CN117625586A - Sn-1,3 specific immobilized lipase, and preparation method and application thereof - Google Patents
Sn-1,3 specific immobilized lipase, and preparation method and application thereof Download PDFInfo
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- CN117625586A CN117625586A CN202311573402.6A CN202311573402A CN117625586A CN 117625586 A CN117625586 A CN 117625586A CN 202311573402 A CN202311573402 A CN 202311573402A CN 117625586 A CN117625586 A CN 117625586A
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- 239000004367 Lipase Substances 0.000 title claims abstract description 136
- 102000004882 Lipase Human genes 0.000 title claims abstract description 136
- 108090001060 Lipase Proteins 0.000 title claims abstract description 136
- 235000019421 lipase Nutrition 0.000 title claims abstract description 136
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 36
- 239000007853 buffer solution Substances 0.000 claims abstract description 25
- 239000000243 solution Substances 0.000 claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 claims abstract description 11
- 238000005303 weighing Methods 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000006228 supernatant Substances 0.000 claims abstract description 5
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 5
- 238000003756 stirring Methods 0.000 claims abstract description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 4
- 238000001291 vacuum drying Methods 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 27
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 26
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 26
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- 239000005642 Oleic acid Substances 0.000 claims description 26
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 26
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 26
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 26
- 239000000047 product Substances 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 24
- PVNIQBQSYATKKL-UHFFFAOYSA-N tripalmitin Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCC PVNIQBQSYATKKL-UHFFFAOYSA-N 0.000 claims description 24
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 claims description 23
- 235000020778 linoleic acid Nutrition 0.000 claims description 23
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 claims description 23
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- 150000004665 fatty acids Chemical class 0.000 claims description 19
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- 238000007036 catalytic synthesis reaction Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 239000003513 alkali Substances 0.000 claims description 3
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- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 3
- 235000020256 human milk Nutrition 0.000 abstract description 13
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- 230000002255 enzymatic effect Effects 0.000 description 18
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 13
- 150000003626 triacylglycerols Chemical class 0.000 description 9
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 8
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- 235000021314 Palmitic acid Nutrition 0.000 description 4
- 239000004519 grease Substances 0.000 description 4
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- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 2
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- ZVRMGCSSSYZGSM-CCEZHUSRSA-N (E)-hexadec-2-enoic acid Chemical compound CCCCCCCCCCCCC\C=C\C(O)=O ZVRMGCSSSYZGSM-CCEZHUSRSA-N 0.000 description 1
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 description 1
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- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 102000019280 Pancreatic lipases Human genes 0.000 description 1
- 108050006759 Pancreatic lipases Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
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- HBOQXIRUPVQLKX-UHFFFAOYSA-N linoleic acid triglyceride Natural products CCCCCC=CCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COC(=O)CCCCCCCC=CCC=CCCCCC HBOQXIRUPVQLKX-UHFFFAOYSA-N 0.000 description 1
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- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
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- 239000013154 zeolitic imidazolate framework-8 Substances 0.000 description 1
- MFLKDEMTKSVIBK-UHFFFAOYSA-N zinc;2-methylimidazol-3-ide Chemical compound [Zn+2].CC1=NC=C[N-]1.CC1=NC=C[N-]1 MFLKDEMTKSVIBK-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses sn-1,3 specific immobilized lipase, a preparation method and application thereof, wherein the preparation method comprises the following steps: weighing free lipase, dispersing in buffer solution, centrifuging at 4deg.C, and collecting supernatant to obtain lipase solution; washing the immobilized carrier with buffer solution, carrying out ultrasonic treatment for 15-20 min, then weighing the immobilized carrier, dispersing in lipase liquid, continuously stirring for 2-10 h at 25-40 ℃ to enable the lipase to fully contact with the immobilized carrier, then rinsing the lipase remained on the surface with the buffer solution, and carrying out vacuum drying for 5h at 35-45 ℃ to obtain the sn-1,3 specific immobilized lipase. Compared with commercial lipase, the sn-1,3 specific immobilized enzyme prepared by the method has better activity of catalyzing and synthesizing OPL; the sn-1,3 specific immobilized lipase has great potential for catalyzing and industrialization production of OPL structural fat, and provides a new strategy for the high-efficiency preparation and application fields of human milk fat substitute fat.
Description
Technical Field
The invention relates to the technical field of lipase immobilization methods and grease synthesis, in particular to sn-1,3 specific immobilized lipase, a preparation method thereof and application thereof in catalytic synthesis of OPL.
