WO2018161631A1 - 一种偏甘油酯脂肪酶及富含pufa的油脂的酶法脱酸方法 - Google Patents

一种偏甘油酯脂肪酶及富含pufa的油脂的酶法脱酸方法 Download PDF

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WO2018161631A1
WO2018161631A1 PCT/CN2017/111105 CN2017111105W WO2018161631A1 WO 2018161631 A1 WO2018161631 A1 WO 2018161631A1 CN 2017111105 W CN2017111105 W CN 2017111105W WO 2018161631 A1 WO2018161631 A1 WO 2018161631A1
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oil
pufa
reaction
partial glyceride
lipase
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PCT/CN2017/111105
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French (fr)
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王永华
李道明
王卫飞
刘楠
杨博
蓝东明
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华南理工大学
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Priority to US16/492,141 priority Critical patent/US11208672B2/en
Publication of WO2018161631A1 publication Critical patent/WO2018161631A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6454—Glycerides by esterification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals

Definitions

  • the invention relates to a method for enzymatic deacidification of a partial glyceride lipase and a PUFA-rich fat.
  • Natural animal and vegetable oils that have not been refined contain a certain amount of free fatty acids. After the free fatty acids need to be removed, the oils can be used for storage, processing, and consumption. Therefore, the deacidification of oils is processed in oils and fats. An indispensable process.
  • the method of deacidification of oil and fat includes chemical deacidification (alkali refining and deacidification), physical deacidification, enzymatic deacidification, etc., wherein physical deacidification needs to be carried out at a relatively high temperature (generally above 200 ° C), PUFA-rich grease Oxidation and transversion are easy to occur at high temperature, so physical deacidification is not suitable for deacidification of PUFA-rich fats; chemical deacidification method is carried out by using the principle of alkali-neutralizing free fatty acids in oils, which is formed by alkali-neutralization of fatty acids.
  • the soap will entrain a large amount of neutral oil, which makes the yield of deacidified oil and fat low, especially suitable for deacidification of oil with higher free fatty acid content, and a large amount of industrial wastewater will be generated in the subsequent processing of chemical deacidification. It will cause great pollution to the environment and has become less and less important.
  • Enzymatic deacidification has the characteristics of mild reaction conditions, high catalytic efficiency, strong specificity and environmental friendliness, and the cost can be greatly reduced by the recycling of enzymes, and the application potential is huge.
  • CN105802730A discloses a method for deacidification of rice bran oil by using glycerol as an acyl acceptor, utilizing the high efficiency and specificity of the enzyme reaction, performing esterification and deacidification under vacuum condition, and reacting the acid of rice bran oil after 6 hours of reaction. The value decreased from 26.8 mgKOH/g to 1.96 mgKOH/g, and the fatty acid removal rate reached 92.69%.
  • CN105419937A discloses an enzymatic deacidification method for wheat germ oil. The acid value of wheat germ oil is reduced from 21.72 mgKOH/g to 2.98 mgKOH/g by using immobilized lipase Novozym 435 at 70 ° C for 5 h.
  • CN105349259A discloses an enzymatic deacidification process of high acid value vegetable oil.
  • the immobilized lipase catalyzes the amidation reaction of free fatty acid with monoethanolamine under a vacuum of 0.075-0.1 MPa, and has high reaction selectivity and high catalytic efficiency; There are problems such as an increase in by-products caused by the enzymatic esterification reaction and consumption of neutral oil.
  • CN101824364A discloses an enzymatic refining and deacidification method for high acid price fish oil, which uses anhydrous ethanol as an acyl acceptor and Novozym 435 as a catalyst.
  • the acid value of tuna oil is lowered by 36.3 mg KOH / g.
  • the fatty acid removal rate is up to To 87.05%.
  • enzymatic deacidification is particularly suitable for deacidification of PUFA-rich fats due to its mild conditions, high catalytic efficiency, strong specificity and green environmental reaction.
  • the existing enzymatic deacidification technology generally uses a lipase (triglyceride lipase) as a catalyst, since lipase can also catalyze the reaction of triglyceride with a hydroxyl donor, which causes a large amount of side reactions to occur, reducing The yield of deacidified oils, especially triglycerides in the product.
  • Partial glyceride lipase is a special lipase that has stringent substrate specificity for partial glycerides (monoglycerides and diglycerides) and does not act on triglycerides.
  • partial glycerides can be used to synthesize diglycerides (Journal of Molecular Catalysis B: Enzymatic, 2012, 77: 87-91), and can also be used to remove partial glycerides from glyceride mixtures to prepare high purity triglycerides. Product (Molecules, 2013, 18:9704-9716).
