Method for synthesizing fructose palmitate by catalysis of yeast display lipase

A technology for synthesizing fructose and palmitate, applied in the field of bioengineering, can solve the problems of limited commercial application, poor selectivity, harsh chemical synthesis reaction conditions, etc., and achieves the effect of improving operational stability and inhibiting side reactions

Inactive Publication Date: 2011-10-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional synthesis process of sugar esters is chemical synthesis, but sugar is a polyhydroxy compound, and the selectivity of chemical synthesis is poor. The esterification of specific hydroxyl groups requires protection and deprotection steps, so the process is more complicated; chemical synthesis also has such as reaction conditions Harsh, many by-products, easy to coke and other shortcomings; and the high selectivity of lipase can overcome the above shortcomings, so it has high research value and good application prospects
Lipase is currently the most widely used enzyme in the synthesis of sugar 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 fructose palmitate by catalysis of yeast display lipase
  • Method for synthesizing fructose palmitate by catalysis of yeast display lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Preparation of yeast display lipase

[0020] Synthesize the lipase gene of Rhizopus oryzae (Genbank number: AF229435) and the cell wall alpha lectin gene of Pichia pastoris GS115 (Genbank number M28164) by artificially synthesized methods, and add the C-terminal of the lipase gene The upper linker peptide sequence GSSGGSGGSGGSGGSGS (linker), the nucleotide sequence pro-ROL-linker-α-agglutinin is obtained after linking, and EcoR I and Not I restriction sites are added 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 pairs,

[0022] Upstream primer: 5’-AAGGAAAAAAGAATTCGTTCCAGTTTCTGG-3’;

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

[0024] The PCR reaction system is: 1μl template DNA, 0.5μl high-fidelity DNA polymerase, 0.4μl dNTP (50mM), 0.5μl each of the upstr...

Embodiment 2

[0028] Example 2 Yeast displays lipase to catalyze the synthesis of fructose palmitate

example 1

[0029] Example 1 Take 0.2 g of fructose and 0.5 g of palmitic acid, add 10 mL of acetone to a cork with a stopper, mix and preheat for 10 minutes, then add 0.01 g of yeast display lipase, and place it on a water bath shaker to start the reaction at 200 rpm. After 12 hours, the reaction temperature was kept at 50℃. After 12 hours of reaction, 0.5g molecular sieve (pore size less than 2nm) was added. After the reaction continued for 12 hours, the yeast display lipase and molecular sieve were removed by centrifugation. After adding n-hexane to crystallize at 4°C to obtain fructose palmitate product, drying and pulverizing.

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Abstract

The invention discloses a method for synthesizing fructose palmitate by catalysis of yeast display lipase. The method comprises the following steps of: dissolving fructose and palmitic acid into an organic solvent, adding the yeast display lipase, reacting for 10 to 14 hours at the temperature of between 50 and 60 DEG C, and performing separation and purification to obtain the fructose 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 fructose 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 present invention relates to the technical field of bioengineering, in particular to a method for yeast display lipase to catalyze the synthesis of fructose palmitate. Background technique [0002] Sugar esters are a class of non-ionic surfactants with a very wide range of applications. They have outstanding advantages such as non-toxic, odorless, non-irritating and biodegradable. They are widely used and good in the fields of food, cosmetics, medicine, chemicals, and agriculture. Prospects. [0003] The traditional synthesis process of sugar esters is chemical synthesis, but sugars are polyhydroxy compounds, and the selectivity of chemical synthesis is poor. The esterification of specific hydroxyl groups requires protection and deprotection steps, so the process is more complicated; chemical synthesis also has conditions such as reaction conditions Harshness, many by-products, easy coking, etc.; and the high selectivity of lipase can overcome the above ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12N15/63C12N1/19C12N9/20C12R1/84
Inventor 阮晖周陈伟迪拉热木徐娟王睿之林吉恒何国庆
Owner ZHEJIANG UNIV
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