CN101200740A - Method for preparing vitamin A fatty acid ester by lipase catalysis - Google Patents
Method for preparing vitamin A fatty acid ester by lipase catalysis Download PDFInfo
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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
Technical field
The present invention relates to a kind of method of producing vitamin A fatty acid ester.More particularly, the invention provides the method for lipase-catalyzed vitamin A of a kind of usefulness and fatty acid response synthesise vitamins A fatty acid ester, particularly in non-aqueous system, use the method for the lipase-catalyzed reaction synthesise vitamins of immobilized microorganism A fatty acid ester.
Background technology
Vitamin A is one of nutrient substance of needed by human, be must indispensable micro-nutrients in the upgrowth and development of children process, it participates in the body various physiological processes, can keep and promote people's bulk-growth, growth, reproduction and cell membrane stability, to the tangible effect of being formed with of vision.Because it can anti-inflammatory, anti-oxidant, regulate immunity, anti aging effect, effect such as anticancer has been widely used in food, medicine, dietary supplements, makeup, the fodder additives.But vitamin A is very unsettled, is easy under air, light, high temperature oxidized.And vitamin A has pungency to skin.Unstable and pungency in order to reduce it can change into Davitin A with vitamin A.But for a long time, industrial vitamin A fatty acid ester all is to produce by chemical method, carries out a series of catalyzed reactions with catalyzer such as sodium methylate, Grignard reagent etc. under High Temperature High Pressure and finishes.Not only total conversion rate is not high, but also has many shortcomings that are difficult to overcome.As: catalyzer is toxic, high-temperature high-voltage reaction energy consumption height, side reaction are many, and the product color and luster is dark, separation and refining difficulty etc., and acid is serious to equipment corrosion, is unfavorable for producing.Along with development of biology, the particularly research of enzyme engineering is for the biological catalyst Acrawax provides new method.Compare with traditional chemical method, enzyme process is synthetic to have the reaction conditions gentleness, high specificity, and side reaction is few, and is environmentally friendly, characteristics such as product quality height.Particularly fatty enzymatic reaction in nonaqueous phase, the range of application and the field of having widened enzyme become the focus of enzymology in recent years.
" Inada, Yuji " discloses the Pseudomonas fluorescens quasi-lipase of modifying with the O-methoxy poly (ethylene glycol) and come catalysis Davitin A synthetic method in " preparation of Davitin A " (JP 62248495,1986).The characteristics of this method are as catalyzer with the lipase after the modification of O-methoxy poly (ethylene glycol), in water saturated benzene solvent, temperature of reaction is 25-30 ℃, utilize and C5-C20 lipid acid to comprise valeric acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, oleic acid etc., mol ratio is a synthesise vitamins A ester under 1: 10 the condition, and the Davitin A yield reaches 80-85%.This method Preparation of catalysts complexity, the cost height; And with an organic solvent benzene is made reaction medium, and benzene is toxic, and environment is had pollution.And, use lipid acid too much can increase the difficulty that later separation is purified, must cause the product cost height.
" Tan Tianwei; Liu Tao; Yin Chunhua " is in " method of the fixed lipase catalyzed synthesise vitamins A of a kind of usefulness fatty acid ester " (Chinese patent application 200310116834.0, the method of the fixed lipase catalyzed synthesise vitamins A of a kind of usefulness fatty acid ester is disclosed 2003), be with substrate and C10-C18 lipid acid or fatty acid ester, in molar ratio 1: 1-1: 7 scope, mix with organic solvent and immobilized lipase, the consumption of immobilized lipase is 0.2-5 a times of quality, under 20-50 ℃ condition, reacted 9-50 hour, take out immobilized lipase, reaction solution is through separating, crystallization obtains vitamin A fatty acid ester; Organic solvent is that water content is the C6-C10 saturated alkane of 0-1%; Immobilized lipase is a carrier with diatomite, resin or fabric membrane, and the yeast quasi-lipase is arranged on the carrier, and the Davitin A yield reaches about 80%.Though this method catalyzer cost is low, has simplified the separation purification step, the Davitin A yield is not high, and the work-ing life of immobilized enzyme is low, has increased difficulty to suitability for industrialized production.
