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Method for expressing and producing phycocyanin beta subunit in yeast

A technology for producing phycocyanin and phycocyanin, which is applied in microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of low yield and high cost, and achieve high yield and low cost. , the effect of simple operation

Active Publication Date: 2019-04-26
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 beta 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 high cost and low yield in the production of phycocyanin in the prior art. The expression and production of phycocyanin β subunit in yeast provides a lot of basic materials for further research on the function of phycocyanin β subunit

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0026] (1) connecting the phycocyanin β 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: 25°C, 280r / min shaking culture for 15h;

[0028] (3) The positive yeast transformant was transferred into the fermentation medium, and fermented at 30° C. and 500 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 30g / L, ammonium sulfate 0.6g / L, YNB 15g / L, leucine 20μg / L and tryptophan 25μg / L, pH 5.0.

[0030] The fermentation medium...

Embodiment 2

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

[0033] (1) connecting the gene encoding the phycocyanin beta subunit 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: 28°C, 300r / min shaking culture for 20h.

[0035] (3) The positive yeast transformant was transferred into the fermentation medium, and fermented and cultured at 38° C. and 800 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 40g / L, ammonium sulfate 0.9g / L, YNB 20g / L, leucine 30μg / L and tryptophan 35μg / L, pH 5.5.

[0040] The fermentation medium includes the following components: yeast powder 25g / L, tryptone 12g / L, glucose 25g / L, galactose 5g / L, K 2 HPO 4 0.3g / L and leucine 20μg / L, pH 6.0.

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PUM

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Abstract

The invention provides a method for expressing and producing a phycocyanin beta subunit in yeast. The method comprises the following steps: (1) connecting a phycocyanin beta subunit coding gene and acarrier to obtain a recombinant plasmid, wherein the recombinant plasmid contains a promoter; (2) preparing competent cells of the yeast; transforming the recombinant plasmid into the competent cellsof the yeast through an electric transformation method; culturing to obtain a yeast transformant and screening a positive yeast transformant; (3) transferring the positive yeast transformant into a fermentation culture medium and culturing. According to the method provided by the invention, the phycocyanin beta subunit is successfully expressed and produced in the yeast; after fermentation is finished, the content of phycocyanin reaches 60.8 to 167.6 mg / L and accounts for 40.5 to 61.3 percent of total protein. According to the method provided by the invention, the yield of the phycocyanin is relatively high; the method has the advantages of simplicity in operation, easiness for obtaining raw materials and low cost; the phycocyanin beta subunit is expressed and produced in the yeast and a lot of foundation materials are provided for further researching the function of the beta subunit of the phycocyanin.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for expressing and producing phycocyanin beta 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,...

Claims

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Application Information

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IPC IPC(8): C07K14/795C12N15/81C12P21/02C12R1/865
CPCC12N15/81C12P21/02C07K14/795
Inventor 连惠勇
Owner HAINAN TROPICAL OCEAN UNIV
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