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Stabilization technology for intact phycobilisomes of red algae and blue algae

A technology of phycobilisomes and red algae, which is applied in the field of marine biology, can solve the problems of easy dissociation, difficulty in long-term storage of phycobilisomes, and restrictions on the application of phycobilisomes, achieving good application prospects

Inactive Publication Date: 2005-08-17
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, phycobilisomes are supramolecular complexes formed by self-assembly of phycobiliproteins under the action of linker proteins and non-covalent bonds such as hydrophobicity and charge. Phycobilisomes can only be maintained in a buffer system with a higher ionic strength. Stable, easy to dissociate at low ionic strength, even at high ionic strength, it is difficult to preserve intact phycobilisomes for a long time, which severely limits the use of phycobilisomes as fluorescent probes in biomedical detection application in

Method used

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  • Stabilization technology for intact phycobilisomes of red algae and blue algae
  • Stabilization technology for intact phycobilisomes of red algae and blue algae
  • Stabilization technology for intact phycobilisomes of red algae and blue algae

Examples

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Embodiment 1

[0026] Embodiment 1, the stabilization technique of the complete phycobilisome of cyanobacteria, the method is as follows:

[0027] 1. Crushing of raw materials: 0.8g of fresh Spirulina platensis, dissolved in 16mL 1mol L -1 In the disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH=6.9), the cells were disrupted by ultrasonic wave at a power of 12-15W, and the disruption was performed 5 times, 1.5 min each time, with an interval of 2 min.

[0028] 2. Dissociation of phycobilisomes: add 2.0% volume percentage concentration of detergent Tween X-100 to the broken algal bodies, and stir slowly for 1 hour to 1.5 hours at room temperature to make phycobilisomes dissociate from the thylakoid membrane After dissociation from the upper part, centrifuge at 13000rpm~15000rpm for 1~2 times to remove the surface detergent Tween X-100, chlorophyll and cell debris at the bottom, and collect the solution in the middle of the centrifuge tube, which is the crude phyc...

Embodiment 2

[0035] Embodiment 2, as described in Embodiment 1, the difference is:

[0036] Step 4 Stability protection of complete phycobilisomes: use formaldehyde cross-linking technology to covalently cross-link phycobilisomes, take the complete phycobilisomes solution of the above step 3, and use 0.75mol / L disodium hydrogen phosphate- Potassium dihydrogen phosphate buffer solution (pH = 6.98) adjusted the protein concentration to 0.28 mg / mL, then took 1.8 mL of the phycobilisome solution, and slowly added 0.75 mol / L disodium hydrogen phosphate-diphosphate diphosphate while shaking. 0.2 mL of concentrated 4% (W / V) formaldehyde solution prepared in potassium hydrogen buffer solution until the formaldehyde concentration is 0.4% (W / V), and keep warm at 20°C for 12h. The resulting phycobilisome solution was diluted with deionized water to a low ion concentration of 0.1mol / L, incubated at 20°C for 30 days, and its fluorescence spectrum was measured. The results showed that the stabilized Ph...

Embodiment 3

[0038] Embodiment 3, the stabilization technology of the complete phycobilisome of red algae, the method is as follows:

[0039] As described in Example 1, the difference is:

[0040] The raw material for step 1 is 0.9 g of the single-celled red alga Porphyrococcus, dissolved in 18 mL of 1mol L -1 In the disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH=6.8), the cells were disrupted by ultrasonic wave at a power of 12-15W, and the disruption was performed 6 times, each time for 2 minutes, with an interval of 3 minutes.

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Abstract

The intact cyanophyta and red algae phycobilisome stabilizing technology belongs to the field of marine biotechnology. The cyanophyta and red algae material has the cells ultrasonic wave crushed, the phycobilisome dissociated from the thylakid membrane with detergent TritonX-100, and detergent TritonX-100, chlorophyll and cell fragment eliminated through conventional centrifugation. The intact cyanophyta or red algae phycobilisome is prepared through ultracentrifugal sucrose density gradient process, and protected integrally via non-covalent bond coating process with polysaccharide and gluicosan or covalent bond formaldehyde crosslinking process. The protected phycobilisome may be preserved at room temperature for 30 integrally without dissociation, and the present invention lays foundation for applying phycobilisome in super-sensitive biomedicine.

Description

(1) Technical field [0001] The invention relates to the stabilization technology of complete phycobilisomes of cyanobacteria and red algae, and belongs to the technical field of marine biology. (2) Background technology [0002] Phycobiliprotein is a kind of photosynthetic light-harvesting pigment-protein complex, which mainly exists in cyanobacteria, red algae, cryptophyta and a few dinoflagellates. It plays a role in capturing and transmitting light energy in photosynthesis, and has strong fluorescence. In cyanobacteria and red algae, the supramolecular structure phycobilisome is composed of 2-3 kinds of phycobiliproteins, forming an efficient energy transfer sequence. In the mid-1980s, American scholars who studied algae photosynthesis proposed to use phycobiliprotein as a fluorescent marker for diagnostic reagents. Compared with commonly used fluorescent markers, phycobiliprotein has the following advantages: the production process is safe, non-toxic, strong light ener...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/12
Inventor 张玉忠张熙颖陈秀兰周百成
Owner SHANDONG UNIV
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