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Method for catalyzing and synthesizing octenyl succinic anhydride modified starch ester through yeast show lipase

A technology of octenyl succinic acid and lipase genes, which is applied in the field of bioengineering, can solve the problems of complicated and time-consuming immobilization process, high production cost, and limited commercial application, so as to achieve improved operation stability, low production cost, and high reaction rate. The effect of shortening the time

Inactive Publication Date: 2013-08-14
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 catalyzing and synthesizing octenyl succinic anhydride modified starch ester through yeast show lipase
  • Method for catalyzing and synthesizing octenyl succinic anhydride modified starch ester through yeast show lipase

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Example 1 Preparation of Yeast Displaying 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×PCR ...

Embodiment 2

[0026] Example 2 Yeast shows lipase catalyzed synthesis of octenyl succinic acid starch ester

example 1

[0027] Example 1 Get 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 absorb water and swell, add 1g of the above-mentioned prepared yeast to display lipase after cooling, then add 10g of starch in batches Alkenyl succinic anhydride, placed in a 85-1 type magnetic stirrer to start the reaction, the rotation speed was 100 rpm, the reaction temperature was kept at 34°C, the stirring was stopped after 1 hour of reaction, the pH value was adjusted to 7.0, after precipitation, washing, Spray drying to obtain powder.

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Abstract

The invention discloses a method for catalyzing and synthesizing octenyl succinic anhydride modified starch ester through yeast show lipase, comprising the following steps of: dissolving grain starch in water; after imbibition, adding octenyl succinic anhydride and the yeast show lipase; stirring and reacting at 34-36 degrees centigrade for 1-1.5 h; and separating and purifying to obtain starch sodium ester sodium octenyl succinate. A method for preparing the yeast show lipase comprises the following steps of: transforming linearly treated recombinant plasmids into 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 octenyl succinic anhydride modified starch ester 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 starch octenyl succinate catalyzed by yeast display lipase. Background technique [0002] Starch octenyl succinate is currently the only starch ester approved by the US FDA and my country as a food additive, and it is the most effective food additive for stabilizing food emulsion systems. At present, starch octenyl succinate is produced by esterification reaction between octenyl succinic anhydride (OSA) and starch slurry under weakly reducing conditions. The main problem is that the speed of the esterification reaction is not fast enough. , the reaction time is too long, cause side reaction product generation (side reaction I) and octenyl succinic acid starch ester to hydrolyze (side reaction II), thereby affect esterification reaction efficiency, and make octenyl succinic acid starch ester finished product Doping side reaction products. [0003...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/04C12N15/63C12N1/19C12N9/20C12R1/84
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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