  • partial glyceride lipase can catalyze the reaction of free fatty acids in PUFA-rich fats with short-chain monohydric alcohols to form fatty acid esters, which can be deacidified with low acid value after separation and purification. grease.
  • the existing partial glyceride lipases Lipase SMG1 and Lipase G "Amano" 50
  • the present invention provides a method for deacidification of partial glyceride lipase.
  • immobilized Lipase SMG1 Phe278Asn is used as a catalyst to catalyze the reaction of free fatty acids with short-chain monohydric alcohols to form fatty acid esters in a solvent system, and the reaction products are separated to obtain deacidified fats and oils.
  • a partial glyceride lipase is a mutant Lipase SMG1 Phe278Asn obtained by mutating Phe at position 278 of Lipase SMG1 to Asn, and the amino acid sequence thereof is SEQ ID NO.
  • An enzymatic deacidification method for PUFA-rich fats and oils comprising the steps of:
  • the immobilized enzyme is recovered, and the organic solvent and monohydric alcohol are recovered to obtain a PUFA-rich fat or oil after deacidification.
  • the mass-to-volume ratio of the oil and fat to the organic solvent in the step 1) is 1: (0.4 to 5) g/ml; and the molar ratio of the free fatty acid to the monool in the oil and fat is 1: (1.1 to 4).
  • Step 1) The partial glyceride lipase is added in an amount of 50 to 200 U/g of the total mass of the reaction substrate.
  • the temperature of the esterification reaction is 25 ° C or lower.
  • the immobilized carrier is an epoxy resin
  • the buffer is a phosphate buffer solution
  • the ratio of the partial glyceride lipase to the epoxy resin is 10 to 50 mg/g resin. Mix and fix.
  • the monohydric alcohol described in the step 1) is methanol, ethanol, propanol or a mixture of two or more thereof.
  • the PUFA-rich fat or oil is marine fish oil, algae oil or a mixture rich in one or more of polyunsaturated fatty acids having 20 carbon atoms or more.
  • the PUFA-rich fat and oil described in the step 1) has an acid value of 20 to 80 mgKOH/g.
  • the nonpolar organic solvent in the step 2) is one of n-hexane, n-heptane and isooctane or a mixture of two or more.
  • the monohydric alcohol is carried out in a stepwise manner, which is 1/3 of the total amount added at the beginning of the esterification reaction, and 1/3 is added after the reaction is carried out for 6 hours, and the remaining 1/3 is added after the reaction is carried out for 12 hours.
  • the recovery method of the immobilized enzyme described in the step 2) is filtration recovery, and the organic solvent and the monohydric alcohol are recovered by vacuum distillation or molecular distillation (thin film evaporation).
  • the immobilized partial glyceride lipase Lipase SMG1 Phe278Asn used in the present invention has a significantly higher selectivity for long-chain polyunsaturated fatty acids EPA and DHA than the wild-type Lipase SMG1, and the catalytic activity is also significantly improved. At the same time, it still maintains stringent substrate specificity for partial glycerides.
  • the use of the above partial glyceride esterase to catalyze the reaction of free fatty acids in PUFA-rich fats and oils with short-chain monohydric alcohols converts almost all of the free fatty acids into fatty acid esters.
  • the esterification reaction can be carried out at a lower temperature, avoiding the oxidation of PUFA in the grease.
  • the inventors' research shows that when the immobilized Lipase SMG1 Phe278Asn catalyzes the reaction of free fatty acids in PUFA-rich fats and oils with short-chain monohydric alcohols, the viscosity of the reaction system is large and the reaction speed is high. The phenomenon of slow rate. Further studies have shown that when a certain amount of non-polar organic solvent is added to the reaction system, the mass transfer of the reaction and the slow reaction rate at a later stage can be significantly improved.
  • the present invention improves the reaction of the free fatty acid in the PUFA-rich fat and oil with the short-chain monohydric alcohol by using the immobilized Lipase SMG1 Phe278Asn, and adding a certain amount of the non-polar organic solvent to improve The removal effect of fatty acids.
  • the present invention has the following advantages:
  • immobilized Lipase SMG1 Phe278Asn is used as a catalyst to avoid side reactions of triglyceride, improve the deacidification efficiency of PUFA, and reduce the risk of PUFA oxidation.
  • the present invention uses n-hexane or isooctane or a mixture of the two as a solvent, and adopts a stepwise addition of a monohydric alcohol to improve the deacidification efficiency, and the removal rate of free fatty acids can reach 99% or more, and the fixation is increased.
  • the number of repeated use of lipase is a monohydric alcohol.
  • the immobilized carrier was ECR8285 epoxy resin, and the partial glyceride lipase was mixed with ECR8285 epoxy resin in a ratio of 20 mg/g resin.
