Preparation Of Phycocyanobilin And Its Preliminary Study As Photosensitizer

Phycocyanobilin(PCB)is a natural pigment with a linear tetrapyrrole structure in cyanobacteria,red algae and some cryptoalgae,having special optical properties and good antioxidant activity.It plays an important role in the absorption and transmission of light energy,inhibition of oxidative damage and optogenetics,and has considerable prospects in the development of photosensitizers.In natural algae,there are various photosynthetic pigments such as PCB,chlorophyll,and lutein.The presence of other photosynthetic pigments complicates the process of separating and purifying PCBs from algae.In this paper,two PCB preparation methods were studied:one was to prepare PCB by fermentation of recombinant Escherichia coli(E.coli),and the other was to extract PCB from phycocyanin(PC),a natural product of algae,and the spectra of the two PCB products are analyzed.In addition,the possibility of PCB as a photosensitizer for photodynamic therapy was preliminary verified.conclusion is as below:(1)In this paper,a recombinant plasmid pET-28a-hox1-pcyA was used to construct and screen a recombinant E.coli"EP-1"that can stably express PCB.Response surface optimization,liquid phase-mass spectrometry(LC-MS)and spectroscopic analysis were used to improve the yield of recombinantly expressed PCB and analyze the purity and spectral properties of the prepared PCB.The results showed that during shake flask fermentation,when the fermentation broth was cultured for about 4.6 h,adding lactose to the fermentation broth with a final concentration of 4 mmol/L as an inducer,inducing expression at 25?for 13.6 h,the PCB concentration in the fermentation broth was about13 mg/L.Compared with literature,the output of PCB had increased by about 3 times.The purity of the recombinantly expressed PCB was about 70%.In addition,the recombinantly expressed PCB will protonate under acidic conditions,and the absorption spectrum will redshift from about 600 nm to about 680 nm.(2)The natural PCB was extracted from PC by a combination of high-temperature ethanol cracking PC and vacuum freeze-drying solidification.In addition,the purity and spectral activity of natural PCB were analyzed and identified by LC-MS and spectral analysis methods,and the absorption and fluorescence spectral properties of natural PCB and recombinant PCB were compared and analyzed.The results showed that when 150mL of 70%ethanol was used as the PC lysate,the yield of natural PCB could reach 160mg/10 g PC after cracking at 78?for 18 h.The purity of the extracted natural PCB was about 62%.The recombinantly expressed PCB have the same absorption and fluorescence spectral properties as the natural PCB.(3)Using natural PCB extracted from PC as experimental material,the uptake of PCB by mouse mononuclear macrophage J774A.1 and human hepatoma cell HepG2 was observed by fluorescence confocal microscope.Subsequently,PCB was used as a photosensitizer to verify the photodynamic killing effect of PCB on HepG2 and J774A.1under 600 nm excitation light source.The experimental results showed that the PCB can be taken up by J774A.1 and HepG2.When irradiated with a 600 nm light source of 150mW/cm~2 for 1.5 h,250?g/mL PCB could cause the complete loss of cell viability of HepG2 and J774A.1.PCB is expected to develop into a new type of photosensitizer for photodynamic therapy of tumors.