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Preparation method and application of recombinant phycocyanobilin

A technology of phycocyanin and algae, which is applied in the fields of genetic engineering and food and medicine, can solve the problems of low growth density, high price of high-purity phycocyanin, and occupying a large area of ​​land, so as to reduce production costs and simplify the purification process , stable effect

Inactive Publication Date: 2011-01-19
CHINA UNIV OF PETROLEUM (EAST CHINA) +1
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Problems solved by technology

[0005] Although phycocyanobilin is abundant in Spirulina (accounting for about 1% of the dry weight of the algal body), the large-scale cultivation technology of Spirulina is relatively mature, but due to the long cultivation period and low growth density of Spirulina, it is necessary to Occupy a large area of ​​land, and the natural extraction process of high-purity free phycocyanobilin is more complicated (McCarty, M.F.Clinical potential of Spirulina as a source of phycocyanobilin, J Med Food 2007, 10, 566-570.), resulting in high prices on the market The price of pure phycocyanocyanin is relatively high (as a photosensitizer, the price of high-purity phycocyanocyanin is about 150-180 US dollars / mg), and currently it mainly depends on imports from the United States, and it is not yet suitable for wide application as a health care product

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  • Preparation method and application of recombinant phycocyanobilin
  • Preparation method and application of recombinant phycocyanobilin
  • Preparation method and application of recombinant phycocyanobilin

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preparation example Construction

[0028] The preparation method of recombinant phycocyanin: clone the key enzyme gene (heme oxidase gene ho1 and ferredoxin oxidoreductase gene pcyA) of phycocyanin synthesis from algae, clone the ho1 and pcyA genes to the appropriate expression The recombinant expression vector is then introduced into E. coli for heterologous expression. E. coli uses its own heme as a substrate to produce recombinant phycocyanin through two-step enzymatic catalytic reactions of Ho1 and PcyA. Finally, after cell disruption, chloroform extraction and gel filtration chromatography, a blue eluate is obtained, which is the recombinant phycocyanin mentioned above. The recombinant phycocyanobilin is a blue compound, soluble in dilute salt solution, with a molecular weight of about 588.69 g / mol. The recombinant phycocyanocyanin can be directly used as medicine, reagent, food additive, active ingredient of health care or functional food and the like.

[0029] Wherein the algae can be cyanobacteria, gre...

Embodiment 1

[0033] (1) The whole genome of Synechocystis sp. PCC 6803 has been sequenced and can be obtained for free from Genebank (Genebank number BA000022). According to the gene sequence of Synechocystis sp. PCC 6803 heme oxidase gene (ho1) and ferredoxin oxidoreductase gene (pcyA) published in Genebank, appropriate primers were designed to synthesize Synechocystis sp. PCC 6803) genomic DNA was used as a template, and genetic engineering was used to clone the key enzyme genes ho1 and pcyA of phycocyanocyanin synthesis. The numbers of the key enzyme genes ho1 and pcyA in the synthesis of phycocyanobilin in Genebank are sll1184 and slr0116, respectively.

[0034] (2) Using genetic engineering methods, add BamH I and Sac I restriction sites at both ends of the ho1 gene respectively, and insert them into the first multiple cloning site of the pETDuet-1 plasmid (purchased from Novagen, Germany) ( BamH I and Sac I restriction sites, hereinafter expressed in the same manner) to obtain the r...

Embodiment 2

[0040] (1) The whole genome of Synechocystis sp. PCC 6803 has been sequenced and can be obtained for free from Genebank (Genebank number BA000022). According to the gene sequence of Synechocystis sp. PCC 6803 (Synechocystis sp. Synechocystis sp. PCC 6803) genomic DNA was used as a template, and genetic engineering was used to clone the key enzyme genes ho1 and pcyA of phycocyanocyanin synthesis. The numbers of the key enzyme genes ho1 and pcyA in the synthesis of phycocyanobilin in Genebank are sll1184 and slr0116, respectively.

[0041] (2) Using genetic engineering methods, Nde I and EcoR V restriction sites were added to both ends of the ho1 gene, and inserted into the multiple cloning site of the pET28a-1 plasmid (Novagen, Germany) to obtain the recombinant vector pET28a- ho1. Add EcoR V and Xho I restriction sites to the two ends of the gene pcyA respectively, and clone into the downstream site of pET28a-ho1 to obtain the recombinant vector pET28a-ho1-pcyA (see attached...

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Abstract

The invention discloses a method for preparing recombinant phycocyanobilin, in particular a method for producing the recombinant phycocyanobilin by gene engineering technology. The method comprises the following steps of: cloning phycocyanobilin synthesizing key enzyme genes (hol and pcyA) from algae and cloning the key enzyme genes to proper expression vectors; inoculating a recombinant vector into an Escherichia coli expression system for heterogenous expression, and synthesizing the recombinant phycocyanobilin through enzymatic catalytic reaction; and finally performing extraction, chromatography and other purification steps to prepare the recombinant phycocyanobilin. The prepared recombinant phycocyanobilin has the advantages of higher capacity of removing DPPH radicals, can be used for treating a plurality of diseases caused by oxidative damage, and also can be used as an additive in functional foods.

Description

technical field [0001] The invention relates to the fields of genetic engineering and food medicine, in particular to a preparation method and application of recombinant phycocyanin. Background technique [0002] Phycocyanobilin (PCB) is a photosynthetic accessory pigment present in red algae and cyanobacteria. Similar to bilirubin, it is connected by 4 pyrrole rings in a straight chain through a methylene group, and its molecular weight is about 588.69g / mol. They are combined in vivo with a soluble protein called phycocyanin (phycocyanin) or allophycocyanin, dissolved in dilute salt solution. They play a role in absorbing and transmitting light energy in photosynthesis. [0003] As a derivative of bilirubin, phycocyanobilin has been shown to have antioxidative properties (Hirata, T., Tanaka, M., Ooike, M., Tsunomura, T., and Sakaguchi, M. Antioxidant activities of phycocyanobilin prepared from Spirulina platensis J Appl Phycol 12, 2000, 435-439.), scavenging hydroxyl rad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/16C12N15/53C12N15/63C12N1/21A23L1/30A61K31/409A61P39/06C12R1/19A23L33/10
Inventor 葛保胜黄方
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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