  • the liquid was added in an amount equal to the volume of the enzyme solution, and after mixing, it was immobilized at room temperature for 7 hours in a water bath shaker at a rotation speed of 200 rpm.
  • the immobilized enzyme was recovered by filtration through a Buchner funnel and dried under vacuum at 30 ° C for 6 h.
  • the immobilized enzyme finally obtained had a protein adsorption amount of 52.7 mg/g, a protein adsorption rate of 82.11%, and an esterase activity of 328 U/g (n-propanol lauric acid method).
  • the total molar ratio of free fatty acid to absolute ethanol in the carp oil was 1:2.
  • the squid oil after deacidification was analyzed. Acid value, the acid value of salmon oil decreased from the initial 26.78mgKOH / g to 0.10mgKOH / g, the removal rate of free fatty acid can reach 99.63%, the peroxide value of the recovered deacidified oil is 3.2meq / Kg (with raw materials Basically consistent), the activity of the immobilized enzyme was not significantly reduced after repeated use of 6 batches.
  • the total molar ratio of free fatty acid to absolute ethanol in the carp oil was 2:3.
  • the squid oil after deacidification was analyzed. Acid value, the acid value of salmon oil decreased from the initial 26.78mgKOH / g to 0.19mgKOH / g, the removal rate of free fatty acid can reach 99.29%, the peroxide value of the recovered deacidified oil is 3.1meq / Kg (with raw materials Basically consistent), the activity of the immobilized enzyme was not significantly reduced after repeated use of 6 batches.
  • the total molar ratio of free fatty acid to absolute ethanol in the tuna oil was 2:5.
  • the deacidified tuna oil was analyzed. Acid value, the acid value of tuna oil decreased from the initial 36.16mgKOH/g to 0.10mgKOH/g, the free fatty acid removal rate can reach 99.72%, and the recovered deacidified oil has a peroxide value of 2.3meq/Kg (with raw materials). Basically consistent), the activity of the immobilized enzyme was not significantly reduced after repeated use of 6 batches.
  • the acid value of the salmon oil decreased from the initial 26.78mgKOH/g to 0.09mgKOH/g, the free fatty acid removal rate reached 99.66%, and the recovered deacidified fat had a peroxide value of 3.0meq/Kg (with The raw materials were basically the same), and the activity of the immobilized enzyme was not significantly reduced after repeated use of 6 batches.
  • the total molar ratio of free fatty acid to absolute ethanol in the tuna oil was 2:5.
  • the deacidified tuna oil was analyzed. The acid value of the tuna oil is reduced from the initial 36.16mgKOH/g to 0.11mgKOH/g, the free fatty acid removal rate can reach 99.70%, and the recovered deacidified fat has a peroxide value of 2.2meq/Kg (and The raw materials were basically the same), and the activity of the immobilized enzyme was not significantly reduced after repeated use of 6 batches.
  • the total molar ratio of free fatty acid to absolute ethanol in the carp oil was 1:2.
  • the squid oil after deacidification was analyzed.
  • the acid value of the salmon oil decreased from the initial 26.78 mg KOH/g to 3.73 mg KOH/g, and the free fatty acid removal rate reached 86.07%.
  • the total molar ratio of free fatty acid to absolute ethanol in the carp oil was 1:2.
  • the deacidification was analyzed.
  • the acid value of the salmon oil the acid value of the salmon oil decreased from the initial 26.78mgKOH / g to 3.41mgKOH / g, the removal rate of free fatty acids can reach 87.27%.
  • the acid value of tuna oil is 36.16mgKOH / g
  • 0.28g of anhydrous methanol the molar ratio of free fatty acid to anhydrous methanol is 3:2
  • 80mL of isooctane in 500mL with stopper In a flask, mix and preheat to 20 ° C
  • 80 U / g anhydrous methanol
  • the gas bath shaker stirring speed is 200 rpm
  • 0.28 g of anhydrous methanol was continuously added (the total molar ratio of free fatty acid to anhydrous methanol in the tuna oil was 1:2 after three additions), and after 30 hours of esterification, the tuna oil after deacidification was analyzed.
  • the acid value of the tuna oil decreased from the initial 36.16 mgKOH/
  • the tuna oil was free.
  • the total molar ratio of fatty acid to absolute ethanol is 1:2).
  • the acid value of the tuna oil after deacidification is analyzed.
  • the acid value of the tuna oil is reduced from the initial 36.16 mgKOH/g to 5.39 mgKOH/g.
  • the removal rate of free fatty acids can reach 85.09%.
