed according to the mass volume ratio of 1:1 (W:V), and dried at room temperature to obtain activated carrier; co-fixative was a mixture of PEG6000, coconut oil, Tween 80, gelatin, lecithin and magnesium sulfate , its mass ratio is gelatin: lecithin: PEG6000: Tween 80: magnesium sulfate: coconut oil=5: 1: 1: 2: 1: 1. The fermented liquid of candida lipolytica lipase is soaked with the activated carrier at the ratio of 8000IU/g activated carrier, mixed and dried at room temperature to prepare the immobilized lipase with an enzyme activity of 8000IU/g.

2. Catalytic synthesis of vitamin A palmitate

Substrate 0.160g (0.56mmol) vitamin A and 0.429g (1.68mmol) palmitic acid and 10ml water content are 0.5% sherwood oil solvent and 0.3g immobilized lipase mixing, add 0.5g silica gel, at temperature 30 ℃ Under conditions, in a shaker (190r/min), reacted for 8 hours, and the conversion rate was 95%; the reaction liquid was removed from immobilized lipase and silica gel, and the solvent petroleum ether was evaporated under reduced pressure to obtain the crude product vitamin A palmitate, and then Wash the crude vitamin A palmitate several times with methanol at 30°C, centrifuge, discard the supernatant, and obtain purified vitamin A palmitate after vacuum drying, and further crystallize to obtain light yellow vitamin A palmitate crystals.

Example 2:

The operation steps are the same as in Example 1, except that: 0.429g (1.68mmol) palmitic acid is changed to 0.143g (0.56mmol), and the substrate molar ratio (vitamin A/palmitic acid) is 1: 1; conversion rate is 93% .

Example 3:

The operating steps are the same as in Example 1, except that: 0.429g (1.68mmol) palmitic acid is changed to 0.286g (1.12mmol), and the substrate molar ratio (vitamin A/palmitic acid) is 1: 2; the conversion rate is 93% .

Example 4:

0.429g (1.68mmol) of palmitic acid was changed to 0.527g (2.24mmol), and the substrate molar ratio (vitamin A/palmitic acid) was 1:4; the conversion rate was 94%.

Example 5:

The operating steps are the same as in Example 1, except that: 0.429g (1.68mmol) palmitic acid is changed to 0.715g (2.8mmol), and the substrate molar ratio (vitamin A/palmitic acid) is 1: 5; conversion rate is 95% .

Embodiment 6:

The operation steps are the same as in Example 1, except that 0.3 g of immobilized lipase is changed to 0.05 g of immobilized lipase; the conversion rate is 91.5%.

Embodiment 7:

The operation steps are the same as in Example 1, except that 0.3 g of immobilized lipase is changed to 0.1 g of immobilized lipase; the conversion rate is 94%.

Embodiment 8:

The operation steps are the same as in Example 1, except that 0.3 g of immobilized lipase is changed to 0.2 g of immobilized lipase; the conversion rate is 95%.

Embodiment 9:

The operation steps are the same as in Example 1, except that 0.3 g of immobilized lipase is changed to 0.4 g of immobilized lipase; the conversion rate is 95%.

Example 10:

The operation steps are the same as in Example 1, except that 0.3 g of immobilized lipase is changed to 0.5 g of immobilized lipase; the conversion rate is 95%.

Example 11:

The operation steps are the same as in Example 1, except that the temperature is changed from 30° C. to 25° C.; the conversion rate is 93%.

Example 12:

The operation steps are the same as in Example 1, except that the temperature is changed from 30° C. to 40° C.; the conversion rate is 89%.

Example 13:

The operation steps are the same as in Example 1, except that the temperature is changed from 30° C. to 50° C.; the conversion rate is 78%.

Example 14:

The operation steps are the same as in Example 1, except that the reaction time is changed from 8h to 0.5h; the conversion rate is 94.7%.

Example 15:

The operation steps are the same as in Example 1, except that the reaction time is changed from 8h to 1h; the conversion rate is 96%.

Example 16:

The operation steps are the same as in Example 1, except that the reaction time is changed from 8h to 2h; the conversion rate is 95%.

Example 17:

The operation steps are the same as in Example 1, except that the reaction time is changed from 8h to 4h; the conversion rate is 95%.

Example 18:

The operation steps are the same as in Example 1, except that the reaction time is changed from 8h to 6h; the conversion rate is 94%.

Example 19:

The operation steps are the same as those in Example 1, except that the addition of 0.5 g of silica gel is changed to 0.3 g of silica gel; the conversion rate is 94%.

