CN102776256B - Method for catalytic synthesis of fructose lauric acid monoester by using immobilized phospholipase A1 - Google Patents
Method for catalytic synthesis of fructose lauric acid monoester by using immobilized phospholipase A1 Download PDFInfo
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- CN102776256B CN102776256B CN201210229312.0A CN201210229312A CN102776256B CN 102776256 B CN102776256 B CN 102776256B CN 201210229312 A CN201210229312 A CN 201210229312A CN 102776256 B CN102776256 B CN 102776256B
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Abstract
The invention discloses a method for catalytic synthesis of the fructose lauric acid monoester by using the immobilized phospholipase A1. According to the method, lauric acid and fructose are subjected to catalytic reaction through enzymes to synthesize the fructose lauric acid monoester. The preparation method includes the steps of immobilizing the phospholipase A1, preparing a dehydration organic solvent, and subjecting the immobilized phospholipase A1 to catalytic synthesis to produce the fructose lauric acid monoester. According to the method for catalytic synthesis of the fructose lauric acid monoester by using the immobilized phospholipase A1, the used phospholipase A1 is cheap in price and only 5% to 10% of the price of lipase, and the price of resin D-201 used for immobilizing the phospholipase is low. The immobilized phospholipase A1 has good catalytic effect, the conversion rate of the fructose lauric acid monoester is high, the product separation is simple, and the immobilized phospholipase A1 can be recycled. Accordingly, products prepared through the method are low in cost and have good application prospect in industry.
Description
Technical field
The present invention relates to a kind of immobilized PLA1 and catalyze and synthesize surgar ester surfactant---fructose lauric acid monoester, belongs to organic synthesis field.
Background technology
Sugar ester is to be generally formed by connecting by ester bond by longer chain fatty acid and sugar.Therefore sugar ester molecule has the poly-hydroxy sugar of nonpolar longer chain fatty acid and polarity simultaneously, thereby has Action of Surfactant.Sugar ester molecule does not have ionization functional group, is typical nonionogenic tenside.It,, as the typical non-ionic type green bio tensio-active agent of a class excellent property, has important use in the industry such as food, makeup, medicine, washing composition, fiber.Fructose ester, as a class nonionogenic tenside, has a wide range of applications field equally.
The synthetic method of sugar ester has chemical method and enzyme process, chemical method carries out often under high temperature, noxious solvent and basic catalyst, this makes, and product color is dark, monoester content is not high, production energy consumption is high and have a potential safety hazard, and esterification position and the gamma value of product are wayward, for product separation and use have brought inconvenience.Therefore from Product Safety and environmental angle, Applied Biotechnology substitutes traditional chemical route of synthesis production compound and meets Overseas Development trend.Enzyme process is synthetic has the features such as high efficiency and specificity, reaction conditions gentleness, by product be less, and development in recent years is very fast.
At present, it is mainly lipase that enzyme process is prepared fructose lauric acid monoester method, and as NOVO435, but business-like lipase is expensive, has therefore just limited its application.
Phospholipase A1 is a kind of microbe-derived Phospholipid hydrolase that Novozymes Company releases, and is usually used in coming unstuck of grease, and this enzyme is a kind of carboxylic ester hydrolase, has lipase activity and phospholipase activity simultaneously.In certain reaction system, its meeting precedence table reveals wherein a kind of specific enzyme lives, such as lipase activity.Do not see at present about adopting immobilized PLA1 catalysis fructose and lauric acid to prepare the report of fructose lauric acid monoester.