  • the stirring speed of the gas bath shaker was 200 rpm, the reaction was 6 h and After 12h, 0.29g of absolute ethanol was continuously added (the total molar ratio of free fatty acid to absolute ethanol in the carp oil was 1:2 after three additions), and the acid value of the squid oil after deacidification was analyzed after esterification for 30 hours.
  • the acid value of the salmon oil decreased from the initial 26.78 mgKOH/g to 1.14 mgKOH/g, and the free fatty acid removal rate reached 95.74%.
  • the immobilized enzyme was used repeatedly for 3 batches, its viability became 48% of the initial viability.

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Abstract

一种偏甘油酯脂肪酶及富含PUFA的油脂的酶法脱酸方法，包括以下步骤：1)将富含PUFA的油脂与非极性有机溶剂、短链一元醇混合，添加固定化偏甘油酯脂肪酶进行酯化反应；所述偏甘油酯脂肪酶是将Lipase SMG1第278位的Phe突变为Asn后得到的突变体；2)回收固定化酶，回收有机溶剂和一元醇，即得到脱酸后的富含PUFA的油脂。所述偏甘油酯脂肪酶不催化甘油三酯发生醇解等副反应，脱酸效率高，反应温度低，避免了PUFA的高温氧化，且固定化酶可多次回收重复利用，在工业上具有良好的应用前景。

Description

一种偏甘油酯脂肪酶及富含PUFA的油脂的酶法脱酸方法 技术领域
本发明涉及一种偏甘油酯脂肪酶及富含PUFA的油脂的酶法脱酸方法。
背景技术
未经精炼加工的天然动、植物油脂中均含有一定量的游离脂肪酸，需要对游离脂肪酸进行脱除后，油脂才能达到存储、加工、食用等利用标准，因此油脂的脱酸处理是油脂加工中不可或缺的加工过程。
油脂脱酸方法有化学脱酸(碱炼脱酸)、物理脱酸、酶法脱酸等，其中物理脱酸需要在较高的温度下进行(一般在200℃以上)，富含PUFA的油脂在高温容易发生氧化与反式化，因此物理脱酸不适用于富含PUFA的油脂脱酸；化学脱酸方法是利用碱中和油脂中游离脂肪酸的原理进行的，由于碱中和脂肪酸后形成的皂会夹带大量的中性油脂，使得脱酸油脂的得率较低，特别不适用于较高游离脂肪酸含量的油脂脱酸，而且化学脱酸的后续加工过程中会产生大量的工业废水，会对环境造成极大的污染，目前已经越来越不被人们重视。酶法脱酸具有反应条件温和、催化效率高、专一性强及环境友好等特点，且通过酶的回收利用可大大降低成本，应用潜力巨大。CN105802730A公布了一种米糠油酶法脱酸的方法，用甘油作为酰基受体，利用酶反应的高效性和专一性，在真空状态下进行酯化脱酸，反应6h后，米糠油的酸值自26.8mgKOH/g降至1.96mgKOH/g，脂肪酸脱除率达到92.69％。CN105419937A公布了一种小麦胚芽油的酶法脱酸方法，利用固定化脂肪酶Novozym 435，在70℃下反应5h后，小麦胚芽油的酸值由21.72mgKOH/g降至2.98mgKOH/g，脂肪酸脱除率达到86.28％。CN105349259A公开了一种高酸价植物油的酶法脱酸工艺，在0.075～0.1MPa真空度下，固定化脂肪酶催化游离脂肪酸与单乙醇胺进行酰胺化反应，反应选择性高，催化效率高；避免了酶法酯化反应造成的副产物增多、中性油消耗等问题。CN101824364A公布了一种高酸价鱼油的酶法精炼脱酸方法，利用无水乙醇作为酰基受体，Novozym 435作为催化剂，在50℃下反应1h后，金枪鱼油的酸价由36.3mgKOH/g降至4.7mgKOH/g，脂肪酸脱除率达 到87.05％。总之，酶法脱酸由于其作用条件温和、催化效率高、专一性强且反应过程绿色环保等优势特别适用于对富含PUFA的油脂进行脱酸处理。
