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Method for catalytic synthesis of glucose laurate by utilizing yeast display lipase

A technology of laurate ester and lipase, which is applied in the field of bioengineering, can solve the problems of limited commercial application, high production cost, complicated and time-consuming immobilization process, and achieve 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 currently commercially available sugar ester products are basically chemically synthesized, and the products are complex mixtures. Complicated separation work is required to obtain high-purity products
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 catalytic synthesis of glucose laurate by utilizing yeast display lipase
  • Method for catalytic synthesis of glucose laurate by utilizing yeast display lipase

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1 Preparation of Yeast Displaying 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 glucose laurate

example 1

[0029] Example 1 Take 0.2g of glucose and 0.5g of lauric acid, add them to a stoppered Erlenmeyer flask containing 10mL of acetone, mix and preheat for 10min, then add 0.01g of yeast-displayed lipase, place in a water bath shaker to start the reaction, and the rotation speed is 200 rpm Minutes, the reaction temperature was kept at 50°C, after 12 hours of reaction, 0.5g molecular sieve (pore size less than 2nm) was added, after the reaction was continued for 12 hours, the yeast display lipase and molecular sieve were removed by centrifugation, and the supernatant was removed by rotary evaporation to remove the organic solvent, washed with water for 3 After the second time, add n-hexane to crystallize at 4°C to obtain the glucose laurate product, which can be dried and crushed.

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Abstract

The invention discloses a method for catalytic synthesis of glucose laurate by utilizing yeast display lipase, and the method comprises the following steps: dissolving glucose and laurate in an organic solvent, adding the yeast display lipase, reacting at 50-60 DEG C for 10-14 hours, and separating and purifying, thus obtaining glucose laurate. 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 glucose laurate, 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 glucose laurate catalyzed by yeast display lipase. Background technique [0002] Sugar esters are non-ionic surfactants with carbohydrates as hydrophilic groups and fatty acids as hydrophobic groups. They have the advantages of non-toxicity, easy biodegradation, wide range of HLB values ​​(1-16) and good surface activity, and It has the functions of dispersing and lubricating, decontaminating, foaming, adjusting viscosity, preventing aging, preventing crystallization, and antibacterial properties, so it is widely used in food, medicine, cosmetics and other industries. It is the United Nations Food and Agriculture Organization (FAO) and the World Health Organization ( WHO) recommended food additives. [0003] The currently commercially available sugar ester products are basically chemically synthesized, and the products are complex mixtures. Compl...

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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