Summary of the invention
The object of this invention is to provide a kind of method that immobilized PLA1 catalyzes and synthesizes fructose lauric acid monoester.The method reaction solvent is nontoxic, and enzyme dosage is few and can recycle, and reaction product all easily separates with lipase with reaction solvent, is conducive to serialization and produces.Employing immobilized PLA1 provided by the present invention catalyzes and synthesizes the method for fructose lauric acid monoester, comprises following content:
(1) immobilization of phospholipase A1: the DA-201 macroporous adsorbent resin of getting 5 ~ 10 parts of activated processing of quality, the phosphate buffered saline buffer of 10 ~ 25 parts of quality, 0.5 ~ 2 part of free phospholipase A1 of quality mixes, whip attachment 2 ~ 6h at 25 ~ 35 DEG C, then filter, obtain macroporous adsorbent resin immobilized phospholipase, by the immobilized enzyme vacuum-drying making, for subsequent use;
(2) prepare dehydrated organic solvent: 3 or 4 molecular sieves are placed in 95～105 DEG C of baking ovens and activate 5～10h, then be placed in moisture eliminator and be cooled to room temperature, for subsequent use; Afterwards, then the amount that 3 or 4 molecular sieves after activation are added to 50～80 parts of quality according to every liter of solvent joins stand-byly in the organic solvent of reaction medium, and 24～72h is placed in sealing, removes by filter molecular sieve, obtains dehydrated organic solvent;
(3) immobilized PLA1 catalyzes and synthesizes fructose lauric acid monoester: get 10～40.6 parts of Quality Month cinnamic acids and 1.8～36.5 parts of quality fructose and be placed in above-mentioned dehydrated organic solvent and mix, wherein lauric acid concentration is 5～24wt%, then add with respect to the immobilized PLA1 of lauric acid quality 8～25wt% start reaction; After reaction 0～2h according to adding 3 after activation or 4 molecular sieves with the water except generating in dereaction with respect to lauric acid quality 0.5-2 amount doubly; Vibration or stirring reaction 8～96h at 35 DEG C～45 DEG C, hunting speed 150 ~ 185r/min; After reaction finishes, adopt filter method that immobilized PLA1, molecular sieve and unreacted sugar are removed; Gained filtrate is removed organic solvent by rotary evaporation, obtains product fructose lauric acid monoester after purification.
In described step (1), the DA-201 macroporous adsorbent resin of activated processing and phosphate buffered saline buffer mass ratio are 1:2; The consumption of phospholipase A1 is the 20wt% of macroporous adsorbent resin quality.
In described step (2), reaction medium organic solvent kind is any one in propyl carbinol, the trimethyl carbinol, tertiary amyl alcohol, chloroform, butanone, acetone or acetonitrile, or two or more mixtures; And described reaction medium organic solvent polarity scope
Log?P=3.0～7.0。
In described step (3), the consumption of immobilized PLA1 is lauric acid quality 20wt%.
In described step (3), consumption and the lauric acid mass ratio of 3 or 4 molecular sieves of activation are 1:1.
advantage of the present invention is as follows
(1) owing to adopting enzymatic synthesis method, there is eco-friendly feature compared with traditional chemical synthesis process; In reaction process, add molecular sieve to be conducive to, except the moisture producing in dereaction, esterification be carried out to positive dirction; Reaction solvent belongs to conventional organic solvent, and toxicity is little, and boiling point is low, is easy to separate with product.
(2) through HPLC-MS(LC-MS) detect prove catalysate be fructose lauric acid monoester; Detect by RP-HPLC-ELSD (RPLC-light scattering detector), the transformation efficiency of immobilized PLA1 catalysis fructose lauric acid monoester reaches 68.05%.
(3) compare with the fixed lipase catalyzed synthetic sugar ester of the employing of reporting, the price of immobilized PLA1 of the present invention only has the 10%-15% of commercialization immobilized lipase, therefore adopt phospholipase A1 to have cost advantage as catalyzer, industrial applications has good prospects.
Brief description of the drawings
Fig. 1 is the esterification equation of immobilized PLA1 catalysis fructose lauric acid monoester of the present invention.
Fig. 2 is the LC-MS collection of illustrative plates that reacts the fructose lauric acid monoester obtaining.
Embodiment
Below in conjunction withspecific examples and drawings the present invention is described in further detail, but implementation method of the present invention is flexible, is not limited only to the described concrete operations mode of this example.
Material therefor in below implementing: commercialization phospholipase A1 (Lecitase
?ultra, Novozymes Company of Denmark), to measure through tributyrin pH-stat method, enzyme activity unit is 3326 U/g; Macroporous adsorbent resin DA-201, Tianjin sea light Chemical Co., Ltd.; D-Fructose, Bai Ao bio tech ltd, Shanghai; Lauric acid, Tianjin great Mao chemical reagent factory; 3 or 4 molecular sieves, Tianjin good fortune occasion chemical reagent factory; Various reaction mediums are AR level.