There are a lot of shortcomings in present biocatalysis vitamin A fatty acid ester production method, and too high such as cost, the vitamin A fatty acid ester productive rate is low etc.Therefore, need provide a kind of easy low cost, the method for produced in high yields vitamin A fatty acid ester.
Summary of the invention
An object of the present invention is to provide a kind of new method for preparing vitamin A fatty acid ester.
The present invention use lipase especially immobilized lipase as catalyzer, catalysis vitamin A and fatty acid response, synthesise vitamins A fatty acid ester.By this method, realized purpose of the present invention.Preferably, described method is carried out in non-aqueous system.
Compare with method of the prior art, in the method disclosed in the present, the lipase consumption is few, and the reaction times is short, vitamin A transformation efficiency height, and the lipid acid consumption is few, advantages such as lipase long service life, thus reduced production cost, help suitability for industrialized production.
Embodiment
In the present invention, when describing the reaction of vitamin A fatty acid ester, " vitamin A " is meant Vogan-Neu (retinol); And in general description, vitamin A general reference can be in vivo or external any material that the Vogan-Neu source directly or indirectly is provided, include but not limited to Vogan-Neu, retinene, vitamin A acid and various derivative thereof, such as retinyl acetate, Vogan-Neu fatty acid ester, Vogan-Neu cetylate etc.
In one aspect, the invention provides the method for preparing vitamin A fatty acid ester with lipase-catalyzed vitamin A and fatty acid response.
In the present invention, lipase is meant and can reacts and the esterolytic enzyme of reversed reaction lipid acid by the catalysis fatty acid esterification.All there is lipase in a lot of organisms.Lipase of the present invention can derive from the biology of any natural expression lipase.Similarly, described lipase also can be recombinant production, and described recombinant lipase can be mutant forms or not mutated bodily form formula.In one embodiment, described lipase can be modified, such as with polyethyleneglycol modified.Therefore, in the present invention, the lipase of all mentioning " lipase " or particular source includes natural form, recombinant forms, mutant form, variant form and the various modified forms of this lipase.Lipase of the present invention can commercially obtain or prepare according to currently known methods, the preparation method of lipase well known to a person skilled in the art, comprise for example separation from natural origin, chemosynthesis, gene recombination preparation, random mutagenesis, site-directed mutagenesis, and recombinant expressed, and the modification technique of range protein.This respect has numerous prior art files can be for reference, comprises for example Maniatis and Sambrook etc., " molecular cloning experiment guide ", the 2nd edition, 1989.
In one embodiment, described lipase derives from microorganism, includes but not limited to bacterium and yeast.In one embodiment, described lipase derives from bacterium, as Rhodopseudomonas (Pseudomonas), and preferred Pseudomonas fluorescens (Pseudomonas fluorescence).In one embodiment, described lipase derives 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 are separated fat yeast (Yarrowia lipolytica), particularly derive from Candida lipolytica.The original origin of lipase gene is expressed in " source " described herein expression.For example, the lipase that obtains for expressing the B biology from the biological lipase gene of A thinks to derive from the A biology among the present invention.For example, lipase of the present invention can derive from Candida lipolytica, antarctic candida, inferior sieve and separate in fat yeast, the Pseudomonas fluorescens etc. one or more.In one embodiment, lipase of the present invention derives from Candida lipolytica.These and much the lipase in other biology be known in the art, their preparation method can find in a lot of documents, and be in those skilled in the art's limit of power.
In one embodiment, described lipase can be free lipase or immobilized lipase.
As the lipase of free form, can use isolating lipase, also can use unsegregated lipase.Isolating lipase is meant has removed the lipase that initially has in the environment part or all contaminants, includes but not limited to through the lipase of purifying in various degree.Unsegregated lipase is meant the lipase that is present in the initial environment, includes but not limited to the fermented liquid and the fermented liquid supernatant of fatty enzyme.
In a preferred embodiment, described lipase is immobilized lipase.