Background
Breast milk is a golden nutrition for infants to grow. The breast milk contains 3-5% of fat, namely milk fat, accounting for 45% -55% of the energy of the breast milk, provides essential fatty acid for infants, promotes the absorption of fat-soluble vitamins, and is vital to the growth and development of the infants and the nutrition and health. Milk fat consists of 98% -99% triglycerides, 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride (OPL) and 1, 3-dioleoyl-2-palmitic acid triglyceride (OPO) are two major triglycerides naturally occurring in breast milk, both belonging to the UPU structure (U: unsaturated fatty acid; P: palmitic acid, PA). Although the composition and content of human milk fat is not constant, its content varies with the eating habits of different regions. Numerous studies have shown that OPL is the most abundant lipid in asian breast milk, next to OPO in the european united states family cream, especially in chinese breast milk (17.85% -33.02%). Therefore, OPL plays an important role in the growth of infants as well. However, due to the lack or deficiency of breast feeding, little OPL is currently contained in vegetable oils or bovine, ovine milk sources and infant formulas. In addition, most of the lipid structure of the milk is occupied in sn-1,3, and long-term intake affects the absorption and utilization of lipid and mineral substances, which is unfavorable for the growth and development of infants, and constipation is easy to cause and increase the demand of human milk to replace lipid OPL. Therefore, the synthesis of the human milk to replace the fat OPL has important significance, and has important application prospect in preparing and developing a human milk fat substitute with similar structure and distribution to the fatty acid of Chinese milk.
The enzymatic preparation of functional grease has recently attracted wide attention due to the advantages of mild reaction conditions, high catalytic efficiency, environmental protection and the like. The method can be divided into an esterification method, an acidolysis method, a transesterification method and the like. The enzymatic acidolysis method has mild reaction and simple process, provides a useful method for combining the required fatty acid into the Triacylglyceride (TAG) at a specific position, and is the most direct way to obtain the TAG rich in the special fatty acid. At present, an acidolysis method is mostly adopted for the enzymatic synthesis of OPL, namely palm stearin and Oleic Acid (OA), linoleic Acid (LA) or vegetable oil rich in OA and LA are adopted as raw materials to synthesize the OPL under the catalysis of immobilized lipase. Immobilized lipase is generally used for enzymatic preparation of OPL. The immobilized technology is utilized to control molecular factors such as space complementarity between a lipase topological structure and a substrate thereof, hydrogen bonds between residues around a catalytic site and tetrahedral intermediates, and the like, so that the selectivity and catalytic properties of the lipase can be effectively controlled, and the catalytic activity of the lipase can be further improved. However, the domestic upscale production of UPU structured lipids is limited by the technical barriers presented by commercial lipases. Meanwhile, the OPL production process is not mature, the high cost of imported commercial lipase and the lack of a grease substrate source rich in sn-2PA limit the industrialization of the domestic human milk to replace grease, so that the price of OPO and OPL products is high.
Disclosure of Invention
In view of the above, the invention aims at breaking through the technical barrier of lipase and saving cost, and provides a sn-1,3 specific immobilized lipase, a preparation method thereof and application thereof in catalytic synthesis of OPL (1-oleic acid-2-palmitic acid-3 linoleic acid triglyceride).
The invention aims to achieve the aim, and the aim is achieved by the following technical scheme:
according to a first aspect of the present invention, there is provided a process for the preparation of an sn-1,3 specific immobilized lipase, comprising the steps of:
weighing free lipase, dispersing in buffer solution, centrifuging at 4deg.C, and collecting supernatant to obtain lipase solution;
washing the immobilized carrier with buffer solution, carrying out ultrasonic treatment for 15-20 min, then weighing the immobilized carrier, dispersing in lipase liquid, continuously stirring for 2-10 h at 25-40 ℃ to enable the lipase to fully contact with the immobilized carrier, then rinsing the lipase remained on the surface with the buffer solution, and carrying out vacuum drying for 5h at 35-45 ℃ to obtain the sn-1,3 specific immobilized lipase.
Further, the free lipase is sn-1, 3-specific lipase.
Further, the buffer solution is one or more of phosphate buffer solution, tris-HCl buffer solution and citric acid-sodium citrate buffer solution, the concentration of the buffer solution is 0.02-0.1 mol/L, and the pH value is 5-9.