By LC-MS, catalysate fructose lauric acid monoester is identified; Through high performance liquid chromatograph-light scattering detector to product in the content of fructose lauric acid monoester detect, to determine the transformation efficiency of fructose lauric acid monoester.
embodiment 1:
A. take the DA-201 macroporous adsorbent resin of the activated processing of 5 g, 10g phosphate buffered saline buffer, the free phospholipase A1 of 1g mixes, and then whip attachment 2h at 25 DEG C filters, and obtains macroporous adsorbent resin immobilized phospholipase, and its enzyme work is 1850 U/g.Vacuum-drying, for subsequent use;
B. 4 molecular sieves are placed in 105 DEG C of baking ovens and activate 10h, then be placed in moisture eliminator and be cooled to room temperature, for subsequent use; Afterwards, then 4 molecular sieves after activation are joined in the trimethyl carbinol of reaction medium according to the amount of every liter of solvent interpolation 80g, 72h is placed in sealing, removes by filter molecular sieve, obtains dehydrated organic solvent;
C. enzymatic esterification: getting 10g lauric acid and 1.8g fructose is to mix in the above-mentioned dehydrated organic solvent of 42ml at volume, then add the immobilized PLA1 of 2g to start reaction; After reaction 2h, add activation 4 molecular sieves of 10g with the water except generating in dereaction; Vibration or stirring reaction 48h at 45 DEG C, hunting speed 160r/min; After reaction finishes, adopt filter method that immobilized PLA1, molecular sieve and unreacted sugar are removed, get gained filtrate, identify that through LC-MS known synthetic product is fructose lauric acid monoester; Measure through high performance liquid chromatograph, the transformation efficiency of fructose lauric acid monoester is 68.05%.
embodiment 2:
A. take the DA-201 macroporous adsorbent resin of the activated processing of 5 g, 20g phosphate buffered saline buffer, the free phospholipase A1 of 1g mixes, and whip attachment 4h at 30 DEG C, then filters, and obtains macroporous adsorbent resin immobilized phospholipase, and its enzyme work is 1462 U/g.Vacuum-drying, for subsequent use;
B. 4 molecular sieves are placed in 100 DEG C of baking ovens and activate 8h, then be placed in moisture eliminator and be cooled to room temperature, for subsequent use; Afterwards, then 4 molecular sieves after activation are joined in the tertiary amyl alcohol of reaction medium according to the amount of every liter of solvent interpolation 60g, 24h is placed in sealing, removes by filter molecular sieve, obtains dehydrated organic solvent;
C. enzymatic esterification: getting 10g lauric acid and 2.3g fructose is to mix in the above-mentioned dehydrated organic solvent of 100ml at volume, then add the immobilized PLA1 of 2g to start reaction; After reaction 1h, add activation 4 molecular sieves of 10g with the water except generating in dereaction; Vibration or stirring reaction 48h at 40 DEG C, hunting speed 160r/min; After reaction finishes, adopt filter method that immobilized PLA1, molecular sieve and unreacted sugar are removed, get gained filtrate, identify that through LC-MS known synthetic product is fructose lauric acid monoester; Measure through high performance liquid chromatograph, the transformation efficiency of fructose lauric acid monoester is 64.68%.
embodiment 3:
A. take the DA-201 macroporous adsorbent resin of the activated processing of 5 g, 20g phosphate buffered saline buffer, the free phospholipase A1 of 1g mixes, and whip attachment 3h at 35 DEG C, then filters, and obtains macroporous adsorbent resin immobilized phospholipase, and its enzyme work is 1344 U/g.Vacuum-drying, for subsequent use;
B. 3 molecular sieves are placed in 95 DEG C of baking ovens and activate 5h, then be placed in moisture eliminator and be cooled to room temperature, for subsequent use; Afterwards, then 3 molecular sieves after activation are joined in the acetone of reaction medium according to the amount of every liter of solvent interpolation 50g, 72h is placed in sealing, removes by filter molecular sieve, obtains dehydrated organic solvent;
C. enzymatic esterification: getting 40.6g lauric acid and 36.5 fructose is to mix in the above-mentioned dehydrated organic solvent of 812ml at volume, then add the immobilized PLA1 of 8.12g to start reaction; After reaction 1h, add activation 3 molecular sieves of 40.6g with the water except generating in dereaction; Vibration or stirring reaction 72h at 40 DEG C, hunting speed 170r/min; After reaction finishes, adopt filter method that immobilized PLA1, molecular sieve and unreacted sugar are removed, get gained filtrate, identify that through LC-MS known synthetic product is fructose lauric acid monoester; Measure through high performance liquid chromatograph, the transformation efficiency of fructose lauric acid monoester is 61.35%.
Fig. 2 is the LC-MS collection of illustrative plates that reacts the fructose lauric acid monoester obtaining, and the molecular weight of fructose lauric acid monoester is 362, and 385.3 peaks that occur in spectrogram are [M+Na] mass signal, and the product therefore catalyzing and synthesizing is for being fructose lauric acid monoester.
