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Method for catalytically synthesizing L-ascorbyl oleate with yeast display lipase

A technology of acid oleate and lipase, which is applied in the field of yeast lipase-catalyzed synthesis of L-ascorbyl oleate, can solve the problems of limited commercial application, harsh conversion conditions, complex products, etc., and achieve the goal of improving operational stability 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

[0003] At present, there are chemical methods and biological enzymatic methods to achieve this "grafting". 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, etc. , the biological enzymatic method is 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 catalytically synthesizing L-ascorbyl oleate with yeast display lipase
  • Method for catalytically synthesizing L-ascorbyl oleate with yeast display lipase

Examples

Experimental program
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Embodiment 1

[0019] Example 1 Preparation of Yeast to Display Lipase

[0020] 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.

[0021] Using the above artificially synthesized sequence as a template, PCR amplification was performed using the following primer pair,

[0022] Upstream primer: 5'-AAGGAAAAAAAGAATTCGTTCCAGTTTCTGG-3';

[0023] Downstream primer: 5'-TTTTCCTTTTGCGGCCGCTAATGAAACG-3'

[0024] 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

[0028] Example 2 Yeast Display Lipase Catalyzed Synthesis of L-Ascorbyl Oleate

example 1

[0029] Example 1 Take 0.176g of L-ascorbic acid and 1.27ml of oleic acid, put them into a ground-neck Erlenmeyer flask containing 10mL of tetrahydrofuran, mix and preheat for 10min, then add 0.5g of the above-mentioned yeast display lipase, fill with N 2 Sealed, placed in a 85-1 type magnetic stirrer and stirred to start the reaction, the rotating speed was 250 rpm, and the reaction temperature was kept at 45°C. After 6 hours of reaction, 0.5g molecular sieve (pore size less than 2nm) was added, and the reaction was continued for 12 hours. After that, the stirring was stopped. Centrifuge and precipitate to remove yeast display lipase and molecular sieves, take the supernatant and carry out rotary evaporation to remove tetrahydrofuran, wash with water for 3 times, add n-hexane and crystallize at 4°C to obtain L-ascorbyl oleate product, which can be dried and pulverized.

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Abstract

The invention discloses a method for catalytically synthesizing L-ascorbyl oleate with yeast display lipase. The method comprises the following steps: dissolving L-ascorbic acid and oleic acid in an organic solvent; adding yeast display lipase to react at 50-60 DEG C for 4-8 hours under anaerobic conditions; and carrying out separation and purification to prepare L-ascorbyl oleate, wherein the yeast display lipase is prepared by transforming recombinant plasmid which is subjected to linearization into Pichia pastoris GS115, inoculating the obtained transformant in a BMMY culture medium, carrying out inducing culture for 72-144 hours, centrifuging to collect a thallus, and carrying out washing, bio-imprinting and freeze-drying on the thallus. By displaying the lipase outside the cell and utilizing the lipase preparation to catalytically synthesize the L-ascorbyl oleate, the invention improves the transformation efficiency, shortens the reaction time and lowers the production cost.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for synthesizing L-ascorbyl oleate catalyzed by yeast display lipase. Background technique [0002] Synthetic antioxidants such as BHA, BHT, PG, and TBHQ are widely used in the food industry. However, due to the safety hazards such as carcinogenicity and toxicity after ingestion in the body, their consumption is increasingly questioned, and natural antioxidants are therefore favored. L-ascorbic acid (vitamin C) is one of the most used natural antioxidants at present. With the continuous maturity of the two-step fermentation technology to produce vitamin C, the production of vitamin C in my country ranks first in the world, and the production cost is relatively low. However, vitamin C is a water-soluble natural antioxidant that can only function in water-phase systems, but food systems are often oil-phase or heterogeneous, which greatly limits the scope of use of v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/04C12N15/63C12N1/19C12N9/20C12R1/84
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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