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Method for catalyzing and synthesizing starch acetate through yeast show lipase

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

Inactive Publication Date: 2014-07-16
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 starch acetate through yeast show lipase
  • Method for catalyzing and synthesizing starch acetate through yeast show lipase

Examples

Experimental program
Comparison scheme
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 display lipase catalyzed synthesis of starch acetate

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 absorb water and swell, add 1g of the above-mentioned prepared yeast to display lipase after cooling, and then add 10g of acetic acid in batches Anhydride, placed in a 85-1 type magnetic stirrer to stir to start the reaction, the rotation speed is 100 rpm, the reaction temperature is kept at 66°C, the stirring is stopped after 2 hours of reaction, the pH value is adjusted to 6.0, the precipitate is collected by centrifugation, and washed with water for several times Precipitate, wash away unreacted acetic anhydride, then dry and crush the precipitate to obtain the finished product.

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Abstract

The invention discloses a method for catalyzing and synthesizing starch acetate through yeast show lipase, comprising the following steps of: dissolving grain starch in water; after imbibition, adding acetic anhydride and the yeast show lipase; stirring and reacting at 65-75 degrees centigrade for 2-2.5 h; and separating and purifying to obtain starch acetate. 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 starch acetate 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 acetate catalyzed by yeast display lipase. Background technique [0002] Starch acetate is an important type of modified starch, which can produce products with better coagulation resistance, transparency and other performance than native starch. Compared with native starch, starch acetate after acetylation shows the following properties Features: easy gelatinization, low gelatinization temperature, stable viscosity, neutral solution, no gel formation even after cooling, good film-forming property, greatly improved thermal stability, and greatly improved comprehensive performance , Has been used in food, paper, textile, adhesives and other industrial sectors. [0003] Compared with the chemical synthesis of starch acetate, the biological enzymatic method can obtain a reaction product with a high degree of substitution in a short period of ...

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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