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Method for catalyzing and synthesizing sucrose stearate through yeast show lipase

A technology of sucrose stearate and lipase, which is applied in the field of bioengineering, can solve the problems of limited commercial application, high production cost, difficult control of esterification reaction sites, etc., so as to improve operation stability and inhibit side reactions. Effect

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] For a long time, the chemical preparation of sucrose esters has mostly used the method of direct esterification of sucrose. Since sucrose has 8 hydroxyl groups with similar reactivity, it is difficult to control the site of the esterification reaction. It is very difficult to obtain a single product with a specified structure. Lengthy protection and deprotection steps
At present, all the technological researches on the preparation of sucrose stearate are still limited to pure chemical methods, focusing on optimizing the relevant factors of the esterification reaction (such as temperature, reaction time, initial molar ratio of raw materials, etc.), so the problem of side reactions cannot be solved all the time. solve
Lipase is currently the most widely used enzyme in the synthesis of sucrose 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 catalyzing and synthesizing sucrose stearate through yeast show lipase
  • Method for catalyzing and synthesizing sucrose stearate through yeast show 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 sucrose stearate

example 1

[0029] Example 1 Take 0.2g of sucrose and 0.5g of stearic acid, add to a stoppered Erlenmeyer flask containing 10mL of tert-butanol, mix and preheat for 10min, then add 0.01g of yeast display lipase, place in a water bath shaker to start the reaction, the rotating speed is 200 rpm, keep the reaction temperature at 50°C, add 0.5g molecular sieve (pore size less than 2nm) after 12 hours of reaction, continue the reaction for 12 hours, remove the yeast-displayed lipase and molecular sieve by centrifugal precipitation, take the supernatant and carry out rotary evaporation to remove the organic solvent , washed with water for 3 times, added n-hexane and crystallized at 4°C to obtain sucrose stearate product, dried and pulverized.

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Abstract

The invention discloses a method for catalyzing and synthesizing sucrose stearate through yeast show lipase, comprising the following steps of: dissolving sucrose and stearic acid in an organic solvent; adding the yeast show lipase; reacting at 50-60 degrees centigrade for 10-14 h; separating and purifying to obtain the sucrose stearate; transforming linearly treated recombinant plasmids into pichia pastoris (Pichia pastoris) GS115; inoculating the obtained transformant into a BMMY (buffered methanol-complex medium) culture medium; after inducing and culturing for 72-144 h, centrifugally collecting thallus; and washing, biologically imprinting, freezing and drying the thallus so as to obtain the yeast show lipase. By showing the lipase outside cells, the sucrose stearate is catalyzed and synthesized through the lipase preparation. By means of the method disclosed by the invention, the transformation efficiency is improved; furthermore, 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 sucrose stearate catalyzed by yeast display lipase. Background technique [0002] Sucrose esters (SE) are a new class of nonionic surfactants. It has good emulsifying, dispersing, lubricating, decontaminating, foaming, adjusting viscosity, preventing aging, preventing crystallization, etc., and is harmless to the human body, easy to biodegrade into substances that are easily absorbed by the human body, so it is widely used in food and pharmaceuticals , cosmetics, washing and other industries. [0003] For a long time, the chemical preparation of sucrose esters has mostly used the method of direct esterification of sucrose. Since sucrose has 8 hydroxyl groups with similar reactivity, it is difficult to control the site of the esterification reaction. It is very difficult to obtain a single product with a specified structure. Lengthy protection an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/12C12N15/63C12N1/19C12N9/20C12R1/84
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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