Study On Optimization And Mechanism Of Purification Of Phycocyanin By Column Chromatography

The problem of the large number of blooms of cyanobacteria after salvage in China every year is difficult to disposal and cause secondary pollution,in order to solve it and overcome the drawback caused by the low value-added and passive digestion of using cyanobacteria,a method for extracting and purifying high-purity phycobiliprotein from cyanobacteria come into being.In this paper,the fresh cyanobacteria of Chaohu Lake was used as experimental material,and the phycocyanin and allophycocyanin were refined by column chromatography using Cellufine A-500 and hydroxyapatite as fillers.According to the characteristics of the elution peaks corresponding to the two kinds of fillers on the elution curve,making full use of the difference in the UV-visible spectral characteristics of phycoerythrin,phycocyanin,allophycocyanin and nucleic acid,carotenoids,general proteins,so the dynamic change of the elution peak composition and content can be judged.UV-visible absorption spectroscopy was used to study the spectral characteristics and variation of phycobiliprotein elution peaks by two kinds of packed column chromatography,the change of composition and content of each elution peak can be qualitatively and quantitatively determined.;combining the characteristics of the two kinds of fillers,the charge characteristics and coordination ability of phycocyanin,allophycocyanin,phycoerythrin,etc.can be analyzed,revealing the intrinsic mechanism and essence of the fractional elution of the two column chromatography materials.In the purification experiment of phycocyanin by Cellufine A-500,through single factor experiment and response surface methodology optimization of eluent pH,ionic strength,elution rate,and injection concentration,the optimum conditions were obtained as follows: pH 7.29,ionic strength 0.23 mol/L,elution rate 5.95 mL/min and injection concentration 1 mg/mL.Under the condition,the highest purity of phycocyanin was 4.42,and the recovery rate of reagent grade phycocyanin was 60.32%.In the purification experiment of phycocyanin by hydroxyapatite,through single factor experiment and response surface methodology optimization of eluent pH,ionic strength,elution rate,and injection concentration,the optimum conditions were obtained as follows: pH 6.32,ionic strength 0.08 mol/L,elution rate 3.56 mL/min and injection concentration 2 mg/mL.Under the condition,the highest purity of phycocyanin was 4.51,and the recovery rate of reagent grade phycocyanin was 38.36%;the highest purity of phycocyanin was 4.48,and the recovery rate of reagent grade allophycocyanin was 10.35%.During the purification of phycobiliprotein by Cellufine A-500,four elution peaks appeared on the elution curve with the replacement of the eluate.After scanning the ultraviolet-visible absorption spectrum of the sampling point,it was found that:The main component of peak I is positively charged or electrically neutral hetero protein and carotenoid;the main component of peak II is phycoerythrin,heteroprotein and nucleic acid with a small amount of negative charge;the main component of the peak III is high-purity phycocyanin with a large negative charge and a small amount of allophycocyanin,and the further improvement of the purity of phycocyanin is restricted due to the incomplete separation of phycocyanin and allophycocyanin;The main component of the peak IV is a hetero protein and a low-purity phycocyanin with a large amount of negative charge.In the process of purifying phycobiliprotein by hydroxyapatite,three elution peaks appeared on the elution curve with the replacement of the eluate.After scanning the ultraviolet-visible absorption spectrum of the sampling point,it was found that: The main component of the peak I is cationic or basic protein,such as hetero protein,nucleic acid,carotenoid,or the like;The main component of the peak II is high-purity phycocyanin which combines with calcium ions to form a weak coordination bond,and the phycocyanin and the allophycocyanin can be completely separated,which is beneficial to the further improvement of the purity of the phycocyanin;The main component of the peak III is a high-purity allophycocyanin which combines with calcium ions to form a strong coordinate bond.The purification effect of the two fillers was comprehensively analyzed by UV-Vis absorption spectroscopy: Cellufine A-500 filler can effectively remove phycoerythrin,nucleic acid,carotenoids and hybrid proteins from phycobiliprotein preliminary purification solution,and finally obtain reagent grade phycocyanin with a purity of 4.2,but can not completely separate phycocyanin and allophycocyanin;Hydroxyapatite filler can not only effectively remove phycoerythrin,nucleic acid,carotenoids and hybrid proteins from phycobiliprotein preliminary purification liquid,but also effectively separate phycocyanin and allophycocyanin,and obtain reagent grade phycocyanin and reagent grade allophycocyanin.