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Method for catalytic synthesis of starch phosphate by utilizing yeast display lipase

A starch phosphate and lipase technology, applied in the field of bioengineering, can solve the problems of complicated and time-consuming immobilization process, limited commercial application and high production cost, and achieve the effects of improving operational stability, shortening reaction time and low production cost.

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

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 catalytic synthesis of starch phosphate by utilizing yeast display lipase
  • Method for catalytic synthesis of starch phosphate by utilizing yeast display lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Preparation of Yeast Displaying Lipase

[0019] 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.

[0020] Using the above artificially synthesized sequence as a template, PCR amplification was performed using the following primer pair,

[0021] Upstream primer: 5'-AAGGAAAAAAAGAATTCGTTCCAGTTTCTGG-3';

[0022] Downstream primer: 5'-TTTTCCTTTTGCGGCCGCTAATGAAACG-3'

[0023] 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

[0027] Example 2 Yeast shows lipase catalyzed synthesis of starch phosphate

example 1

[0028] Example 1 Take 350g corn starch and prepare 35% starch milk (starch dry base) with water, absorb water at 65°C for 15min 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 NaH in batches 2 PO 4 , placed in a 85-1 type magnetic stirrer and stirred to start the reaction, the rotation speed was 200 rpm, the reaction temperature was kept at 60°C, the stirring was stopped after 1 hour of reaction, the pH value was adjusted to 7.0, the starch phosphate was precipitated by ethanol, and washed , suction filtration, vacuum drying, and the resulting powder is the finished product.

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Abstract

The invention discloses a method for catalytic synthesis of starch phosphate by utilizing yeast display lipase, and the method comprises the steps: dissolving grain starch in water, adding NaH2PO4 and the yeast display lipase after imbibition, reacting at 55-65 DEG C for 1-1.5 hours, and separating and purifying, thus obtaining the starch phosphate. 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 thallus, thus obtaining the yeast display lipase. Through displaying lipase outside cells, and utilizing the lipase to conduct catalytic synthesis of the starch phosphate, 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 starch phosphate catalyzed by yeast display lipase. Background technique [0002] Starch phosphate is a kind of starch derivative produced by the esterification of native starch and phosphate. It is an anionic modified starch with the characteristics of high transparency, easy gelatinization, strong adhesiveness, good stability and weak retrogradation. , especially in food technology, as a food additive, starch phosphate is a good emulsification stabilizer, a clear and stable thickener, and has good freeze-thaw stability. [0003] At present, starch phosphate is mostly composed of inorganic phosphate (sodium dihydrogen phosphate NaH 2 PO 4 , sodium tripolyphosphate STP: Na 5 P 3 o 10 , sodium trimetaphosphate: STMP (NaPO 3 ) 3 etc.) and starch slurry are produced by esterification reaction under weakly alkaline conditions. The main problem ...

Claims

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Application Information

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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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