Recombinant Production Of ? Subunits Of C-phycocyanin From Spirulina Subsalsa In Escherichia Coli And Antioxidant Studies

Phycocyanin has been mianly prepared by the extraction from cyanobacteria,and can also be produced by heterogenous production.Recombinant phycocyanin had similar spectral characteristics to natural phycocyanin,but exhibited stronger antioxidant activity.This process of eparation and purification phycocyanin from cyanobacteria is rather complicated and rawmaterial wasting and had a low yield.Thus,it is a better choice to produce phycocyanin from Escherichia coli.Five genes,cpcB encoding phycocyanin-?-subunit,cpcS encoding phycocyanin-?-subunit phycocyanin lyase S,cpcT encoding phycocyanin-?-subunit phycocyanin lyase T,ho1 encoding hemeoxygenase,pcyA encoding phycocyanbilin:ferredoxino-reductase,were amplified by PCR from Spirulina subsalsa FACHB351.When the plasmid pETDuet harbouring two cassettes was constructed:cpcB in one cassette;the fusion gene cpcS::ho1::pcyA in the other cassette,and expressed in Escherichia coli,the screened transformant acquired a pronounced blue colour.The results showed that a fluorescent?subunit phycocyanin,PCB-CpcB?C-82?,was biosynthesized in E.coli,exhibiting an absorption maximum at 620 nm and a fluorescence emission maximum at 640 nm.When cpcS was replaced by cpcT,another fluorescent?subunit phycocyanin,PCB-CpcB?C-153?,was produced and given a maximum absorption at 590 nm and fluorescence emission at 620 nm.The strategy of single expression plasmid was adopted to avoid the defects of multi-plasmid transformation,to reduce the environmental pollution caused by the use of antibiotics and the production cost,and increasing the stability of the expression of exogenous protein.Escherichia coli has a periplasmic space,targeting protein to the periplasmic space or to the culture medium has several advantages over intracellular productionIn this study,the coding region of PelB signal peptide coding region was added to the upstreamof cpcB,coexpressing with ho1,pebS and cpcS in E.coil.Recombinant phycocyanin PelB::CpcB?C-82?-PEB was synthesized and secreted into the culture medium.The spectral characteristics of extracellular phycocyanin are basically the same as those expressed in cells,indicating that their physicochemical properties have not changed.Phycocyanin does not require cell disruption to secrete directly into the medium,which will save costs.In order to study the antioxidant characteristics of different algae-homologous recombinant phycocyanin,the genes(cpcB_Sp,cpcB_Ma and cpcB_No)encoding C-PC were cloned from three cyanobacteria species,Spirulina subsalsa,Mastigocladus laminosus and Nostoc PCC 7120.The current recombinant products,CpcB_Sp,CpcB_Ma and CpcB_No,respectively,were obtained from Escherichia coli.The antioxidant capacities of recombinant CpcB_Sp,CpcB_Ma and CpcB_No were evaluated by measuring their hydroxyl radical scavenging activity and DPPH radical scavenging activity.The results revealed that the hydroxyl radical and DPPH radical scavenging activity of recombinant proteins increased with increasing protein concentration.At the tested concentrations,the results demonstrated that the anti-oxidant capacities are CpcB_Sp<CpcB_Ma<CpcB_No,both in scavenging hydroxyl radicals and in scavenging DPPH radicals.The results showed that recombinant apo-phycocyanin has a high antioxidant activity,while it showed different antioxidant abilities due to the difference of cyanobacteria species,Two fluorescent biliproteins showed a stronger scavenging activity on hydroxyl and DPPH free radicals than apo-CpcB.The IC50 values for hydroxyl radical scavenging of His-PCB-CpcB?C-82?,His-PCB-CpcB?C-153?and His-apo-CpcB were 38.72±2.48?g/mL,51.06±6.74?g/mL and 81.82±0.67?g/mL respectively,and for DPPH radical scavenging were201.00±5.86?g/mL,240.34±4.03?g/mL and 352.93±26.30?g/mL,respectively.The antioxidant capacities are apo-CpcB?CpcB?C-153?-PCB?CpcB?C-82?-PCB,on account of bilin binding.The results of this study suggest that fluorescent phycocyanin?subunits of Spirulina subsalsa were reconstructed by one expression vector in E.coli and could be developed as an excellent antioxidant agent for therapeutic and nutritional values.