Further, the final concentration of the prepared lipase solution is 3-15 mg/mL.
Further, the method also comprises the step of preprocessing the immobilized carrier, and comprises the following steps: soaking the immobilized carrier in 95% ethanol for 2-4h;
after decompression and filtration, washing off ethanol by using deionized water until no obvious ethanol smell exists, and storing in a refrigerator to obtain the treated immobilized carrier;
wherein the immobilization carrier is one or a mixture of a plurality of weak-polarity macroporous resin, medium-polarity macroporous acrylic resin and polar macroporous resin.
According to a second aspect of the present invention, there is provided an sn-1, 3-specific immobilized lipase prepared by the above-described process for preparing an sn-1, 3-specific immobilized lipase.
According to a third aspect of the present invention there is provided the use of an immobilized lipase specific to sn-1,3 as described above in the catalytic synthesis of OPL comprising:
the sn-1,3 specific immobilized lipase is applied to the immobilized lipase catalytic acidolysis method for synthesizing the 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride under the condition of no solvent system.
Further, the method specifically comprises the following steps:
the tripalmitin triglyceride, oleic acid and linoleic acid are taken as substrate raw materials, the sn-1,3 specific immobilized lipase is added, and the reaction is carried out for 1 to 10 hours at the reaction temperature of 45 to 70 ℃;
and after the reaction is finished, separating and removing the sn-1,3 specific immobilized lipase, removing fatty acid in the product, and enriching to obtain a lipid product rich in an OPL structure.
Further, the molar ratio of the oleic acid to the linoleic acid is 1: (0.5-2), wherein the molar ratio of the tripalmitin triglyceride to the total amount of the oleic acid and the linoleic acid is 1: (6-16), wherein the dosage of the sn-1,3 specific immobilized lipase is 6-16% of the total mass of tripalmitin triglyceride and oleic acid and linoleic acid.
Further, the method for removing fatty acid is one of alkali neutralization, solvent extraction and molecular distillation, and the content of OPL in the lipid product is 35-50%.
Compared with the prior art, the invention has the following beneficial effects:
(1) The immobilized lipase with sn-1,3 specificity, which is prepared by immobilizing lipase ANL on weak-polarity-nonpolar macroporous resin, has excellent biocatalytic activity and sn-1,3 site specificity, is easy to recycle, overcomes the defects of poor lipase separability and poor stability in the free enzyme catalytic reaction process, and is applied to the enzymatic catalytic synthesis of OPL to improve the yield of OPL.
(2) According to the invention, the sn-1,3 specific immobilized lipase is subjected to enzymatic catalysis synthesis of 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride (OPL) in a solvent-free system, so that the OPL content can reach 47.93%, the sn-2 palmitic acid relative content is 71.69%, the synthesis effect of the immobilized lipase is better than that of commercial lipase Lipozyme435, the catalysis effect consistent with that of OPL synthesized by commercial enzyme NS40086 can be achieved, the production cost can be reduced by adopting ANL immobilized lipase, and the immobilized lipase has good industrialized application prospect.
Drawings
FIG. 1 shows the synthetic OPL process route of the ANL immobilized lipase enzymatic acidolysis method.
FIG. 2 shows the changes in the IR spectrum before and after the immobilization of ANL on lipase.
FIG. 3 shows the enrichment of PPP and LC-ELSD liquid phase analysis.
FIG. 4 is a comparison of ANL immobilized lipase to commercial enzyme catalyzed synthesis of OPL.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to specific embodiments, but they should not be construed as limiting the scope of the present invention.
According to a first aspect of the present invention, there is provided a process for the preparation of an sn-1,3 specific immobilized lipase, comprising the steps of:
weighing free lipase (ANL 02), dispersing in buffer solution, centrifuging at 4deg.C, and collecting supernatant to obtain lipase solution;
washing the immobilized carrier with buffer solution, carrying out ultrasonic treatment for 15-20 min, then weighing the immobilized carrier, dispersing in lipase liquid, continuously stirring for 2-10 h at 25-40 ℃ to enable the lipase to fully contact with the immobilized carrier, then rinsing the lipase remained on the surface with the buffer solution, and carrying out vacuum drying for 5h at 35-45 ℃ to obtain the sn-1,3 specific immobilized lipase.
Further, the free lipase is sn-1, 3-specific lipase derived from Aspergillus niger Aspergillus niger.
