A method for expressing and producing phycocyanin alpha subunit in yeast

A technology for the production of phycocyanin and α subunits, applied in the biological field, can solve the problems of high cost and low yield, and achieve the effects of low cost, high yield and shortened production time

Active Publication Date: 2022-01-25
HAINAN TROPICAL OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of this, the purpose of the present invention is to provide a method for expressing and producing phycocyanin α subunit in yeast, which not only solves the problems of high cost and low yield in the production of phycocyanin in the prior art, but also solves the problems of the present invention in the production of phycocyanin. The expression and production of phycocyanin α subunit in yeast provides a lot of basic materials for further research on the function of phycocyanin α subunit

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] A method for expressing and producing phycocyanin alpha subunit in yeast, comprising the following steps:

[0026] (1) connecting the phycocyanin α subunit encoding gene of Spirulina maxima to the vector pYES2 to obtain a recombinant plasmid, which contains the promoter GAL1;

[0027] (2) Prepare Saccharomyces cerevisiae competent cells, transfer the recombinant plasmids into yeast competent cells by electroporation, then cultivate yeast transformants in YNB medium, and screen positive yeast transformants; the culture conditions are: 20°C, 280r / min shaking culture for 15h;

[0028] (3) The positive yeast transformant was then transferred into the fermentation medium according to the volume fraction of 8% inoculum, and fermented and cultivated at 30° C. and 200 r / min for 36 hours. During the fermentation, the DO value in the fermenter was kept at 25%.

[0029] The YNB medium includes the following components: glucose 20g / L, ammonium sulfate 0.5g / L, YNB 10g / L, leucine 20...

Embodiment 2

[0032] A method for expressing and producing phycocyanin alpha subunit in yeast, comprising the following steps:

[0033] (1) connecting the phycocyanin α subunit encoding gene of Spirulina maxima to the vector pYES2 to obtain a recombinant plasmid, which contains the promoter GAL10;

[0034] (2) Prepare Saccharomyces cerevisiae competent cells, transfer the recombinant plasmids into yeast competent cells by electroporation, then cultivate yeast transformants in YNB medium, and screen positive yeast transformants; the culture conditions are: 25°C, 300r / min shaking culture for 20h;

[0035] (3) The positive yeast transformant was transferred into the fermentation medium according to the volume fraction of 10% inoculum, and fermented at 38° C. and 230 r / min for 48 hours. During the fermentation, the DO value in the fermenter was kept at 30%.

[0036] Described YNB medium, fermentation medium are identical with embodiment 1.

Embodiment 3

[0038] The difference between embodiment 3 and embodiment 1 is that

[0039] The YNB medium includes the following components: glucose 25g / L, ammonium sulfate 0.7g / L, YNB 15g / L, leucine 30μg / L and histidine 35μg / L, pH 5.5.

[0040] The fermentation medium includes the following components: yeast powder 25g / L, tryptone 15g / L, glucose 25g / L, galactose 5g / L, glycerol 5g / L and leucine 20μg / L, pH 6.0.

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Abstract

The present invention provides a method for expressing and producing phycocyanin α subunit in yeast, comprising the following steps: (1) connecting a phycocyanin α subunit encoding gene with a carrier to obtain a recombinant plasmid, wherein the recombinant plasmid contains a promoter (2) preparing yeast competent cells, transferring recombinant plasmids into yeast competent cells by electrotransformation, culturing to obtain yeast transformants, and screening positive yeast transformants; (3) transferring positive yeast transformants into fermentation medium nourish. The invention successfully realizes the expression and production of phycocyanin α subunit in yeast, and after the fermentation, the content of phycocyanin reaches 70.8-174.6 mg / L, accounting for 43.5-66.8% of the total protein. The method of the invention not only has high yield of phycocyanin, simple operation, easily available raw materials, and low cost, but also expresses and produces phycocyanin α subunit in yeast, which provides a method for further studying the α subunit function of phycocyanin. Plenty of base material.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for expressing and producing phycocyanin α subunit in yeast. Background technique [0002] Phycocyanin (PC for short, absorption spectrum at 610-640nm) is an important light-harvesting pigment protein, not only can be used as industrial raw materials and food additives. And because phycocyanin is easy to combine with biotin, antibody and other proteins, so phycocyanin as a fluorescent probe has been paid more and more attention. At present, phycocyanin is mainly separated and purified from spirulina, but it is difficult to cultivate spirulina and break the wall. The cost of separating and purifying phycocyanin from spirulina is high, the process is complicated, the yield is low, and the waste of raw materials is serious. Yeast expression system is the most successful high-efficiency expression system so far. Its cell reproduction speed is fast, the growth cycle is short, th...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C07K14/795C12N15/81
CPCC12N15/81C07K14/795
Inventor连惠勇
OwnerHAINAN TROPICAL OCEAN UNIV