Study On Phycoerythrin Purification By A Novel Aqueous Two-Phase System And Its Detection Of Mercury And Copper

There are abundant marine algae resources in China,which contain abundant bioactive substances.Among them,Porphyra yezoensis is a kind of common red algae and rich in high value active substance R-PE.So far,the purification method of R-PE owns some disadvantages,such as complex steps,long time consuming or low yield,which leads to high cost.In this study,a purification technique for R-phycoerythrin（R-PE）based on DES-ATPS was established,which provided a new method with lower cost and simpler operation for the extraction and purification of R-PE.Besides,R-PE and R-PE silver nanoparticles（R-PE-AgNPs）were used to detect mental ions in the environment,which provided non-toxic and low-cost fluorescent probes to this field.In this study,R-PE was purified from Porphyra yezoensis by freeze-thaw-salting out-ATPS.Firstly,the crude protein was obtained by repeatedly freezing and thawing,and then the dialyzed R-PE was obtained by salting out using 10%+50%ammonium sulfate.DEA-ATPSs were constructed and choline chloride-urea（Ch Cl-U）/K₂HPO₄ was selected as the most suitable system for R-PE extraction.Under optimal conditions,the purity(A₅₆₅/A₂₈₀)of the obtained R-PE reached 3.83,which reached drug grade(A₅₆₅/A₂₈₀>3.0)and the yield reached 69.99%（w/w）after the whole purification process,indicating the good purification effect.The UV-vis absorption spectra showed characteristic absorption peaks at 495 nm,540 nm,and 565 nm and the spectra of R-PE before and after purification followed a similar trend.Besides,R-PE displayed an emission wavelength（E_m）at 570 nm when excited at 495 nm and the spectra of R-PE before and after purification also be similar.Furthermore,the spectra followed a similar trend before and after purification,which demonstrated that the spatial structure of R-PE was not destroyed.Fourier transform infrared spectra suggested that the functional groups of R-PE were maintained well,indicating that the structure of the R-PE was not changed.The circular dichroism spectra indicated that the secondary structure of R-PE was maintained well during the purification process.All spectra results could prove the effectiveness of this method.Transmission electron microscopy showed that the complex formed by DES micelle and R-PE was the driving force for R-PE extraction.After obtaining R-PE in the above research,the application of R-PE in the detection of metal ion was further studied.In order to establish a method for the detection of metal ions using R-PE as a fluorescent probe,it was selected that R-PE had good selectivity and anti-interference for Hg²⁺among a series of metal ions.Single factor experiment was conducted to optimize the conditions.Under the optimal conditions,the standard curve was established and the limit of detection（LOD）was 0.00261 mg/L（0.0130μM）.The method above was adopted to detect Hg²⁺in tap water and sea water（Weihai）.The recovery was 92.0%～108.0%with relative standard deviation（RSD）of less than 1.60%.A fluorescence test paper based on R-PE was prepared,whose detection time was only 2min and the minimum visual concentration was 0.050μM（0.010 mg/L）.It could be also applied to detect Hg²⁺in tap water and sea water（Weihai）.The fluorescence quenching mechanism was investigated preliminarily through fluorescence microscope imaging and time-resolved fluorescence lifetime,which indicated that the binding of R-PE and Hg²⁺led to static fluorescence quenching.Fourier transform infrared spectroscopy and peak fitting were used to characterize the influence on the secondary structures of R-PE by Hg²⁺.It was showed that Hg²⁺could affect the secondary structures of R-PE,leading to the reduction ofα-helix and random coil contents and the increase ofβ-sheet andβ-turn contents.Molecular docking simulation results concluded that Hg²⁺had two optimal binding sites on R-PE monomer,namely 78LYS and 82CYS onαchain and 73CYS and82CYS onβchain.It was speculated that Hg²⁺was bound to sulfydryl（-SH）group of amino acids.The above results provided a new natural fluorescent probe to the field of detecting Hg²⁺rapidly and a fast and convenient semi-quantitative detection tool for the targeting rapidly the concentration range of metal ions.Then,R-PE-AgNPs were synthesized by biological method to modify R-PE.R-PE-AgNPs had good selectivity to Cu²⁺.Single factor experiment was conducted and the standard curve was established.LOD was 0.00122 mg/L（0.0190μM）.Cu²⁺in tap water and lake water（Weihai）were determined by recovery experiment.The recoveries ranged from 91.6%to 102.2%with RSD less than 1.6%.R-PE-AgNPs-based fluorescence test paper was prepared,and the minimum visual concentration was 1.0μM（0.064 mg/L）.It was found that Cu²⁺could cause aggregation phenomenon of R-PE-AgNPs,accompanied by the greatly increased particle size.The Fourier transform infrared spectra showed that the peak transferred from 1647 cm^-1 to 1641 cm^-1.Cu²⁺caused a red shift to the localized surface plasmon resonance peak of R-PE-AgNPs.Based on the results above,it was inferred that the reaction mechanism may be the formation of coordination complex between the carbonyl（-C=O）group of peptide chain and Cu²⁺,which resulted in static fluorescence quenching.This R-PE-AgNPs enriched AgNPs types synthesized by biological method and expanded the application field of AgNPs.The prepared fluorescent test paper could be used for rapid detection of Cu²⁺in water,providing a convenient tool for rapid in situ detection of Cu²⁺pollution.