但是现有的酶法脱酸技术一般采用脂肪酶(甘油三酯脂肪酶)为催化剂，由于脂肪酶也可以催化甘油三酯与羟基供体进行反应，这就使得会有大量副反应发生，降低了脱酸油脂特别是产物中甘油三酯的得率。偏甘油酯脂肪酶是一种特殊的脂肪酶，其对偏甘油酯(甘油单酯和甘油二酯)具有严格的底物专一性，而不能作用于甘油三酯。研究表明偏甘油酯可以用于合成甘油二酯(Journal of Molecular Catalysis B:Enzymatic,2012,77:87-91)，也可以用于去除甘油酯混合物中的偏甘油酯制备高纯度的甘油三酯产品(Molecules,2013,18:9704-9716)。
进一步的研究表明，利用偏甘油酯脂肪酶的底物特异性，可以催化富含PUFA的油脂中的游离脂肪酸与短链一元醇反应生成脂肪酸酯，分离纯化后可以得到低酸价的脱酸油脂。但是，现有的偏甘油酯脂肪酶(Lipase SMG1和Lipase G“Amano”50)其对长链多不饱和脂肪酸特别是EPA和DHA的催化效率较低，应用于富含PUFA的油脂脱酸后，脱酸效果差，产物中脂肪酸含量高。
发明内容
为克服现有的富含长链PUFA油脂酶法脱酸工艺中催化效率较低、反应时间长、反应不稳定等问题，本发明提供一种偏甘油酯脂肪酶脱酸方法。
研究表明，偏甘油酯脂肪酶的催化功能特点也是由其分子结构特别是一级结构决定的；进一步的研究发现将Lipase SMG1 278位的Phe突变为Asn后，得到的Lipase SMG1 Phe278Asn，在用于富含长链PUFA的油脂脱酸时，具有良好的脱酸效果，并且不催化原料中的甘油三酯发生副反应，进而形成了本发明。
在本发明中，采用固定化Lipase SMG1 Phe278Asn作为催化剂，在溶剂体系下催化游离脂肪酸与短链一元醇反应生成脂肪酸酯，反应产物经过分离后得到脱酸后的油脂。
本发明的技术方案如下：
一种偏甘油酯脂肪酶，是将Lipase SMG1第278位的Phe突变为Asn后得到的突变体Lipase SMG1 Phe278Asn，其氨基酸序列为SEQ ID NO.1。
一种富含PUFA的油脂的酶法脱酸方法，包括以下步骤：
1)将富含PUFA的油脂与非极性有机溶剂、短链一元醇混合，添加固定化 偏甘油酯脂肪酶进行酯化反应；所述偏甘油酯脂肪酶的氨基酸序列为SEQ ID NO.1；
2)回收固定化酶，回收有机溶剂和一元醇，即得到脱酸后的富含PUFA的油脂。
步骤1)所述的油脂与有机溶剂的质量体积比为1：(0.4～5)g/ml；所述油脂中游离脂肪酸与一元醇的摩尔比1:(1.1～4)。
步骤1)所述偏甘油酯脂肪酶的添加量为50～200U/g反应底物总质量。
所述酯化反应的温度为25℃以下。
步骤1)中所述固定化偏甘油酯脂肪酶的制备：固定化载体为环氧树脂，缓冲液为磷酸盐缓冲液，偏甘油酯脂肪酶与环氧树脂按10～50mg/g树脂的比例混合进行固定化。
所述固定化载体为ECR8285环氧树脂，缓冲液的浓度为1.5moL/L，pH＝6.0；固定化时间为7h。
步骤1)中所述的一元醇为甲醇、乙醇、丙醇或者其中两种以上的混合。
所述的富含PUFA的油脂为海洋鱼油、藻油或富含具有20个碳原子以上多不饱和脂肪酸中的一种或两种以上的混合物。
步骤1)中所述的富含PUFA的油脂的酸值为20～80mgKOH/g。
步骤2)所述的非极性有机溶剂为正己烷、正庚烷、异辛烷中的一种或两种以上的混合物。
所述一元醇采用分步添加的方式进行，分别为酯化反应开始时添加总量的1/3，反应进行6h后添加1/3，反应进行12h后添加余下的1/3。
步骤2)中所述的固定化酶的回收方式为过滤回收，有机溶剂和一元醇采用减压蒸馏或分子蒸馏(薄膜蒸发)的方式回收。本发明所采用的固定化偏甘油酯脂肪酶Lipase SMG1 Phe278Asn对长链多不饱和脂肪酸EPA和DHA的选择性较野生型Lipase SMG1有显著提高，催化活力也有明显提高。与此同时，其仍保持对偏甘油酯严格的底物特异性。应用上述偏甘油酯酶催化富含PUFA的油脂中的游离脂肪酸与短链一元醇反应，几乎可以将全部的游离脂肪酸转化为脂肪酸酯。酯化反应可以在较低的温度下进行，避免了油脂中PUFA的氧化。
发明人研究表明，利用固定化Lipase SMG1 Phe278Asn催化富含PUFA的油脂中的游离脂肪酸与短链一元醇反应时，存在反应体系粘度大、反应后期速 率慢的现象。进一步研究表明，当反应体系中加入一定量的非极性有机溶剂时，能显著改善反应的传质及后期反应速率慢的问题。因此，综合考虑反应速率和脂肪酸的去除效果，本发明在利用固定化Lipase SMG1 Phe278Asn催化富含PUFA的油脂中的游离脂肪酸与短链一元醇反应时，加入一定量的非极性有机溶剂来改善脂肪酸的去除效果。
与现有技术相比，本发明具有如下优点：
(1)本发明中，采取固定化Lipase SMG1 Phe278Asn为催化剂，避免了甘油三酯发生副反应，提高了PUFA的脱酸效率，降低了PUFA氧化的风险。
