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Method for catalytically synthesizing starch sodium alkenylsuccinate (SSAS) with yeast display lipase

A technology of dodecenylsuccinic acid and lipase gene, applied in the field of bioengineering, can solve the problems of limited commercial application, high production cost, complicated and time-consuming immobilization process, etc., and achieves improved operation stability, low production cost, The effect of shortened reaction time

Inactive Publication Date: 2013-10-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lipase is currently the most widely used enzyme in esterification synthesis, 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 starch sodium alkenylsuccinate (SSAS) with yeast display lipase
  • Method for catalytically synthesizing starch sodium alkenylsuccinate (SSAS) with yeast display lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Preparation of Yeast Surface Display Lipase

[0018] 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.

[0019] Using the above artificially synthesized sequence as a template, PCR amplification was performed using the following primer pair,

[0020] Upstream primer: 5'-AAGGAAAAAAAGAATTCGTTCCAGTTTCTGG-3';

[0021] Downstream primer: 5'-TTTTCCTTTTGCGGCCGCTAATGAAACG-3'

[0022] 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...

Embodiment 2

[0026] Example 2 Synthesis of starch dodecenyl succinate catalyzed by lipase displayed on the surface of yeast

example 1

[0027] Example 1 Take 350g of corn starch and prepare 35% starch milk (starch dry basis) with water, absorb water at 65°C for 15 minutes to make the starch milk swell with water, add 1g of the yeast surface-displayed lipase prepared above after cooling, and then add 10ml in batches 15% (volume) ethanol solution of dodecenyl succinic anhydride, placed in a 85-1 type magnetic stirrer and stirred to start the reaction, the rotating speed was 200 rpm, the reaction temperature was kept at 60°C, and the stirring was stopped after 1 hour of reaction, and the pH was adjusted When the value reaches 7.0, the product is obtained after dehydration, alcohol washing, drying, crushing and sieving.

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Abstract

The invention discloses a method for catalytically synthesizing starch sodium alkenylsuccinate (SSAS) with yeast surface display lipase. The method comprises the following steps: dissolving starch of grain in water; adding dodecenly succinic anhydride ethanol solution and yeast surface display lipase after imbibition; stirring the mixture to react at 55-65 DEG C for 1-1.5 hours; and carrying out separation and purification to prepare the SSAS. The yeast surface 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 SSAS, 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 starch dodecenyl succinate ester catalyzed by lipase on the surface of yeast. Background technique [0002] Starch Sodium Alkenylsuccinate (SSAS) is starch modified by dodecenyl succinic anhydride (Dodecenly Succinic Anhydride, DDSA), and a hydrophilic and lipophilic amphoteric group is introduced into the starch molecular chain. , so that starch molecules have both hydrophilic and hydrophobic functions, and can be used as thickeners, emulsifiers, sizing agents and microcapsule wall materials in food, textile, paper and pharmaceutical industries. At present, SSAS is mostly produced by esterification reaction between DDSA and starch slurry under weakly alkaline conditions. In terms of preparation methods, compared with the dry process, it has disadvantages such as high equipment requirements, uneven reaction, and high impurity content. The wet proc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/04C12R1/84
Inventor 阮晖王睿之古再丽努尔徐娟周陈伟林吉恒何国庆
Owner ZHEJIANG UNIV
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