Preparation Of New Type Of Phycobiliproteins And Application In Biosensors And Dye-sensitized Solar Cells

Photosynthesis is an ancient and important conversion of solar light to biological energy in photosynthetic organisms.This highly efficient process starts from the light capturing by light-harvesting antenna of photosynthetic RCs.Phycobilisomes system,in cyanobacteria and red algae is the one of two major light-harvesting systems of photosynthetic oxygen organisms.Phycobilisomes are aggregations of water-soluble phycobiliproteins and linker polypeptides.Phycobiliproteins are multi-subunit complex bearing covalently attached open-chain tetrapyrroles,known as phycobilins,orderly assembled into phycobilisomes system and enabling them to harvest light in the visible region of the spectrum.The absorbed energy can be transferred at almost 95% efficiency to the reaction center.Phycobiliproteins have been applied in the field biomedicine and DSSC for their unique molecular structures and spectral characterizations.In recent years,the reconstruction of phycobiliproteins by gene engineering technology,not only provided a new way for the research on the energy transfer process of phycobilisomes,but also provide conditions for the development and application of biosensors.Based on above facts,this study investigated the preparation of natural and recombinant phycobiliproteins,and then study the spectral characteristics and explore the application.Detailed results will be introduced in the following sections:1.In this thesis,we discussed the isolation and efficient purification of B-phycoerythrin?B-PE?from microalga Porphyridium cruentum.Phycobiliproteins in Porphyridium cruentum was extracted by osmotic shock and initially purified by ultrafiltration,ammonium sulphate precipitation and chitosan adsorbent respectively.Further purification was carried out with a SOURCE 15 Q exchange column Analytical grade B-PE was obtained with a purity ratio?absorbance ratio,A545/A280?of 5.1 and a yield of 68.5%.Our protocol provides attractive alternative to consider for the purification procedure to obtain analytical grade B-PE at commercial level.The analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis?SDS–PAGE?showed a bulky band between 18 and 20 kDa which could be assigned to subunits ? and ? and a low intensity band of 27 kDa assigned to ? subunit.It showed a double absorption peaks at 545 nm and 565 nm and a shoulder peak at 498 nm,and displayed a fluorescence emission maximum at 580 nm and 620 nm.CD spectrum of B-PE in 250⁷50 nm was obtained and resolved for the first time.The three CD spectrum peaks of B-PE: 260 nm,305 nm and 330-380 nm,were considered to correlated to Phe,Trp and phycobilin respectively.Phe and Tyr might be in a conservative hydrophobic microenvironment PEB139?/ PEB158? and PEB82?/ PEB82? were consisted as two exciton-coupled bilin pairs.Energy transfer within exciton-coupled pairs was by exciton splitting,while between exciton-coupled pairs by F?rster resonance.Potential energy transfer was obtained finally.2.In this study,we optimized the culture conditions of the engineering strain MAC;10L fermentation was performed 3 times to collect a large number of cells with recombinant protein of MAC.The expression rate of MAC reached 43% of the total soluble proteins and the final optical cell density of the broth was 12.5,the total wet weight of MAC is around 400 g.The process of ultrasonication was optimized by orthogonal test and then the MAC was purified by chromatography.The analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis?SDS–PAGE?showed a bulky 76 kDa band as well as expected.It showed three absorption peaks at 628 nm,340 nm,370 nm and a shoulder peak at 575 nm,and displayed a fluorescence emission maximum at 640 nm.The CD spectrum peaks was the same with the absorption spectrum.We found that when aromatic amino acids and phycobilins were excited,fluorescence arose in visible region of the spectra,suggesting there is energy transfer pathway from aromatic amino acid residues and phycobilins to chromophores.Spectra results showed that the MAC has the correct conformation and good optical activity.3.Glucose Biosensor Based on MF0?? subunit of recombinant phycocyanin?and Graphene Oxide was designed.A novel graphene oxide?GO?nano-material conjugated with low molecular chitosan?CS?was synthesized through a rapid and simple covalent conjugation.Synthesized GO-CS was employed to detect glucose in a low concentration level based on the competitive binding with Maltose-binding protein?MBP?labeled MF0.In this designed biosensor system,the MF0 emission is quenched through the binding between GO-CS and MBP,while,in the present of glucose,MF0 fluorescence released after glucose competitively bind to MBP.Based on fluorescence signal recovery measurement,the target glucose was detected sensitively and selectively with the linear detection range from 0.1 mg/mL to 1 mg/m L.The limit of detection?LOD?for glucose is around 0.05 mg/m L.This biosensor system exhibits sensitivity and specificity properties for glucose.4.Dye-sensitized solar cells were assembled using 7 different phycobiliprotein as sensitizer,TiO₂ thin films as photoelectrodes,and their photoelectrical properties were studied.The result showed that the sensitization of B-PE was superior to other phycobiliprotein,eg could significantly improve the photoelectrical properties of DSSC.The short-circuit current,open circuit voltage,fill factor and photoelectric conversion efficiency of DSSC assembled with TiO₂ thin films as photoelectrode were 0.809 A/cm2,0.545 V,0.569 and 1% respectively.Coupling extended spectral response range and increased the photoelectric conversion efficiency of DSSC.The composite gel was syntheticed and had been proved to be stable and conducive to encapsulate.The composite gel can not only improve the photoelectric conversion efficiency,but also improve the short-circuit current of cell and can be used as an approximate solid electrolyte.The IPCE and ICE provided a new way for the research on the structure and energy transfer process of phycobiliprotein.