Method for catalytic synthesis of vitamin E succinate by utilizing yeast display lipase

A succinate and lipase technology, applied in the field of bioengineering, can solve the problems of high production cost, limited commercial application, difficult separation, etc., achieve high esterification reaction efficiency and yield, improve operational stability, and reduce production. cost effect

Inactive Publication Date: 2013-08-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are chemical methods and biological enzymatic methods to achieve this transformation. Due to the disadvantages of chemical methods such as harsh conversion conditions (strong acid, alkali catalysis, high temperature and pressure), non-specificity, complex products, many by-products, difficult separation, and poor safety, biological Enzymatic methods are becoming more and more popular
Lipase is currently the most widely used enzyme in the synthesis of vitamin esters, but its commercial application is greatly limited by the high production cost and complicated and time-consuming immobilization process.

Method used

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  • Method for catalytic synthesis of vitamin E succinate by utilizing yeast display lipase
  • Method for catalytic synthesis of vitamin E succinate by utilizing yeast display lipase

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Example 1 Preparation of Yeast Displaying Lipase

[0018] Synthesize the lipase gene (Genbank number: AF229435) of Rhizopus oryzae and the cell wall α-lectin gene of Pichia pastoris GS115 (Genbank number: M28164) by artificial synthesis, and add Connect the peptide sequence GSSGGSGGSGGSGGSGS(linker), and get the nucleotide sequence pro-ROL-linker-α-agglutinin after connection, and add EcoR I and Not I restriction sites at both ends of the sequence, where pro-ROL is the lipase gene , α-agglutinin is the cell wall α lectin gene.

[0019] Using the above artificially synthesized sequence as a template, PCR amplification was performed using the following primer pair,

[0020] Upstream primer: 5'-AAGGAAAAAAAGAATTCGTTCCAGTTTCTGG-3';

[0021] Downstream primer: 5'-TTTTCCTTTTGCGGCCGCTAATGAAACG-3'

[0022] The PCR reaction system is: 1 μl of template DNA, 0.5 μl of high-fidelity DNA polymerase, 0.4 μl of dNTP (50 mM), 0.5 μl of upstream and downstream primers, 5 μl of 10×PCR ...

Embodiment 2

[0026] Example 2 Yeast shows lipase-catalyzed synthesis of vitamin E succinate

example 1

[0027] Example 1 Take 0.472g of vitamin E and 0.5g of succinic anhydride, add them into a ground-neck Erlenmeyer flask containing 10mL of n-hexane, mix and preheat for 10min, then add 0.5g of yeast lipase, and fill with N 2 Seal it, put it in a 85-1 type magnetic stirrer and stir to start the reaction, the speed is 250 rpm, the reaction temperature is kept at 45°C, stop stirring after 12 hours of reaction, centrifuge to remove the lipase displayed by yeast, take the supernatant and rotate Evaporate to remove the organic solvent, wash with water for 3 times, add n-hexane to crystallize at 4°C to obtain vitamin E succinate product, dry and pulverize.

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Abstract

The invention discloses a method for catalytic synthesis of vitamin E succinat by utilizing yeast display lipase, and the method comprises the following steps: dissolving vitamin E and succinic anhydride in an organic solvent, adding the yeast display lipase, reacting at 50-60 DEG C for 10-14 hours under an anaerobic condition, and separating and purifying, thus obtaining the vitamin E succinate.The preparation method of the yeast display lipase comprises the following steps: transforming the recombinant plasmid subjected to linear treatment into a Pichia Pastoris yeast GS115, inoculating the obtained transformants into a BMMY (buffered methanol-complex medium), inducing and culturing for 72-144 hours, carrying out centrifugal collection on thallus, washing, and carrying out organism printing and frozen drying on the washed thallus, thus obtaining the yeast display lipase. Through displaying lipase outside cells, and utilizing the lipase to conduct catalytic synthesis of the vitamin E vitamin E succinate, the conversion efficiency can be improved, the reaction time is shortened, and the production cost is lowered.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for synthesizing vitamin E succinate catalyzed by yeast display lipase. Background technique [0002] Natural vitamin E (tocopherol) is an important fat-soluble vitamin and natural antioxidant with various physiological functions in the human body. It has significant antioxidant properties, eliminates free radicals in the body, eliminates sexual disorders, promotes blood circulation, prevents premature aging, and It is an extremely important medicine and health care raw material for the functions of cancer occurrence and improving the body's immunity. However, natural vitamin E is easily oxidized, and most vitamin E products on the market are stable derivatives after esterification. Vitamin E acetate and ester Vitamin E succinic acid monoester are the main vitamin E ester derivatives. Among them, vitamin E succinate (vitamin E succinate) not only has the functio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/06C12N15/63C12N1/19C12N9/20C12R1/84
Inventor 阮晖徐娟地里热巴周陈伟王睿之林吉恒何国庆
Owner ZHEJIANG UNIV
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