Can use the methods known in the art immobilized lipase.For example, can use that any prepares immobilized lipase of the present invention in following two kinds of methods: (A) with carrier and altogether fixing agent press mass volume ratio 1: 1-1: 3 scope mixing, room temperature is dried and is obtained activated carrier; Fixing agent is PEG6000, Oleum Cocois, tween 80, gelatin, Yelkin TTS and sal epsom mixture altogether, and its mass ratio is a gelatin: Yelkin TTS: PEG6000: tween 80: sal epsom: Oleum Cocois=5: 1: 1: 2: 1: 1, magnesium salts was magnesium chloride or sal epsom; With the aqueous solution of lipase by 1000-30000 units/gram activated carrier with the activated carrier immersion, mix, room temperature dries, make enzyme and live and be the immobilized lipase of about 8000IU/g; Perhaps the fermented liquid of (B) usefulness lipase or fermented liquid supernatant are soaked carrier, mix, and room temperature is dried, and make enzyme work and are the immobilized lipase of 8000IU/g.The enzyme work of lipase is defined as among the present invention: under 40 ℃ of conditions, the enzyme amount that hydrolysis sweet oil in the phosphate buffer solution of pH8.0, per minute discharge 1 μ mol lipid acid is an enzyme unit alive.The present invention can make the level of about 8000IU/g carrier when the preparation immobilized lipase, can certainly the flat preparation of other enzyme running water immobilized lipase.This is those skilled in the art's a common practise.
In addition, the preparation of immobilized enzyme of the present invention can be referring to the applicant's common pending trial Chinese invention patent application No.200510112638.5.In addition, in following document, also instructed the preparation of the fermented liquid etc. of the thick enzyme, immobilized enzyme, modifying enzyme and the enzyme that use in the present invention: 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, TianweiTan, Purification and characterization of the extracellular lipase Lip2from Yarrowia lipolytica, Process Biochemistry 42 (2007) 384-391; KailiNie, Feng Xie, Fang Wang, Tianwei Tan, Lipase catalyzed methanolysisto produce biodiesel:Optimization of the biodiesel production.Journalof Molecular Catalysis B:Enzymatic 43 (2006) 142-147; And Chinese invention patent application number 02117614.0, its 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 and Purification 53 (2007) 255-263.The content of above-mentioned document is all incorporated this paper by reference in full into.
In addition, it will be understood by those skilled in the art that fermentation, transformation and the immobilization of enzyme is not limited to the method that above-mentioned document provides, those of ordinary skill can very easily be determined the preparation method of suitable enzyme according to the general knowledge in this area.
In lipase immobilization of the present invention, can adopt any suitable carriers.Described carrier can be a solid particulate for example, as silica gel, diatomite or molecular sieve, and preferred silica gel.Perhaps, described carrier can be membranous fabric or non-woven, for example natural fabric such as cotton or man made fiber fabric such as polyester textile, especially membranous fabric, be preferably cotton, nylon, silk, polyester or cellulosic fabric, it has, and surface-area is big, adsorptivity is strong, low price, good stability and the characteristics that can reuse.
Used carrier diatomite and resin also are known common carrier, and fabric membrane is the fabric of selling on the market.The used fermented liquid of the present invention for example can be a yeast quasi-lipase fermented liquid, with commercially available or all can of preparation certainly.For example: adopt the Candida lipolytica fermentative preparation, fermentation condition can be that 26 ℃ of temperature, pH are natural, mixing speed is that 300rpm, air flow are 1VVM, and the substratum composition can be: compositions such as organic nitrogen source, organic carbon source, inorganic salt.
In one embodiment, the immobilized lipase of fabric membrane of the present invention can be used for stirred-tank reactor, and immobilized lipase is fixed on the cylindrical rack, and rack is fixed in the rotating shaft and does axially rotation as stirring rake in the lump.As an alternative, also can use immobilized lipase by alternate manner known in the art.
In one embodiment, the immobilized lipase of fabric membrane of the present invention can be used for fixed bed type reactor, and immobilized lipase is fixed on the cylindrical rack, and cylindrical rack is fixed in the reactor.As an alternative, also can use immobilized lipase by alternate manner known in the art.