(2)本发明采用正己烷或异辛烷或两者的混合物为溶剂，并采用分步添加一元醇的方法，提高了脱酸效率，游离脂肪酸的去除率可以达到99％以上，增加了固定化脂肪酶的重复使用次数。
具体实施方式
以下通过实施例更详细地介绍本发明的实施。在所述实施例中，所有百分比均以质量计。
固定化Lipase SMG1 Phe278Asn的制备：固定化载体为ECR8285环氧树脂，偏甘油酯脂肪酶与ECR8285环氧树脂按20mg/g树脂的比例混合，缓冲液为1.5moL/L pH＝6.0的磷酸盐缓冲液且添加量与酶液体积相等，混匀后于转速为200rpm的水浴摇床中室温下固定化7h。固定化酶通过布氏漏斗过滤回收，于30℃下真空干燥6h。最终得到的固定化酶的蛋白吸附量为52.7mg/g,蛋白吸附率为82.11％，酯化酶活为328U/g(正丙醇月桂酸法)。
实施例1
取20g脱色后的鱿鱼油，鱿鱼油的酸值为26.78mgKOH/g，加入0.29g无水乙醇(游离脂肪酸和无水乙醇的摩尔比为3:2)和80mL正己烷置于500mL具塞三角瓶中，混匀并预热至25℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1 Phe278Asn，在25℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.29g无水乙醇(三次添加后，鱿鱼油中游离脂肪酸与无水乙醇的总摩尔比为1:2)，酯化反应30h后，分析脱酸后鱿鱼油的酸值，鱿鱼油的酸值由初始的26.78mgKOH/g降至0.10mgKOH/g，游离脂肪酸的去除率可以达到99.63％，回收得到的脱酸油脂的过氧化值为3.2meq/Kg(与原料基本一致)，固定化酶重复使用6个批次后，其活力没有明显降低。
实施例2
取20g脱色后的鱿鱼油，鱿鱼油的酸值为26.78mgKOH/g，加入0.22g无水乙醇(游离脂肪酸和无水乙醇的摩尔比为2:1)和60mL正己烷置于500mL具塞三角瓶中，混匀并预热至20℃后，加入100U/g(占反应底物总质量)固定化Lipase SMG1 Phe278Asn，在20℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.22g无水乙醇(三次添加后，鱿鱼油中游离脂肪酸与无水乙醇的总摩尔比为2:3)，酯化反应30h后，分析脱酸后鱿鱼油的酸值，鱿鱼油的酸值由初始的26.78mgKOH/g降至0.19mgKOH/g，游离脂肪酸的去除率可以达到99.29％，回收得到的脱酸油脂的过氧化值为3.1meq/Kg(与原料基本一致)，固定化酶重复使用6个批次后，其活力没有明显降低。
实施例3
取20g脱色后的金枪鱼油，金枪鱼油的酸值为36.16mgKOH/g，加入0.5g无水乙醇(游离脂肪酸和无水乙醇的摩尔比为6:5)和80mL正己烷置于500mL具塞三角瓶中，混匀并预热至25℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1 Phe278Asn，在25℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.5g无水乙醇(三次添加后，金枪鱼油中游离脂肪酸与无水乙醇的总摩尔比为2:5)，酯化反应30h后，分析脱酸后金枪鱼油的酸值，金枪鱼油的酸值由初始的36.16mgKOH/g降至0.10mgKOH/g，游离脂肪酸的去除率可以达到99.72％，回收得到的脱酸油脂的过氧化值为2.3meq/Kg(与原料基本一致)，固定化酶重复使用6个批次后，其活力没有明显降低。
实施例4
取20g脱色后的金枪鱼油，金枪鱼油的酸值为36.16mgKOH/g，加入0.28g无水甲醇(游离脂肪酸和无水甲醇的摩尔比为3:2)和60mL正己烷置于500mL具塞三角瓶中，混匀并预热至20℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1 Phe278Asn，在20℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.28g无水甲醇(三次添加后，金枪鱼油中游离脂肪酸与无水甲醇的总摩尔比为1:2)，酯化反应30h后，分析脱酸后金枪鱼油的酸值，金枪鱼油的酸值由初始的36.16mgKOH/g降至0.14mgKOH/g，游离脂肪酸的去除率可以达到99.61％，回收得到的脱酸油脂 的过氧化值为2.2meq/Kg，固定化酶重复使用6个批次后，其活力没有明显降低。
实施例5