Example 20:

The operation steps are the same as those in Example 1, except that the addition of 0.5 g of silica gel is changed to 0.8 g of silica gel; the conversion rate is 95%.

Example 21:

The operation steps are the same as those in Example 1, except that 0.5 g of silica gel is added instead of silica gel; the conversion rate is 91%.

Example 22:

The operation steps are the same as those in Example 1, except that: instead of adding 0.5 g of silica gel, 0.3 g of immobilized lipase is replaced by 0.05 g of immobilized lipase; the conversion rate is 61.5%.

Example 23:

The operation steps are the same as those in Example 1, except that: instead of adding 0.5 g of silica gel, 0.3 g of immobilized lipase is replaced by 0.1 g of immobilized lipase; the conversion rate is 74.2%.

Example 24:

The operation steps are the same as those in Example 1, except that: instead of adding 0.5 g of silica gel, 0.3 g of immobilized lipase is replaced by 0.2 g of immobilized lipase; the conversion rate is 86%.

Example 25:

The operation steps are the same as those in Example 1, except that: instead of adding 0.5 g of silica gel, 0.3 g of immobilized lipase is replaced by 0.4 g of immobilized lipase; the conversion rate is 90%.

Example 26:

The operation steps are the same as those in Example 1, except that the addition of 0.5 g of silica gel is changed to no addition of silica gel, and the reaction time of 8 h is changed to 0.5 h; the conversion rate is 70%.

Example 27:

The operation steps are the same as those in Example 1, except that the addition of 0.5 g of silica gel is changed to no addition of silica gel, and the reaction time of 8 hours is changed to 1 hour; the conversion rate is 84%.

Example 28:

The operation steps are the same as those in Example 1, except that the addition of 0.5 g of silica gel is changed to no addition of silica gel, and the reaction time of 8 hours is changed to 2 hours; the conversion rate is 90%.

Example 29:

The operation steps are the same as those in Example 1, except that the addition of 0.5 g of silica gel is changed to no addition of silica gel, and the reaction time of 8 hours is changed to 4 hours; the conversion rate is 90%.

Example 30:

The operation steps are the same as those in Example 1, except that the addition of 0.5 g of silica gel is changed to no addition of silica gel, and the reaction time of 8 hours is changed to 6 hours; the conversion rate is 91%.

Example 31:

The operation steps are the same as those in Example 1, except that the addition of 0.5 g of silica gel is changed to no addition of silica gel, and the reaction time of 8 hours is changed to 1 hour; the conversion rate is 84%.

Example 32:

The fermented liquid of Candida lipolytica lipase is immobilized by the method of embodiment 1, and the immobilized lipase that makes enzyme activity is 8000IU/g; Substrate 0.160g (0.56mmol) vitamin A and 0.429g ( 1.68mmol) of palmitic acid and 10ml water content are 0.5% sherwood oil solvent and 1g immobilized lipase mixing, add 0.8g silica gel, under the condition of temperature 30 ℃, in the shaker (190r/min), react 4 hours, The conversion rate is 95%; take out the immobilized lipase, dry it and put it into the new above-mentioned reaction system, react for 4 hours, the conversion rate is 96%; repeat the above operation 50 times, the conversion rate remains above 92% all the time, that is, immobilized lipase The lipase can be reused 50 times, while the conversion rate remains above 92%.

Comparative Example 1

The fermented liquid of M. lipolytica lipase is immobilized by the method of Example 1, and the obtained enzyme activity is the immobilized lipase of 8000IU/g; Substrate 0.100g (0.3mmol) vitamin A acetate and 0.234 g (0.9mmol) of palmitic acid is mixed with 10ml of petroleum ether solvent with a water content of 0.5% and 0.3g of immobilized lipase, and is reacted in a shaking table (190r/min) at a temperature of 30°C for 24 hours, and the conversion rate 80%; the immobilized lipase was taken out, and the reaction solution was separated and crystallized to obtain light yellow vitamin A palmitate crystals.

Comparative Example 2

The operating steps are the same as in Comparative Example 1, except that 0.234g of palmitic acid is changed to 0.078g, and the substrate molar ratio (vitamin A acetate/palmitic acid) is 1: 1; the conversion rate is 49%.

Comparative Example 3

The operating steps are the same as in Comparative Example 1, except that 0.234g of palmitic acid is changed to 0.156g, and the substrate molar ratio (vitamin A acetate/palmitic acid) is 1:2; the conversion rate is 60%.

Comparative Example 4

The operation steps are the same as those in Comparative Example 1, except that the reaction time is 1 hour instead of 24 hours; the conversion rate is 25%.

