Method of catalytically synthesizing alpha-monolinolenin by using immobilization lipase
A technology of immobilized lipase and linolenic acid monoglyceride, which is applied to the direction of immobilization on/in organic carrier, fermentation, etc., can solve the problem of hindering the wide application of α-linolenic acid monoglyceride and the excessive residual organic solvent in medicine , Difficult purification of extracts and other issues, to achieve the effect of easy continuous production, conducive to large-scale industrial production, and low cost
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Embodiment 1
[0027] Example 1 Pore expansion modification and characterization of mesoporous material SBA-15
[0028] Dissolve 0.8 g of the triblock copolymer compound P123 (Pluronic P123) in deionized water, add it into hydrochloric acid after the P123 is completely dissolved, and stir to dissolve. The mixture was stirred at a constant temperature of 50° C. to 60° C. for 30 to 45 minutes, then 3 ml of ethanol was added, stirred, put into a reaction kettle, and placed in an oven at 100° C. for 48 hours. The product was filtered, washed and dried. Then burn at 500°C-550°C for 6-8 hours to completely remove the surfactant. The product name is: SBA-15 / 80.
[0029] The mesoporous material SBA-15 / 80 was dissolved in deionized water with a mass:volume ratio of 1g:30ml, heated to reflux for 3.5h, the mixture was cooled and centrifuged to remove water, and the white solid was dried in air for 6 days. Take the hydrated mesoporous material and add 130ml of toluene and 15ml of APTES successively, ...
Embodiment 2
[0036] The assembly of embodiment 2 immobilized lipase and determination of its optimum conditions
[0037] Get the mesoporous material NH that embodiment 1 makes 2 -SBA-15 / 80 and glutaraldehyde, mass:volume ratio 1g:30ml, reacted for 3h under stirring, then washed with phosphate buffer to remove unreacted glutaraldehyde. Add 30ml Candida lipase solution to the mesoporous material and react for 5 hours, finally wash with PBS and centrifuge (5000rpm, 13min) until no protein can be detected in the solution.
[0038] In this example, the Candida lipase was replaced by Pseudomonas aeruginosa lipase, the usage is the same, and the effect of the two is also the same.
[0039] For the optimum temperature, optimum pH, Michaelis constant (Km) and thermal stability of the immobilized lipase, see accompanying drawings 5 to 8 of the description.
[0040] Figure 5 shows the change of optimum temperature before and after lipase immobilization, that is, the optimum temperature of lipase ...
Embodiment 3
[0045] Example 3 Synthesis of α-linolenic acid monoglyceride in organic phase by immobilized enzyme
[0046] Take 1 g of the immobilized enzyme synthesized in Example 2, add the organic solvent n-hexane, and stir evenly. Add linolenic acid and glycerin (20ml each), and the initial concentrations are 25mmol / ml and 30.0mmol / ml respectively. The mixture was incubated in a constant temperature water bath shaker at 20-50°C for a certain period of time. Finally, the reaction mixture was filtered to remove the immobilized enzyme, and n-hexane was evaporated under reduced pressure. The residue is the product alpha-linolenic acid monoglyceride, a coixenolide. Qualitatively and quantitatively determined by high-performance liquid chromatography, the yield is 75-80%.
[0047] In this embodiment, the organic solvent n-hexane can be replaced by ethyl acetate.
[0048] In this example, there is a slight increase or decrease in the amount of each raw material (as within the scope of the ...
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