取20g脱色后的鱿鱼油，鱿鱼油的酸值为26.78mgKOH/g，加入0.29g无水乙醇(游离脂肪酸和无水乙醇的摩尔比为3:2)和80mL异辛烷置于500mL具塞三角瓶中，混匀并预热至25℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1 Phe278Asn，在25℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.29g无水乙醇(三次添加后，鱿鱼油中游离脂肪酸与无水乙醇的总摩尔比为1:2)，酯化反应30h后，分析脱酸后鱿鱼油的酸值，鱿鱼油的酸值由初始的26.78mgKOH/g降至0.09mgKOH/g，游离脂肪酸的去除率可以达到99.66％，回收得到的脱酸油脂的过氧化值为3.0meq/Kg(与原料基本一致)，固定化酶重复使用6个批次后，其活力没有明显降低。
实施例6
取20g脱色后的金枪鱼油，金枪鱼油的酸值为36.16mgKOH/g，加入0.5g无水乙醇(游离脂肪酸和无水乙醇的摩尔比为6:5)和60mL异辛烷置于500mL具塞三角瓶中，混匀并预热至20℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1 Phe278Asn，在20℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.5g无水乙醇(三次添加后，金枪鱼油中游离脂肪酸与无水乙醇的总摩尔比为2:5)，酯化反应30h后，分析脱酸后金枪鱼油的酸值，金枪鱼油的酸值由初始的36.16mgKOH/g降至0.11mgKOH/g，游离脂肪酸的去除率可以达到99.70％，回收得到的脱酸油脂的过氧化值为2.2meq/Kg(与原料基本一致)，固定化酶重复使用6个批次后，其活力没有明显降低。
对比实施例1
取20g脱色后的鱿鱼油，鱿鱼油的酸值为26.78mgKOH/g，加入0.29g无水乙醇(游离脂肪酸和无水乙醇的摩尔比为3:2)和80mL正己烷置于500mL具塞三角瓶中，混匀并预热至25℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1，在25℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.29g无水乙醇(三次添加后，鱿鱼油中游离脂肪酸与无水乙醇的总摩尔比为1:2)，酯化反应30h后，分析脱酸后鱿鱼油的 酸值，鱿鱼油的酸值由初始的26.78mgKOH/g降至3.73mgKOH/g，游离脂肪酸的去除率可以达到86.07％。
对比实施例2
取20g脱色后的鱿鱼油，鱿鱼油的酸值为26.78mgKOH/g，加入0.29g无水乙醇(游离脂肪酸和无水乙醇的摩尔比为3:2)和80mL正己烷置于500mL具塞三角瓶中，混匀并预热至25℃后，加入80U/g(占反应底物总质量)固定化Lipase G“Amano”50，在25℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.29g无水乙醇(三次添加后，鱿鱼油中游离脂肪酸与无水乙醇的总摩尔比为1:2)，酯化反应30h后，分析脱酸后鱿鱼油的酸值，鱿鱼油的酸值由初始的26.78mgKOH/g降至3.41mgKOH/g，游离脂肪酸的去除率可以达到87.27％。
对比实施例3
取20g脱色后的金枪鱼油，金枪鱼油的酸值为36.16mgKOH/g，加入0.28g无水甲醇(游离脂肪酸和无水甲醇的摩尔比为3:2)和80mL异辛烷置于500mL具塞三角瓶中，混匀并预热至20℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1，在20℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.28g无水甲醇(三次添加后，金枪鱼油中游离脂肪酸与无水甲醇的总摩尔比为1:2)，酯化反应30h后，分析脱酸后金枪鱼油的酸值，金枪鱼油的酸值由初始的36.16mgKOH/g降至4.89mgKOH/g，游离脂肪酸的去除率可以达到86.48％。
对比实施例4
取20g脱色后的金枪鱼油，金枪鱼油的酸值为36.16mgKOH/g，加入0.5g无水乙醇(游离脂肪酸和无水乙醇的摩尔比为6:5)和80mL正己烷置于500mL具塞三角瓶中，混匀并预热至25℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1，在25℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.5g无水乙醇(三次添加后，金枪鱼油中游离脂肪酸与无水乙醇的总摩尔比为2:5)，酯化反应30h后，分析脱酸后金枪鱼油的酸值，金枪鱼油的酸值由初始的36.16mgKOH/g降至5.69mgKOH/g，游离脂肪酸的去除率可以达到84.26％。