Comparative Example 5

The operation steps are the same as those in Comparative Example 1, except that the reaction time is 3 hours instead of 24 hours; the conversion rate is 55%.

Comparative Example 6

The operating steps are the same as in Comparative Example 1, except that the reaction time is 12 hours instead of 24 hours; the conversion rate is 79%.

Comparative Example 7

The fermented liquid of Candida lipolytica lipase is immobilized by the method of Example 1, and the obtained enzyme activity is 8000IU/g immobilized lipase; then substrate 0.100g (0.3mmol) vitamin A acetate and 0.234g (0.9mmol) palmitic acid and 10ml water content are the sherwood oil solvent of 0.5% and 0.3g immobilized lipase mixing, under the condition of temperature 30 ℃, in the shaker (190r/min), reaction 12 hours, conversion rate 80%; take out the immobilized lipase, dry it and put it into the new above-mentioned reaction system, react for 12 hours, the conversion rate is 80%; repeat the above-mentioned operation, the 5th time, the conversion rate is 75%; repeat the above-mentioned operation, the 6th Repeat the above operation, the 7th time, the conversion rate is 55%; repeat the above operation, the 8th time, the conversion rate is 45%.

Those skilled in the art can make various supplements, modifications, changes and substitutions to the technical solutions disclosed in the present invention on the basis of understanding the disclosed content of the present invention. These supplements, modifications, changes and substitutions are also included in the present invention. within the scope of the claims.

Claims (12)

1. method for preparing vitamin A fatty acid ester, it comprises the step of utilizing lipase-catalyzed vitamin A and fatty acid response.

2. the process of claim 1 wherein that described lipase is immobilized lipase.

3. the method for claim 2; the fixation support of wherein said immobilized lipase is selected from solid particulate or membranaceous natural or non-natural fabric or non-woven; such as silica gel, diatomite, resin, cotton and polyester, nylon, silk and cellulosic fabric and non-woven, preferably be selected from cotton and nylon, silk, polyester or cellulosic fabric and non-woven.

4. the method for claim 1, wherein said lipase derives from microorganism, for example derive from bacterium, as Rhodopseudomonas (Pseudomonas), preferred Pseudomonas fluorescens (Pseudomonas fluorescence), perhaps derive from yeast, especially derive from yeast belong (Saccharomyces), mycocandida (Candida) and/or inferior sieve yeast belong (Yarrowia), preferably derive from and be selected from following yeast: Candida lipolytica (Candida lipolytica), antarctic candida (Candida antarctica), inferior sieve is separated fat yeast (Yarrowia lipolytica), particularly derives from Candida lipolytica.

5. the method for claim 1, wherein said lipid acid is selected from C4-C18 lipid acid and composition thereof, preferred C5-C18 lipid acid and composition thereof, more preferably C6-C16 lipid acid and composition thereof, especially preferred C8-C16 lipid acid and composition thereof, fatty acid mixt or isolating lipid acid, most preferably palmitinic acid such as lauric acid, tetradecanoic acid, palmitinic acid, stearic acid and composition thereof, animal/Vegetable oil lipoprotein hydrolysis generation.

6. the process of claim 1 wherein that described being reflected in the non-aqueous system carry out.

7. the method for claim 6, the water content of wherein said non-aqueous system is 0-5%, preferred 0-3%, more preferably 0-2%, 0-1% most preferably, such as be 0,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% or therebetween any number.

8. the method for claim 6, wherein said non-aqueous system is an organic solvent, preferred described organic solvent is selected from: benzene,toluene,xylene, methyl chloride, methylene dichloride, trichloromethane, tetrachloromethane, monochloroethane, ethylene dichloride, normal hexane, normal heptane, octane, n-decane, hexanaphthene, sherwood oil etc. or its mixture more preferably are selected from normal hexane or sherwood oil.

9. the process of claim 1 wherein that the mol ratio of vitamin A and lipid acid can be the ratio of any appropriate, preferably be no more than 1: 1, more preferably 1: 1-1: in 5 scopes, most preferably 1: 1-1: in 3 scopes.

10. the process of claim 1 wherein the consumption of lipase be the vitamin A quality 0.2-5 doubly, for example 0.3-4 doubly, 0.5-3 is doubly.

11. the method for claim 6 wherein adds solid particulate carrier in described non-aqueous system, for example silica gel, diatomite or molecular sieve, preferably silica gel.

12. the process of claim 1 wherein and carry out under the described 20-50 of being reflected at ℃, preferred 25-45 ℃, more preferably 30-40 ℃, especially preferred 30-37 ℃ of condition, for example carry out at 30 ℃.