Further, the buffer solution is one or more of phosphate buffer solution, tris-HCl buffer solution and citric acid-sodium citrate buffer solution, the concentration of the buffer solution is 0.02-0.1 mol/L, and the pH is 5-9.
Further, the final concentration of the prepared lipase solution is 3-15 mg/mL.
Further, the method also comprises the step of preprocessing the immobilized carrier, and comprises the following steps: soaking the immobilized carrier in 95% ethanol for 2-4 hr;
after decompression and filtration, washing off ethanol by using deionized water until no obvious ethanol smell exists, and storing in a refrigerator to obtain the treated immobilized carrier;
wherein the immobilization carrier is one or a mixture of a plurality of weak-polarity macroporous resin, medium-polarity macroporous acrylic resin and polar macroporous resin.
According to a second aspect of the present invention, there is provided an sn-1, 3-specific immobilized lipase prepared by the above-described process for preparing an sn-1, 3-specific immobilized lipase.
According to a third aspect of the present invention there is provided the use of an immobilized lipase specific to sn-1,3 as described above in the catalytic synthesis of OPL comprising:
the sn-1,3 specific immobilized lipase is applied to the immobilized lipase catalytic acidolysis method for synthesizing the 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride (OPL) under the condition of no solvent system.
FIG. 1 shows the synthetic OPL process route of the ANL immobilized lipase enzymatic acidolysis method.
Further, as shown in fig. 1, the method specifically includes the following steps:
tripalmitic acid triglyceride (PPP), oleic acid and linoleic acid are used as substrate raw materials, sn-1,3 specific immobilized lipase is added, and the reaction is carried out for 1 to 10 hours at the reaction temperature of 45 to 70 ℃;
and after the reaction is finished, separating and removing sn-1,3 specific immobilized lipase, removing fatty acid in the product, and enriching to obtain a lipid product rich in OPL structure.
Wherein PPP is tripalmitin; 2-MP, 2-palmitoleic acid monoglyceride, O, oleic acid; l is linoleic acid; OPL 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride.
Further, separating palm stearin by a solvent method, and enriching to obtain tripalmitin triglyceride (PPP) which is used as a donor source of sn-2PA in a subsequent enzymatic synthesis process; the solvent comprises one or more of acetone, petroleum ether and ethyl acetate.
Further, the melting point of palm stearin is 58 ℃, and the content of tripalmitin triglyceride (PPP) in the obtained triglyceride is 75-95%
Further, the molar ratio of oleic acid to linoleic acid is 1: (0.5-2), wherein the mole ratio of the tripalmitin triglyceride to the total amount of oleic acid and linoleic acid is 1: (6-16), the dosage of the sn-1,3 specific immobilized lipase is 6-16% of the total mass of tripalmitin triglyceride and oleic acid and linoleic acid.
Further, the reaction temperature in S2 is 50 to 70℃and preferably 50 to 60 ℃.
Further, the reaction time in S2 is 2 to 8 hours, preferably 4 to 6 hours.
Preferably, the molar ratio of oleic acid to linoleic acid in S2 is 1: (0.5 to 1.5), more preferably 1: (0.5-1).
Preferably, the molar ratio of the PPP to the total amount of oleic acid and linoleic acid in S2 is 1: (8 to 16), more preferably 1: (10-16).
Preferably, the amount of the immobilized lipase in S2 is 6 to 14% by weight, more preferably 10 to 14% by weight of the total mass of tripalmitin (PPP) and oleic acid and linoleic acid.
Further, the method for removing fatty acid is one of reduced pressure filtration, alkali neutralization, solvent extraction and molecular distillation, and the content of OPL in the lipid product is 35-50%.
Further, when the method for removing fatty acid in the product is a molecular distillation method, the preheating temperature is 50-70 ℃, the evaporating temperature is 180-200 ℃, the condensing temperature is 30-50 ℃, the feeding rate is 1-4 mL/min, the film scraping rotating speed is 200-250 rpm, and the absolute pressure is 0.1MP.
Measurement of protein adsorption Rate: protein content was determined using BCA method. The calculation formula of the protein adsorption rate of the immobilized enzyme is as follows:
note that: x is X 1 -the amount of protein in the immobilized pre-adsorption lipase solution (mg);
X 2 -amount of residual protein (mg) in the immobilized enzyme filtrate and wash after immobilized adsorption.