对比实施例5
取20g脱色后的金枪鱼油，金枪鱼油的酸值为36.16mgKOH/g，加入0.4g 无水乙醇(游离脂肪酸和无水乙醇的摩尔比为3:2)和80mL异辛烷置于500mL具塞三角瓶中，混匀并预热至25℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1，在25℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.4g无水乙醇(三次添加后，金枪鱼油中游离脂肪酸与无水乙醇的总摩尔比为1:2)，酯化反应30h后，分析脱酸后金枪鱼油的酸值，金枪鱼油的酸值由初始的36.16mgKOH/g降至5.39mgKOH/g，游离脂肪酸的去除率可以达到85.09％。
对比实施例6
取20g脱色后的鱿鱼油，鱿鱼油的酸值为26.78mgKOH/g，一次性加入0.87g无水乙醇(游离脂肪酸和无水乙醇的总摩尔比为1:2)和80mL正己烷置于500mL具塞三角瓶中，混匀并预热至25℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1 Phe278Asn，在25℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，酯化反应30h后，分析脱酸后鱿鱼油的酸值，鱿鱼油的酸值由初始的26.78mgKOH/g降至1.70mgKOH/g，游离脂肪酸的去除率可以达到93.65％。
对比实施例7
取20g脱色后的鱿鱼油，鱿鱼油的酸值为26.78mgKOH/g，加入0.29g无水乙醇(游离脂肪酸和无水乙醇的摩尔比为3:2)后置于100mL具塞三角瓶中，混匀并预热至25℃后，加入80U/g(占反应底物总质量)固定化Lipase SMG1 Phe278Asn，在25℃下开始进行酯化反应，气浴摇床搅拌速度为200rpm，反应6h和12h后分别继续加入0.29g无水乙醇(三次添加后，鱿鱼油中游离脂肪酸与无水乙醇的总摩尔比为1:2)，酯化反应30h后，分析脱酸后鱿鱼油的酸值，鱿鱼油的酸值由初始的26.78mgKOH/g降至1.14mgKOH/g，游离脂肪酸的去除率可以达到95.74％。固定化酶重复使用3个批次后，其活力变为最初活力的48％。

Claims (10)

  1. 一种偏甘油酯脂肪酶，其特征在于，其氨基酸序列如SEQ ID NO.1所示。
  2. 一种富含PUFA的油脂的酶法脱酸方法，其特征在于，包括以下步骤：
    1)将富含PUFA的油脂与非极性有机溶剂、短链一元醇混合，添加固定化偏甘油酯脂肪酶进行酯化反应；所述偏甘油酯脂肪酶的氨基酸序列如SEQ ID NO.1所示；
    2)回收固定化酶，回收有机溶剂和一元醇，即得到脱酸后的富含PUFA的油脂。
  3. 根据权利要求2所述的方法，其特征在于，步骤1)所述的油脂与有机溶剂的质量体积比为1：(0.4～5)g/ml；所述油脂中游离脂肪酸与一元醇的摩尔比1:(1.1～4)。
  4. 根据权利要求2所述的方法，其特征在于，步骤1)所述偏甘油酯脂肪酶的添加量为50～200U/g反应底物总质量，所述酯化反应的温度为25℃以下。
  5. 根据权利要求2或3或4所述的方法，其特征在于，步骤1)中所述固定化偏甘油酯脂肪酶的制备：固定化载体为环氧树脂，缓冲液为磷酸盐缓冲液，偏甘油酯脂肪酶与环氧树脂按10～50mg/g树脂的比例混合进行固定化。
  6. 根据权利要求2或3或4所述的制备方法，其特征在于，步骤1)中所述的一元醇为甲醇、乙醇、丙醇或者其中两种以上的混合；所述的富含PUFA的油脂为海洋鱼油、藻油或富含具有20个碳原子以上多不饱和脂肪酸中的一种或两种以上的混合物。
  7. 根据权利要求6所述的方法，其特征在于，步骤1)中所述的富含PUFA的油脂的酸值为20～80mgKOH/g。
  8. 根据权利要求2或3或4所述的方法，其特征在于，步骤2)所述的非极性有机溶剂为正己烷、正庚烷、异辛烷中的一种或两种以上的混合物。
  9. 根据权利要求2或3或4所述的方法，其特征在于，所述一元醇采用分步添加的方式进行，分别为酯化反应开始时添加总量的1/3，反应进行6h后添加1/3，反应进行12h后添加余下的1/3。
  10. 根据权利要求2或3或4所述的制备方法，其特征在于，步骤2)中所述的固定化酶的回收方式为过滤回收，有机溶剂和一元醇采用减压蒸馏或分子蒸馏的方式回收。
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