Study On Biosynthesis And Stability Of Allophycocyanin

As one of the phycobiliproteins, allophycocyanin was mainly present inCyanobacteria and red algae, it is a secondary pigment protein, held mainly energytransfer and part of the energy storage function in the organism, and it’s alsofluorescent. Allophycocyanin is very promising, in addition to the applications inareas such as food, cosmetics, fluorescent dyes, it’s also can be made into fluorescentreagents, used in fields such as medicine, chemistry, and biology.Currently, allophycocyanin is mainly naturally extrcted from algae. But theacquiration of natural allophycocyanin goes through algae cultivation, extraction andpurification. The long preparation cycle, difficult separation and purification process,and high cost largely limit the applications of allophycocyanin, and become the majorconstraints of allophycocyanin’s fluorescence probe reagent and commercialization.In order to get allophycocyanin more quickly and easily, molecular biological methodwhich is simple, convenient and low-cost become the current popular preparationmethod. But recombinant allophycocyanin obtained through biological cloningmeans, which is easily denatured in heating, pH value changing, and long time laying,has a poor fluorescent stability, and it limits its application in fluorescence reagentdirectly.This test first optimized the fermentation conditions of allophycocynin in E. coli.using response surface method, and obtained a best fermentation conditions for:induced temperature28℃, fermentation medium initial pH8.5, induced timing3h,and IPTG end concentration0.1mM. The obtained protein expression in optimizatedfermentation conditions turned to be20.4mg/L, which is2.3times of the expressionin original fermentation conditions(which is9mg/L), and increased70%-580%compared to the expression of rAPC-α which is3-12mg/L in reported documents[54]. In the premise of efficient and stable fermentation, we had an experimentalresearch and analysis to the stability of allophycocyanin from the molecular level andstructure level. In the analysis of its molecular level, we found amino acidsdifferences in14points through matching amino acids sets of two different algaeswhich is Synechocystis sp. PCC6803and Thermosynechococcus elongatus BP-1.Through site-directed mutation, fermentation and hot stability analysis, we obtainedsite-directed mutants (which contains11single-site mutants and3double-sitesmutants) in11different sites, and proved that this11points had a big association withthe stability of allophycocynin, guess this11points participated in the regulation ofstructural stability of the allophycocyanin.In the analysis of its structure level, we had a analysis on the allophycocyaninstability related factors by tracing the entire bio-reaction process. We compared theeffects of different crack synthase, and found that different crack synthase has aremarkably different effect on catalyzing the combination of phycocyanobilin withapoprotein; While through genetic engineering means, we obtained a sole materials ofphycocyanobilin, crack synthase and apoprotein, and then achieved the combinationof phycocyanobilin with apoprotein and the self-assembly of rAPC-(αβ)3in vitro. Wegroped the test conditions of allophycocynin’s catalytic synthesis and self-assembly invitro, and obtained a phycocyanobilin bonding ratio of about90%for both rAPC-αand rAPC-β through catalytic synthesis in vitro, which is respectively3times and6times of the original phycocyanobilin bonding ratio (rAPC-α and rAPC-β for28.6%and14.3%respectively). The raising rate of phycocyanobilin bonding ratio isremarkable. Through3different ways: extracting directly from algae, directexpression, and assembling in vitro after expression, we obtained3kinds ofAPC-(αβ)3which had a difference on phycocyanobilin bonding ratio(phycocyanobilin bonding ratio: natural-APC＞assembled-APC＞directexpressed-APC). And by temperature and pH stability analysis on3APC-(αβ)3, wefound the temperature and pH stability trend of3APC-(αβ)3are all like this:natural-APC＞assembled-APC＞direct expressed-APC, it proved that thephycocyanobilin bonding ratio had a heavily influence on the protein stability, the higher phycocyanobilin bonding ratio, the more complete protein structure, so thebetter protein stability.The study provides a reference for allophhycocynin large scale production, andlays foundation for a in-depth study on the internal structure mechanism and stabilityof allophycocyanin.