Immobilized enzyme Activity detection 4mL of 4% polyvinyl alcohol-olive oil emulsion (v: v) and 5mL of 0.025mol/L phosphate buffer (pH 7.5) are added into a 50mL triangular flask, then a proper amount of lipase is added for reaction for 15min at 37 ℃, 15mL of 95% ethanol solution (v: v) is added for stopping reaction, 0.05mol/L NaOH solution is used for titration, phenolphthalein is used as an indicator, and a blank group is simultaneously made.
Analysis of lipid composition of enzymatic product: detection using a liquid phase-evaporative light scattering detector (LC-ELSD) was performed as follows: air is used as atomizing gas; the ELSD detector temperature was 50deg.C, carrier gas pressure 3.5bar, gain 6, and column DionexC30 (250 mm. Times.4.6 mm,5 μm); binary gradient elution was performed with acetonitrile and isopropanol, the elution procedure is as in Table 1, flow rate 1mL/min, column temperature 30 ℃. The oil sample was dissolved in n-hexane (10 mg/mL), and the sample amount was 20. Mu.L. The TAG is characterized by using a standard substance, and the relative content of OPL to the total TAG is calculated by adopting a peak area normalization method.
TABLE 1LC elution procedure
Enzymatic product fatty acid composition analysis: in n-hexane/diethyl ether/acetic acid solution (45:25:1, v: v) in order to develop the agent, the agent is, the reaction product was separated by thin layer silica gel chromatography to give about 50. Mu.L. The plates were then sprayed with 0.2%2, 7-dichlorofluorescein in methanol (w: v) and observed under a UV lamp. The bands corresponding to TAG were scraped off, methyl esterified, and the total fatty acid composition of the analyzed product was detected by GC-MS. GC conditions using a DB-WAX capillary column (60 m. Times.0.25 mm. Times.0.25 μm); the detector temperature is 250 ℃, the sample inlet temperature is 250 ℃, the carrier gas is helium, the flow rate is 1.0mL/min, the split ratio is 1:20, and the sample injection is 1 mu L. Heating program: first at 50 ℃ for 1min, then at 22.5 ℃/min to 175 ℃, finally at 4 ℃/min to 230 ℃ and for 20min. MS analysis conditions: the EI ionization source energy was 70eV and the ion source and transmission line temperatures were 230℃and 150℃respectively. The scanning range is 30-500m/z, and the solvent delay is 6min. Qualitative analysis of fatty acids was performed according to standard and spectral library (nist.08) and quantitative analysis used peak area normalization.
sn-2 fatty acid composition analysis: to the isolated TAG product was added 1mL of 1mol/LTris-HCl buffer (pH 8.0), 0.25mL of 0.05% sodium cholate (w: v), 0.1mL of 2.2% CaCl 2 (w: v), 20mg pancreatic lipase. The mixture was vigorously shaken at 40℃for 3min, then 1mL of 6mol/L HCl and 2mL of diethyl ether were added, and centrifuged. The ether was dried over anhydrous sodium sulfate and evaporated to 200 μl with nitrogen. The hydrolyzate was separated on a thin layer silica gel chromatography plate with the developing solvent n-hexane/diethyl ether/acetic acid (50:50:1, v: v). The sn-2 monoglyceride band was scraped off, methyl esterified and analyzed by the GC-MS method as described above. The relative sn-2PA content is calculated as follows:
note that: % sn-2PA, the mass fraction (%) of palmitic acid at position 2 to total palmitic acid;
sn-2 PA-palmitic acid content (%) at position 2;
PA-palmitic acid content (%).
Example 1
Preparation of ANL immobilized lipase:
0.5g of lipase ANL02 was dispersed in 20mmol/L phosphate buffer (pH 5.6), centrifuged at 4℃and 10000g for 10min, and the supernatant was collected to prepare a lipase solution of 0.50 mg/mL. 10g of macroporous acrylic resin rinsed by buffer salt solution is weighed and dispersed in lipase liquid, and continuously stirred for 6 hours at the speed of 150r/min, so that the lipase and the resin are fully contacted. And finally, rinsing the residual lipase on the surface of the immobilized lipase by using a buffer solution, and drying in vacuum at 45 ℃ for 5 hours to obtain the ANL immobilized lipase.
Characterization of ANL immobilized lipase:
FIG. 2 shows the changes in the IR spectrum before and after the immobilization of ANL on lipase. In FIG. 2, (A) is a blank resin and (B) is an immobilized enzyme resin.