Claims (2)
1. utilize immobilized PLA1 catalysis to prepare a method for fructose lauric acid monoester, it is characterized in that lauric acid and fructose, through enzymic catalytic reaction acrose lauric acid monoester, comprising the following steps:
(1) immobilization of phospholipase A1: the DA-201 macroporous adsorbent resin of getting the activated processing of 5 ~ 10g, the phosphate buffered saline buffer of 10 ~ 25g, the free phospholipase A1 of 0.5 ~ 2g mixes, whip attachment 2 ~ 6h at 25 ~ 35 DEG C, then filter, obtain macroporous adsorbent resin immobilized phospholipase, by the immobilized enzyme vacuum-drying making, for subsequent use;
(2) prepare dehydrated organic solvent: 3 or 4 molecular sieves are placed in 95～105 DEG C of baking ovens and activate 5～10h, then be placed in moisture eliminator and be cooled to room temperature, for subsequent use; Afterwards, then the amount that 3 or 4 molecular sieves after activation are added to 60～80g according to every liter of solvent joins stand-byly in the organic solvent of reaction medium, and 24～72h is placed in sealing, removes by filter molecular sieve, obtains dehydrated organic solvent; Described reaction medium organic solvent kind is any one in the trimethyl carbinol, tertiary amyl alcohol, acetone, or two or more mixtures;
(3) immobilized PLA1 catalyzes and synthesizes fructose lauric acid monoester: get 10 ~ 40.6g lauric acid and 1.8 ~ 36.5g fructose and be placed in above-mentioned dehydrated organic solvent and mix, wherein the mass volume ratio of lauric acid and organic solvent counts 5%～24% with gram every milliliter, then adds with respect to the immobilized PLA1 of lauric acid quality 8～25% and start reaction; After reaction 1～2h according to adding 3 after activation or 4 molecular sieves with the water except generating in dereaction with respect to lauric acid quality 0.5-2 amount doubly; Vibration or stirring reaction 8～96h at 35 DEG C～45 DEG C, hunting speed 150 ~ 185r/min; After reaction finishes, adopt filter method that immobilized PLA1, molecular sieve and unreacted sugar are removed; Gained filtrate is removed organic solvent by rotary evaporation, obtains product fructose lauric acid monoester after purification.
2. method according to claim 1, is characterized in that, in described step (1), phospholipase A1 is a kind of microbe-derived Phospholipid hydrolase of Novozymes Company, and commodity are called Lecitase Ultra; Its consumption is 5 ~ 20% of macroporous adsorbent resin quality.
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| CN104293764A (en) * | 2013-07-19 | 2015-01-21 | 丰益(上海)生物技术研发中心有限公司 | Phosphatidase A1 immobilization method |
| CN105462952B (en) * | 2015-12-31 | 2018-06-29 | 内蒙古金达威药业有限公司 | A kind of method of fixed fat enzyme B in fermented supernatant fluid from antarctic candida |
| CN106727025A (en) * | 2017-01-13 | 2017-05-31 | 华南理工大学 | A kind of camellia skin cream and preparation method thereof |
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| CN101845466A (en) * | 2010-05-10 | 2010-09-29 | 暨南大学 | Method for preparing diglyceride from phosphatidase A1 through catalytic esterification |
| CN102199634A (en) * | 2011-04-14 | 2011-09-28 | 华南理工大学 | Preparation method of diglyceride-enriched functional oil |
| CN102212580A (en) * | 2011-04-28 | 2011-10-12 | 浙江大学 | Method for catalytic synthesis of glucose laurate by utilizing yeast display lipase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101845466A (en) * | 2010-05-10 | 2010-09-29 | 暨南大学 | Method for preparing diglyceride from phosphatidase A1 through catalytic esterification |
| CN102199634A (en) * | 2011-04-14 | 2011-09-28 | 华南理工大学 | Preparation method of diglyceride-enriched functional oil |
| CN102212580A (en) * | 2011-04-28 | 2011-10-12 | 浙江大学 | Method for catalytic synthesis of glucose laurate by utilizing yeast display lipase |
Non-Patent Citations (4)
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| "无溶剂体系中酶促合成糖酯";胡俊 等;《分子催化》;20080630;第22卷(第3期);第260-264页 * |
| 付敏 等."吸附－涂层法固定化磷脂酶Lecitase Ultra的研究".《食品工业科技》.2011,第32卷(第10期),第277-280页. |
| 付敏 等."吸附-涂层法固定化磷脂酶Lecitase Ultra的研究".《食品工业科技》.2011,第32卷(第10期),第277-280页. * |
| 胡俊 等."无溶剂体系中酶促合成糖酯".《分子催化》.2008,第22卷(第3期),第260-264页. |
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