In the method for the invention, described lipid acid can be selected from: lipid acid, and composition thereof.Described lipid acid can be selected from C4-C30 lipid acid, C5-C26 lipid acid, and C6-C24 lipid acid, C6-C20 lipid acid, C8-C18 lipid acid, C8-C16 lipid acid, C10-C18 lipid acid, C10-C16 lipid acid, C12-C16 lipid acid, and composition thereof.Such as it can be lauric acid, tetradecanoic acid, palmitinic acid, stearic acid etc., or its mixture.In the present invention, described lipid acid can be saturated or unsaturated fatty acids, preferred saturated fatty acid.
In one embodiment, described lipid acid can be fatty acid mixt or the isolating lipid acid that animal/Vegetable oil lipoprotein hydrolysis produces.
In a preferred embodiment, described lipid acid is palmitinic acid.
In one aspect, the present invention prepares being reflected in the non-aqueous system of vitamin A fatty acid ester and carries out.This non-aqueous system is well known by persons skilled in the art.For example, described non-aqueous system can be an organic solvent, perhaps can also be ionic liquid.Ionic liquid as herein described is meant the material that is constituted and all be liquid state by positive charged ions and electronegative ion between-100 ℃ to 200 ℃.In ionic liquid, do not have electroneutral molecule in theory, and all be negatively charged ion and positively charged ion.
Enzyme catalysis in non-aqueous system is better than the enzyme in the water.Organic solvent can keep lower water-activity, can reduce the thermodynamics and kinetics obstacle of esterification and hydrolysis reaction.And thereby organic substrates has higher solubleness and can improve speed of response in organic solvent.Therefore, the esterification in non-aqueous system can realize high conversion, simplifies separating technology.
In one embodiment, described organic solvent can be aromatic hydrocarbon and derivative, halogenated alkane and derivative thereof, straight chain and branched paraffin, naphthenic hydrocarbon etc. or its mixture.For example, described organic solvent is 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.In a preferred embodiment, described organic solvent is normal hexane or sherwood oil.Sherwood oil is the material that gets from petroleum fractionating, and it is dissimilar to be divided into 30-60,60-90 etc. by the fractionation boiling range, and its main component is lower aliphatic alkane such as pentane and hexane.
In some embodiments, non-aqueous system of the present invention can be moisture or not moisture.For example, the water content of described non-aqueous system can be 0-10%, preferred 0-5%, more preferably 0-2%, most preferably 0-1%.
In a preferred embodiment, described organic solvent is the sherwood oil of water content 0-1%.For example, can be that water content is 0,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% sherwood oil.
In one aspect, can add water-retaining agent in the reaction system of the present invention to remove moisture, such as silica gel, molecular sieve, water-absorbing resin etc.Silica gel as herein described is meant inorganic silica gel, and it mainly is made up of silicon-dioxide, has very strong adsorptive power, is a kind of chromatographic separation carrier commonly used.Silica gel is divided into macroporous silica gel, silochrom, mesoporous silica gel, Kiselgel A etc. according to the big I in its aperture.Molecular sieve is a kind of silico-aluminate compound with cubic(al)grating, mainly connects to form spacious skeleton structure by sial by oxo bridge, and uniform duct, a lot of apertures and marshalling, hole that internal surface area is very big are arranged in structure.These small hole diameter are even, can be than the inside of the little molecular adsorption of channel diameter to the hole, and handle is than the big molecule exclusion that gets in duct, thereby can be the different molecule of shape diameter, the molecule that polarity degree is different, the molecule that boiling point is different, the molecular separation that degree of saturation is different comes, promptly has the effect of " screening " molecule, so be called molecular sieve.The molecular sieve wettability power is extremely strong, should avoid during preservation being directly exposed in the air.Water-absorbing resin is meant various high molecular polymers with water-retaining capacity, such as polyacrylic acid, Mierocrystalline cellulose and derivative thereof etc.
In vitamin A fatty acid ester preparation feedback of the present invention, the mol ratio of vitamin A and lipid acid can be any available ratio, for example considers raw materials cost and preferably is no more than 1: 1.In one embodiment, the mol ratio of vitamin A and lipid acid can be 1: 1-1: 5 scope, and for example 1: 1-1: 3 scope.But in the present invention, the mol ratio of vitamin A and lipid acid is not limited to this scope, and for example its mol ratio can be greater than 1: 1 or less than 1: 5.In the method for the invention, 1: 1 and even more the vitamin A of high molar ratio and lipid acid still can obtain excellent vitamin A transformation efficiency.Such as, in embodiment 2, the vitamin A of 1: 1 mol ratio and palmitinic acid obtain 90% vitamin A transformation efficiency.
