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Method for synthesizing L-ascorbyl palmitate by catalysis of yeast display lipase

A technology of ascorbyl palmitate and lipase, applied in the field of bioengineering, can solve the problems of limited commercial application, difficult separation, poor safety, etc., and achieve the effect of improving operational stability

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

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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 synthesizing L-ascorbyl palmitate by catalysis of yeast display lipase
  • Method for synthesizing L-ascorbyl palmitate by catalysis of yeast display lipase

Examples

Experimental program
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Effect test

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 Palmitate

example 1

[0029] Example 1 Take 0.176g of L-ascorbic acid and 2.308g of palmitic 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 to remove yeast-displayed lipase and molecular sieves, take the supernatant to remove tetrahydrofuran by rotary evaporation, wash with water for 3 times, add n-hexane to crystallize at 4°C to obtain L-ascorbyl palmitate product, dry and pulverize.

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Abstract

The invention discloses a method for synthesizing L-ascorbyl palmitate by catalysis of yeast display lipase. The method comprises the following steps of: dissolving L-ascorbic acid and palmitic acid into an organic solvent, adding the yeast display lipase, reacting for 4 to 8 hours in the absence of oxygen at the temperature of between 50 and 60 DEG C, and performing separation and purification to obtain the L-ascorbyl palmitate, wherein the yeast display lipase is prepared by the following steps of: transferring linearly treated recombinant plasmids to pichia pastoris GS115, inoculating the obtained transformant to a buffered methanol complex (BMMY) medium, performing induced culture for 72 to 144 hours, performing centrifugal collection on bacteria, and performing flushing, biological imprinting and freeze drying on the bacteria to obtain the yeast display lipase. The lipase is displayed outside cells, and the L-ascorbyl palmitate is synthesized by catalysis of the enzyme preparation, so that the transformation efficiency is improved, the reaction time is shortened and the production cost is reduced.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for synthesizing L-ascorbyl palmitate 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 o...

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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