Analysis of ANL Lipase immobilization Using Fourier transform Infrared SpectrometryThe functional groups of the resin were varied to analyze the degree of immobilization of the lipase on the resin, and the results are shown in fig. 2. Overall, the resin had similar characteristic absorption peaks before and after ANL lipase immobilization, indicating that the secondary structure of the resin was not destroyed after enzyme immobilization. Wherein the enzyme-immobilized resin is in the form of 3448, 2927, 2855.09, 1733 and 1152cm -1 The peak area increases at the wavelength. With increasing content of immobilized enzyme ANL on resin, absorption peak 3448cm -1 The strong absorption peak shown nearby is-OH and the stretching vibration generated by-NH-specific to the enzyme. 2927. 2855.09cm -1 Absorption peak is-CH 2 or-CH 3 Stretching vibration peaks indicate successful attachment of the ANL to the resin. Absorption peak 1733cm -1 is-C=O-stretching vibration peak, 1152cm -1 The above results confirm that ANL may successfully bind to the resin through amide and hydrogen bonding, as a C-N stretch vibration peak.
Enzyme Activity of ANL immobilized lipase:
the protein load and the enzyme activity result of the ANL immobilized lipase are shown in Table 2, the protein load of the ANL immobilized lipase is 13.97mg/g, the protein adsorption rate is as high as 92.37%, and the ANL immobilization efficiency is high. Lipase loading and enzyme adsorption efficiency depend on the specific surface area and hydrophobicity of the resin. The resin in this study has a higher ANL adsorption performance than the previous report that ANL has a 83.79% protein adsorption rate on chitosan-coated MP-64 resin. The specific activity of the ANL immobilized lipase reaches 79.52U/mg, which is higher than the enzyme activity of the immobilized enzyme prepared by the ANL on the microporous ZIF-8 material reported by the prior study. In conclusion, the ANL immobilized lipase has good enzyme activity and can be used for subsequent enzyme catalytic reaction.
TABLE 2 protein content and enzymatic Activity of ANL immobilized Lipase
Example 2
Preparation of PPP:
FIG. 3 shows the enrichment of PPP and LC-ELSD liquid phase analysis.
Fully dissolving palm stearin, mixing acetone with palm stearin at a ratio of 5mL/g, standing at 38 ℃ for 4 hours, rapidly filtering after PPP is separated out, and removing solvent from the obtained stearin by rotary evaporation to obtain a high-purity PPP product. The fraction product LC-ELSD was tested for PPP content and GC-MS for fatty acids, the results are shown in fig. 2 and table 3. In FIG. 3, (A) LC-ELSD chromatogram of palm stearin; (B) LC-ELSD chromatogram of lipid product after enrichment by fractionation; and (C) the PPP content changes before and after fractionation and enrichment.
TABLE 3 fatty acid and sn-2 fatty acid composition before and after PPP fractionation enrichment
Note that: FA: fatty acid relative content; sn-2: relative sn-2 fatty acid content.
Example 3
The ANL immobilized lipase catalyzes the synthesis of OPL:
6.055g PPP,16.945g oleic acid, 12.620g linoleic acid, and 4.280g of the ANL immobilized lipase were placed in a flask and reacted for 4 hours at a temperature of 50℃and a rotation speed of 150r/min in a water bath rotator. After the reaction is finished, filtering to remove enzyme, obtaining an enzymatic synthesis product, carrying out molecular distillation, enrichment and purification, wherein the evaporation temperature is 200 ℃, the condensation temperature is 30 ℃, the feeding rate is 3mL/min, the scraping rotation speed is 240rpm, the absolute pressure is 0.1MP, obtaining a heavy phase component, and detecting, the OPL content is 49.31%, and the sn-2PA relative content is 73.25%.
Example 4
The ANL immobilized lipase catalyzes the synthesis of OPL:
6.055g of PPP,16.942g of oleic acid, 16.986g of linoleic acid and 4.798g of the ANL immobilized lipase are taken in a flask and placed in a water bath rotator to react for 4 hours at a temperature of 50 ℃ and a rotating speed of 150 r/min. After the reaction is finished, filtering to remove enzyme, obtaining an enzymatic synthesis product, carrying out molecular distillation, enrichment and purification, wherein the evaporation temperature is 200 ℃, the condensation temperature is 30 ℃, the feeding rate is 2mL/min, the scraping rotation speed is 200rpm, the absolute pressure is 0.1MP, obtaining a heavy phase component, and detecting, the OPL content is 43.36%, and the sn-2PA relative content is 68.73%.