In vitamin A fatty acid ester preparation feedback of the present invention, the consumption of lipase can be the amount of any appropriate.The consumption of lipase is relevant with multiple factor, and these factors include but not limited to specific activity, lipase source of mol ratio, reaction system, temperature of reaction, reaction times, the lipase self of the amount of the amount of vitamin A and concentration, lipid acid and concentration, two kinds of substrates etc.In one embodiment, the consumption of immobilized lipase be the vitamin A quality 0.2-5 doubly.For example, can 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 embodiments, the consumption of immobilized lipase is not limited thereto.
In the preparation feedback system of vitamin A fatty acid ester of the present invention, its temperature of reaction can be any temperature that can react.Optimal reactive temperature is relevant with concrete reaction conditions, such as lipase source, lipase concentration, substrate type, organic solvent, reaction times etc.For example, temperature of reaction can be 20-50 ℃, 25-45 ℃, and 30-40 ℃, 30-37 ℃ etc.In a specific embodiments, temperature of reaction can be 15 ℃, and 20 ℃, 25 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃ etc. for example is 30 ℃.During reaction, it is constant that temperature of reaction can keep, and perhaps changes according to certain rule.In general, during reaction keep temperature of reaction constant.
In the preparation feedback system of vitamin A fatty acid ester of the present invention, its reaction times can be any time length that can react, and this can set according to actual needs.For transformation efficiency and other desired effects that expectation reaches, the concrete reaction times is relevant with multiple reaction conditions, such as amount and concentration, substrate type and amount of substrate, the organic solvent etc. of lipase source, lipase.For example, the reaction times can be 1 minute-1 week, 5 minutes-2 days, or 10 minutes-1 day etc.In a specific embodiments, the reaction times can be 1 minute, 2 minutes, and 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 invention provides a kind of method that in non-aqueous system, prepares vitamin A fatty acid ester with fixed lipase catalyzed vitamin A and fatty acid response.
In one embodiment, with substrate vitamin A and lipid acid, in molar ratio 1: 1-1: 5 scope, mix with organic solvent and immobilized lipase, the consumption of immobilized lipase is 0.3-5 a times of vitamin A quality, under 20-50 ℃ condition, reacts 1-24 hour, take out immobilized lipase, reaction solution obtains vitamin A fatty acid ester through separation, crystallization; Organic solvent is that water content is the sherwood oil of 0-1%; Immobilized lipase is the yeast quasi-lipase that is fixed on the carrier, for example: Candida lipolytica lipase, antarctic candidia lipase etc.
Shallow by the product color that the present invention obtains, the quality height, cost is low.For the suitability for industrialized production of fatty acid ester provides a kind of novel method.
The present invention compares with traditional chemical synthesis, and method reaction conditions gentleness of the present invention, energy consumption are low, greatly reduce production cost.The present invention simultaneously uses biological catalyst, and atopic is strong, by product is few.Product has lighter color, characteristics that quality is high.
The present invention compares with the synthetic method of using to make substrate, has the following advantages: 1, higher transformation efficiency is arranged; 2, the reaction times shortens; 3, lipase can be reused such as immobilized enzyme, thereby prolongs its work-ing life; 4, the mol ratio of vitamin A and lipid acid can be littler, reduces the amount of excess fatty acids.
Embodiment
Further describe the present invention below in conjunction with some specific embodiments.It should be noted that the just explanation as an example of these specific embodiments, and should not be construed as restriction the claimed scope of the present invention.In addition, also provide comparing embodiment, further to illustrate the advantage of the present invention with respect to currently used method.
Used lipase is application reference people's Chinese invention patent application No.200510112638.5 and Tan TW in the embodiment of the invention, Zhang M, Wang BW, Ying CH, Deng L.Screening of high lipase producing Candida sp.and production of lipaseby fermentation.Process Biochem 2003; 39 (4): the method preparation among the 459-65.
