Synthetic method of vitamin A palmitate

A technology of palmitate and synthesis method, applied in the direction of recombinant DNA technology, immobilized on/in organic carrier, using carrier to introduce foreign genetic material, etc., can solve separation difficulties, conversion rate can not meet the requirements of industrial production, etc. problem, to achieve the effect of less dosage, short catalytic reaction time and high immobilization effect

Inactive Publication Date: 2016-03-30
ZHEJIANG UNIV OF TECH
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0005] "Ruan Hui, Xu Juan" disclosed a method for synthesizing vitamin A palmitate catalyzed by yeast lipase in "Yeast-displayed lipase-catalyzed synthesis of vitamin A palmitate" (Chinese patent application 201110110428.8, 2011) , in a 10mL n-hexane reaction system, 0.328g vitamin A acetate and 1.282g palmitic acid (substrate molar ratio 1:5) were used as substrates, 0.5g yeast display lipase was added, under anaerobic conditions, 250rpm / min, 45 After 12 hours of reaction at ℃, the conversion rate reaches 90%, and the substrate concentration is only 32.8g / L. The conversion rate of 90% cannot meet the requirements of industrial production, and subsequent separation will be very difficult.

Method used

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  • Synthetic method of vitamin A palmitate
  • Synthetic method of vitamin A palmitate
  • Synthetic method of vitamin A palmitate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: macroporous adsorption resin carries out pretreatment

[0036] Rinse the macroporous resin AB-8 (purchased from Tianjin Nankai Hecheng Technology Co., Ltd.) with distilled water, soak in distilled water for 24 hours, remove organic impurities and insoluble matter; soak in 95% ethanol aqueous solution for 24 hours, then rinse with distilled water Resin until there is no alcohol smell, the resin after pretreatment is preserved in distilled water for subsequent use.

Embodiment 2

[0037] The preparation of embodiment 2 enzyme powder

[0038] (1) Construction and cultivation of engineering bacteria

[0039] The lipase exists in Pichia pastoris cells, and the lipase gene is derived from a strain of Candida antarctica (Candida antarctica) ZJB09193, which is now preserved in the Wuhan General Culture Collection Center with a preservation number of CCTCCM2010263, which has been published in the literature "non- Screening of aqueous lipase and its application in the synthesis of vitamin C palmitate, Zhang Qiuhua, 2012, published in "Master's Thesis".

[0040] The expression vector pPICZαA-CALB (the nucleotide sequence of the Candida antarctica ZJB09193 lipase B gene is shown in SEQ ID NO: 1) containing the gene encoding Candida antarctica ZJB09193 lipase B was linearized by SacI , electrotransformed into Pichia pastoris (Pichiapastoris) X33 (purchased from Invitrogen), spread on a YPD plate containing Zeocin (bleomycin) at a final concentration of 100 μg / μL,...

Embodiment 3

[0066] Embodiment 3: the preparation of immobilized lipase

[0067] Prepare Na at pH 6.0 with distilled water 2 HPO 4 - citric acid buffer (Na 2 HPO 4 and the molar concentration of citric acid are respectively 0.05M and 0.025M), take by weighing the lipase enzyme powder that 16g embodiment 2 method prepares and dissolve to 1LNa 2 HPO 4 - Prepare the enzyme solution in citrate buffer. Accurately weigh 0.6g (wet weight) of the macroporous adsorption resin AB-8 pretreated in Example 1 and mix it with the enzyme solution at a mass volume ratio of 1:20, and shake and adsorb in a water bath shaker at 35°C and 180rpm. After 2 hours of immobilization (i.e., adsorption saturation), the supernatant was removed by suction filtration, and the adsorbed macroporous resin was washed twice with distilled water (to remove unadsorbed free enzymes on the surface of the carrier), and dried at room temperature (22°C). 0.3 g of the immobilized enzyme product was obtained, and stored in a ref...

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Abstract

The invention relates to a synthetic method of vitamin A palmitate. The vitamin A palmitate is synthesized through a chemical enzyme technology, and the method mainly comprises the following steps: 1, hydrolyzing vitamin A acetate by using an alkali solution with an organic solvent as a cosolvent to generate vitamin A alcohol; and 2, extracting the vitamin A alcohol by using an organic solvent, washing the obtained extract liquid with water to remove the cosolvent reacting with palmitic acid, and reacting the vitamin A alcohol with the palmitic acid in a non-aqueous system under the catalysis of immobilized lipase to generate the vitamin A palmitate. The synthetic method provided by the invention adopts the vitamin A alcohol to substitute vitamin A acetate as a substrate in the biological catalysis process, and the vitamin A palmitate to be synthesized through esterification, so the substrate concentration is improved, the conversion rate is high, the reaction time is shortened, the molar ratio of the substrate is reduced, the service life of the immobilized lipase is prolonged, the cost is reduced, industrial production requirements are met, and the gap in the enzyme production of the vitamin A palmitate in the current market is filled.

Description

(1) Technical field [0001] The invention relates to a new process for synthesizing vitamin A palmitate. The vitamin A palmitate is synthesized by a chemical and enzymatic method, that is, an organic solvent is used as a cosolvent, and vitamin A acetate is hydrolyzed with lye to generate vitamin A alcohol; extracted with an organic solvent Vitamin A alcohol, washing the extract with water to remove the co-solvent that can react with palmitic acid, immobilized lipase catalyzing the reaction of vitamin A alcohol and palmitic acid in the non-aqueous phase system to synthesize vitamin A palmitate. (2) Background technology [0002] The chemical name of vitamin A is retinol, which is the earliest discovered vitamin, a natural fat-soluble vitamin, and one of the essential nutrients for the human body. In 1913, four scientists including Davis in the United States discovered that cod liver oil could cure dry eye disease and A yellow viscous liquid is purified from cod liver oil. In ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P23/00C12N15/81C12N9/20C12N11/02
Inventor 郑裕国柳志强胡忠策刘珊珊
Owner ZHEJIANG UNIV OF TECH
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