Example 5
The ANL immobilized lipase catalyzes the synthesis of OPL:
6.055g of PPP,14.523g of oleic acid, 10.909g of linoleic acid and 3.149g of the ANL immobilized lipase are taken in a flask, placed in a water bath rotator, and reacted for 4 hours at a temperature of 50 ℃ and a rotating speed of 150 r/min. After the reaction is finished, filtering to remove enzyme, obtaining an enzymatic synthesis product, carrying out molecular distillation, enrichment and purification, wherein the evaporation temperature is 180 ℃, the condensation temperature is 30 ℃, the feeding rate is 2mL/min, the scraping rotation speed is 200rpm, the absolute pressure is 0.1MP, obtaining a heavy phase component, and detecting, the OPL content is 39.16%, and the sn-2PA relative content is 64.76%.
Example 6
The ANL immobilized lipase catalyzes the synthesis of OPL:
6.055g of PPP,14.800g of oleic acid, 14.726g of linoleic acid and 4.981g of the ANL immobilized lipase are taken in a flask and placed in a water bath rotator to react for 4 hours at a temperature of 50 ℃ and a rotating speed of 150 r/min. After the reaction is finished, filtering to remove enzyme, obtaining an enzymatic synthesis product, carrying out molecular distillation, enrichment and purification, wherein the evaporation temperature is 180 ℃, the condensation temperature is 30 ℃, the feeding rate is 3mL/min, the scraping rotation speed is 240rpm, the absolute pressure is 0.1MP, obtaining a heavy phase component, and detecting, the OPL content is 44.32%, and the sn-2PA relative content is 67.65%.
Example 7
The ANL immobilized lipase catalyzes the synthesis of OPL:
6.055g PPP,16.945g oleic acid, 12.620g linoleic acid, and 4.280g of the ANL immobilized lipase were placed in a flask and reacted for 6 hours at a temperature of 50℃and a rotation speed of 150r/min in a water bath rotator. After the reaction is finished, filtering to remove enzyme, obtaining an enzymatic synthesis product, carrying out molecular distillation, enrichment and purification, wherein the evaporation temperature is 200 ℃, the condensation temperature is 30 ℃, the feeding rate is 3mL/min, the scraping rotation speed is 240rpm, the absolute pressure is 0.1MP, obtaining a heavy phase component, and detecting, the OPL content is 49.92%, and the sn-2PA relative content is 72.12%.
Example 8
The ANL immobilized lipase catalyzes the synthesis of OPL:
6.055g PPP,16.945g oleic acid, 12.620g linoleic acid, and 4.280g of the ANL immobilized lipase were placed in a flask and reacted for 4 hours at a temperature of 60℃and a rotational speed of 150r/min in a water bath rotator. After the reaction is finished, filtering to remove enzyme, obtaining an enzymatic synthesis product, carrying out molecular distillation, enrichment and purification, wherein the evaporation temperature is 200 ℃, the condensation temperature is 30 ℃, the feeding rate is 3mL/min, the scraping rotation speed is 240rpm, the absolute pressure is 0.1MP, obtaining a heavy phase component, and detecting, the OPL content is 47.13%, and the sn-2PA relative content is 70.55%.
Example 9
Comparison of ANL immobilized lipase with commercial enzyme catalyzed synthesis of OPL:
FIG. 4 is a comparison of ANL immobilized lipase to commercial enzyme catalyzed synthesis of OPL.
The current commercialized sn-1,3 specific immobilized lipase NS40086 and Lipozyme435 widely used for lipid modification have better catalytic activity under similar reaction conditions. Thus, the catalytic activity of the ANL immobilized lipase for the catalytic synthesis of OPL was compared with the commercial enzymes NS40086 and Lipozyme435 under optimized reaction conditions and the results are shown in fig. 4. In FIG. 4, (A) a comparison of the relative amounts of synthetic OPL; comparison of the relative content of sn-2 PA. The alphabetical differences represent significant differences in data (P < 0.05). The OPL content and the sn-2PA relative content in the 3 lipase-catalyzed reactions increased with the increase of the reaction time and tended to stabilize after 4 hours. Wherein, the ANL immobilized lipase and NS40086 have no significant difference in synthetic OPL content and sn-2PA relative content (P > 0.05), and the sn-2PA relative content is significantly higher than that obtained by Lipozyme435 reaction (P < 0.05). And Lipozyme435 had even decreased OPL content at 4h, indicating that there may be an acyl shift during the reaction. These results demonstrate that the catalytic activity and sn-1,3 specificity of the ANL immobilized lipase are significantly higher than Lipozyme435 and can reach comparable capacity to synthesize OPL by NS40086 catalysis.