Calculate the transformation efficiency of vitamin A according to following method.At first the Vitamin A Palmitate 1.7 M.I.U/Gram that generates in the reaction is carried out HPLC and analyze, chromatographic condition is as follows: chromatographic column is Alltech C18 (250 * 4.6mm, 4.5 μ m); Moving phase is 100% methyl alcohol; Detector is Tianjin, island 10A UV-detector; Detect wavelength 327nm; Flow velocity 1ml/min.With Vitamin A Palmitate 1.7 M.I.U/Gram standard substance production standard curve, calculate to generate the quality of Vitamin A Palmitate 1.7 M.I.U/Gram according to typical curve with external standard method, then according to the transformation efficiency of following Equation for Calculating vitamin A:
The transformation efficiency of vitamin A=(m/M)/(m
0/ M
0) * 100%
Wherein, m is for generating the quality (g) of Vitamin A Palmitate 1.7 M.I.U/Gram; M is the molecular weight 524.9 (g/mol) of Vitamin A Palmitate 1.7 M.I.U/Gram; m
0Quality (g) for the substrate vitamin A; M
0Molecular weight 328.4 (g/mol) for vitamin A.
Embodiment 1:
1. lipase immobilization
Cotton and common fixing agent are pressed 1: 1 mixed of mass volume ratio, and (W: V), room temperature is dried, and obtains activated carrier; Fixing agent is PEG6000, Oleum Cocois, tween 80, gelatin, Yelkin TTS and sal epsom mixture altogether, and its mass ratio is a gelatin: Yelkin TTS: PEG6000: tween 80: sal epsom: Oleum Cocois=5: 1: 1: 2: 1: 1.With the fermented liquid of Candida lipolytica lipase in the ratio of 8000IU/g activated carrier with the activated carrier immersion, mix, room temperature dries, make enzyme and live and be the immobilized lipase of 8000IU/g.
2. catalysis synthesise vitamins A cetylate
With substrate 0.160g (0.56mmol) vitamin A and 0.429g (1.68mmol) palmitinic acid and 10ml water content is that 0.5% petroleum ether solvent and 0.3g immobilized lipase mix, add 0.5g silica gel, under the condition of 30 ℃ of temperature, in the shaking table (190r/min), reacted 8 hours, transformation efficiency is 95%; Reaction solution is removed immobilized lipase, silica gel, the solvent removed by evaporation at reduced pressure sherwood oil, obtain the crude product Vitamin A Palmitate 1.7 M.I.U/Gram, under 30 ℃, repeatedly wash the crude product Vitamin A Palmitate 1.7 M.I.U/Gram with methyl alcohol again, centrifugal, abandoning supernatant obtains the Vitamin A Palmitate 1.7 M.I.U/Gram of purifying after vacuum-drying, further crystallization obtains light yellow Vitamin A Palmitate 1.7 M.I.U/Gram crystal.
Embodiment 2:
Operation steps is with embodiment 1, and difference is: 0.429g (1.68mmol) palmitinic acid changes 0.143g (0.56mmol) into, and substrate mol ratio (vitamin A/palmitinic acid) is 1: 1; Transformation efficiency is 93%.
Embodiment 3:
Operation steps is with embodiment 1, and difference is: 0.429g (1.68mmol) palmitinic acid changes 0.286g (1.12mmol) into, and substrate mol ratio (vitamin A/palmitinic acid) is 1: 2; Transformation efficiency is 93%.
Embodiment 4:
0.429g (1.68mmol) palmitinic acid changes 0.527g (2.24mmol) into, substrate mol ratio (vitamin A/palmitinic acid) is 1: 4; Transformation efficiency is 94%.
Embodiment 5:
Operation steps is with embodiment 1, and difference is: 0.429g (1.68mmol) palmitinic acid changes 0.715g (2.8mmol) into, and substrate mol ratio (vitamin A/palmitinic acid) is 1: 5; Transformation efficiency is 95%.
Embodiment 6:
Operation steps is with embodiment 1, and difference is: the 0.3g immobilized lipase changes the 0.05g immobilized lipase into; Transformation efficiency is 91.5%.
