Extracellular Expression Of Recombinant CotA Using Type? Secretion System In Escherichia Coli

CotA is located in the outside of the spore with four copper ion binding sites,which is a typical bacterial laccase.CotA could catalyze and oxidate a wide variety of substrates with high temperature,high pH,salt and alkali resistance.It will minimize inclusion bodies and lead to correct protein folding and purification.The study obtained cotA gene from B.amyloliquefaciens strain LC02 through primers designing,PCR,carriers connecting and conversing.CotA gene from LC02 strain contains 1539 bp and 512 amino acids.By analysis of biological software CotA has three conservative copper binding sites with multiple transmembrane regions.The molecular weight of CotA is 58.14 kDa and its isoelectric point is 4.95 as the hydrophilic protein.Recombinant vector of cotA-HlyAs/pET-20b(+)and hlyBD/pACYC-Duet-1 are constructed by double enzyme digestion and its expression strains are obtained by transformed into competent Escherichia coli cells BL21(DE3).CotA from B.amyloliquefaciens strain LC02 would successfully be secreted by cultivating at low temperature and micro oxygen(table static).The best secretory temperature of LC02 strain CotA is 25? and the secretory protein increases rapidly at 20 h and tends to be stable after 38 h.The best concentration of inducer IPTG is 1 mM and the optimal concentration of Cue2+ is 2.2 mM,in which condition the laccase activity of LC02 strain CotA is highest.SDS-PAGE shows a single band of molecular mass of 65 kDa after screening anion exchange chromatography DEAE Sepharose Fast Flow and G75 of Bacillus amyloliquefaciens LC02.The optimal pH of LC02 strain CotA is 4.0 with ABTS as substrates and is 6.7-6.7 with SGZ as substrates.Besides,LC02 strain CotA would maintain high laccase activity under alkaline conditions.The optimal reaction temperature of LC02 strain CotA is respectively 60?and 50? when using ABTS and SGZ as substrates,which proves to possess notable thermal stability as maintaining high activity at high temperature.Mg2+ facilitate the activity but Hg2+has inhibitions to the enzyme of LC02 strain CotA.Moreover,low concentration of organic solvent has weak inhibitory effect to the enzyme and high concentration demonstrate strong inhibitory effect.