As can be seen from the above examples, the preparation method of sn-1,3 specific immobilized lipase (ANL immobilized lipase) provided by the invention is applied to catalytic synthesis of OPL, and the ANL immobilized lipase has better activity of catalytic synthesis of OPL. Tripalmitic acid triglyceride, oleic acid and linoleic acid are used as substrate raw materials, ANL immobilized lipase accounting for 6-14% of the total mass of the substrate is added, and the mixture is reacted for 1-10 hours at 50-70 ℃. After enrichment and purification, the OPL content in the product can reach 35-50%. The invention has simple process, and compared with commercial lipase, the prepared ANL immobilized lipase has better activity of catalyzing and synthesizing OPL; the ANL immobilized lipase has great potential for catalyzing and producing OPL structural fat in an industrialized mode, and provides a new strategy for the high-efficiency preparation and application fields of human milk fat substitute fat.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the sn-1,3 specific immobilized lipase is characterized by comprising the following steps of:
weighing free lipase, dispersing in buffer solution, centrifuging at 4deg.C, and collecting supernatant to obtain lipase solution;
washing the immobilized carrier with buffer solution, carrying out ultrasonic treatment for 15-20 min, then weighing the immobilized carrier, dispersing in lipase liquid, continuously stirring for 2-10 h at 25-40 ℃ to enable the lipase to fully contact with the immobilized carrier, then rinsing the lipase remained on the surface with the buffer solution, and carrying out vacuum drying for 5h at 35-45 ℃ to obtain the sn-1,3 specific immobilized lipase.
2. The method of manufacturing according to claim 1, characterized in that: the free lipase is sn-1, 3-site specific lipase.
3. The method of manufacturing according to claim 1, characterized in that: the buffer solution is one or more of phosphate buffer solution, tris-HCl buffer solution and citric acid-sodium citrate buffer solution, the concentration of the buffer solution is 0.02-0.1 mol/L, and the pH value is 5-9.
4. The method of manufacturing according to claim 1, characterized in that: the final concentration of the prepared lipase solution is 3-15 mg/mL.
5. The method of manufacturing according to claim 1, characterized in that: further comprising pretreating the immobilization support, comprising: soaking the immobilized carrier in 95% ethanol for 2-4h;
after decompression and filtration, washing off ethanol by using deionized water until no obvious ethanol smell exists, and storing in a refrigerator to obtain the treated immobilized carrier;
wherein the immobilization carrier is one or a mixture of a plurality of weak-polarity macroporous resin, medium-polarity macroporous acrylic resin and polar macroporous resin.
6. An sn-1, 3-specific immobilized lipase prepared by the process for preparing an sn-1, 3-specific immobilized lipase of any one of claims 1-5.
7. Use of an sn-1, 3-specific immobilized lipase of claim 6 in the catalytic synthesis of OPL, comprising:
the sn-1,3 specific immobilized lipase is applied to the immobilized lipase catalytic acidolysis method for synthesizing the 1-oleic acid-2-palmitic acid-3-linoleic acid triglyceride under the condition of no solvent system.
8. The use according to claim 7, characterized in that it comprises in particular the following steps:
the tripalmitin triglyceride, oleic acid and linoleic acid are taken as substrate raw materials, the sn-1,3 specific immobilized lipase is added, and the reaction is carried out for 1 to 10 hours at the reaction temperature of 45 to 70 ℃;
and after the reaction is finished, separating and removing the sn-1,3 specific immobilized lipase, removing fatty acid in the product, and enriching to obtain a lipid product rich in an OPL structure.
9. The use according to claim 8, wherein the molar ratio of oleic acid to linoleic acid is 1: (0.5-2), wherein the molar ratio of the tripalmitin triglyceride to the total amount of the oleic acid and the linoleic acid is 1: (6-16), wherein the dosage of the sn-1,3 specific immobilized lipase is 6-16% of the total mass of tripalmitin triglyceride and oleic acid and linoleic acid.
10. The use according to claim 8, wherein the method for removing fatty acids is one of alkali neutralization, solvent extraction and molecular distillation, and the OPL content of the lipid product is 35-50%.
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