Embodiment 7:
Operation steps is with embodiment 1, and difference is: the 0.3g immobilized lipase changes the 0.1g immobilized lipase into; Transformation efficiency is 94%.
Embodiment 8:
Operation steps is with embodiment 1, and difference is: the 0.3g immobilized lipase changes the 0.2g immobilized lipase into; Transformation efficiency is 95%.
Embodiment 9:
Operation steps is with embodiment 1, and difference is: the 0.3g immobilized lipase changes the 0.4g immobilized lipase into; Transformation efficiency is 95%.
Embodiment 10:
Operation steps is with embodiment 1, and difference is: the 0.3g immobilized lipase changes the 0.5g immobilized lipase into; Transformation efficiency is 95%.
Embodiment 11:
Operation steps is with embodiment 1, and difference is: temperature changes 25 ℃ of temperature into for 30 ℃; Transformation efficiency is 93%.
Embodiment 12:
Operation steps is with embodiment 1, and difference is: temperature changes 40 ℃ of temperature into for 30 ℃; Transformation efficiency is 89%.
Embodiment 13:
Operation steps is with embodiment 1, and difference is: temperature changes 50 ℃ of temperature into for 30 ℃; Transformation efficiency is 78%.
Embodiment 14:
Operation steps is with embodiment 1, and difference is: reaction times 8h changes reaction times 0.5h into; Transformation efficiency is 94.7%.
Embodiment 15:
Operation steps is with embodiment 1, and difference is: reaction times 8h changes reaction times 1h into; Transformation efficiency is 96%.
Embodiment 16:
Operation steps is with embodiment 1, and difference is: reaction times 8h changes reaction times 2h into; Transformation efficiency is 95%.
Embodiment 17:
Operation steps is with embodiment 1, and difference is: reaction times 8h changes reaction times 4h into; Transformation efficiency is 95%.
Embodiment 18:
Operation steps is with embodiment 1, and difference is: reaction times 8h changes reaction times 6h into; Transformation efficiency is 94%.
Embodiment 19:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and adds 0.3g silica gel; Transformation efficiency is 94%.
Embodiment 20:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and adds 0.8g silica gel; Transformation efficiency is 95%.
Embodiment 21:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel; Transformation efficiency is 91%.
Embodiment 22:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and the 0.3g immobilized lipase changes the 0.05g immobilized lipase into; Transformation efficiency is 61.5%.
Embodiment 23:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and the 0.3g immobilized lipase changes the 0.1g immobilized lipase into; Transformation efficiency is 74.2%.
Embodiment 24:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and the 0.3g immobilized lipase changes the 0.2g immobilized lipase into; Transformation efficiency is 86%.
Embodiment 25:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and the 0.3g immobilized lipase changes the 0.4g immobilized lipase into; Transformation efficiency is 90%.
Embodiment 26:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and reaction times 8h changes reaction times 0.5h into; Transformation efficiency is 70%.
Embodiment 27:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and reaction times 8h changes reaction times 1h into; Transformation efficiency is 84%.
Embodiment 28:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and reaction times 8h changes reaction times 2h into; Transformation efficiency is 90%.
Embodiment 29:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and reaction times 8h changes reaction times 4h into; Transformation efficiency is 90%.
Embodiment 30:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and reaction times 8h changes reaction times 6h into; Transformation efficiency is 91%.
Embodiment 31:
Operation steps is with embodiment 1, and difference is: adding 0.5g silica gel changes into and does not add silica gel, and reaction times 8h changes reaction times 1h into; Transformation efficiency is 84%.
Embodiment 32:
With the method immobilization that the fermented liquid of Candida lipolytica lipase is pressed embodiment 1, make enzyme work and be the immobilized lipase of 8000IU/g; Be that 0.5% petroleum ether solvent and 1g immobilized lipase mix again with substrate 0.160g (0.56mmol) vitamin A and 0.429g (1.68mmol) palmitinic acid and 10ml water content, add 0.8g silica gel, under the condition of 30 ℃ of temperature, in the shaking table (190r/min), reacted 4 hours, transformation efficiency is 95%; Take out immobilized lipase, dry and put into new above-mentioned reaction system, reacted 4 hours, transformation efficiency is 96%; Repeat aforesaid operations 50 times, transformation efficiency keeps more than 92% always, and promptly immobilized lipase can be reused 50 times, and transformation efficiency keeps more than 92%.
Comparing embodiment 1
To separate of the method immobilization of the fermented liquid of fat first silk yeast fat enzyme, make enzyme work and be the immobilized lipase of 8000IU/g by embodiment 1; Be that 0.5% petroleum ether solvent and 0.3g immobilized lipase mix again with substrate 0.100g (0.3mmol) and 0.234g (0.9mmol) palmitinic acid and 10ml water content, under the condition of 30 ℃ of temperature, in the shaking table (190r/min), reacted 24 hours, transformation efficiency is 80%; Take out immobilized lipase, reaction solution obtains light yellow Vitamin A Palmitate 1.7 M.I.U/Gram crystal through separation, crystallization.
Comparing embodiment 2
Operation steps is with comparing embodiment 1, and difference is: the 0.234g palmitinic acid changes 0.078g into, substrate mol ratio (/ palmitinic acid) 1: 1; Transformation efficiency is 49%.
Comparing embodiment 3
Operation steps is with comparing embodiment 1, and difference is: the 0.234g palmitinic acid changes 0.156g into, substrate mol ratio (/ palmitinic acid) 1: 2; Transformation efficiency is 60%.
Comparing embodiment 4
Operation steps is with comparing embodiment 1, and difference is: react to change in 24 hours and reacted 1 hour; Transformation efficiency is 25%.
Comparing embodiment 5
Operation steps is with comparing embodiment 1, and difference is: react to change in 24 hours and reacted 3 hours; Transformation efficiency is 55%.
Comparing embodiment 6
Operation steps is with comparing embodiment 1, and difference is: react to change in 24 hours and reacted 12 hours; Transformation efficiency is 79%.
Comparing embodiment 7
The fermented liquid of Candida lipolytica lipase is pressed embodiment 1 method immobilization, make enzyme work and be the immobilized lipase of 8000IU/g; Be that 0.5% petroleum ether solvent and 0.3g immobilized lipase mix again with substrate 0.100g (0.3mmol) and 0.234g (0.9mmol) palmitinic acid and 10ml water content, under the condition of 30 ℃ of temperature, in the shaking table (190r/min), reacted 12 hours, transformation efficiency is 80%; Take out immobilized lipase, dry and put into new above-mentioned reaction system, reacted 12 hours, transformation efficiency is 80%; Repeat aforesaid operations, the 5th, transformation efficiency is 75%; Repeat aforesaid operations, the 6th time, transformation efficiency 60%; Repeat aforesaid operations, the 7th time, transformation efficiency is 55%; Repeat aforesaid operations, the 8th time, transformation efficiency is 45%.
Persons skilled in the art can be on the basis of understanding the disclosed content of the present invention; to technical scheme disclosed in this invention carry out variously replenishing, modification, variation and replacement etc., these replenish, modification, variation and replacement etc. are also all in the protection domain of claim of the present invention.
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 ℃.
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| CNA2007101798080A CN101200740A (en) | 2007-12-18 | 2007-12-18 | Method for preparing vitamin A fatty acid ester by lipase catalysis |
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| CNA2007101798080A CN101200740A (en) | 2007-12-18 | 2007-12-18 | Method for preparing vitamin A fatty acid ester by lipase catalysis |
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| CN102363606A (en) * | 2011-11-03 | 2012-02-29 | 厦门金达威集团股份有限公司 | Method for synthesizing vitamin A palmitate |
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| CN105441521A (en) * | 2014-09-01 | 2016-03-30 | 浙江工业大学 | Synthetic method of vitamin A palmitate |
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| CN109234262A (en) * | 2018-09-17 | 2019-01-18 | 北京化工大学 | A kind of method that carrier granulating technique prepares immobilised enzymes |
| CN109234262B (en) * | 2018-09-17 | 2022-07-08 | 北京化工大学 | Method for preparing immobilized enzyme by carrier granulation technology |
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| CN109777793B (en) * | 2019-03-15 | 2020-12-08 | 常熟理工学院 | GDSL lipase, genetically engineered bacterium and application thereof |
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