The Induction, Purification And Characterization Of Protein Cota From Escherichia Coil

CotA laccase is important for its applications in industries such as environmental protection, food industry, and paper biobleaching.Expression vector pET-22b(+)/cotA containing gene cotA which was constructed and preserved from the microbe laboratory of Northeast Forestry University was transferred into Escherichia coli BL21(DE3). The effects of induction expression conditions and the fermentation medium on the production of CotA laccase by recombinant E. coli were investigated using one factor method and orthogonal design. The expressed CotA proteins were released from periplasmic space of the expressed strain by osmotic pressure breaking method. Finding out the main characterizations of the purifted CotA laccase and degradation on dyes.(1) The strain was cultured first under the following conditions:initial pH7.5,150mL culture medium per500mL flask,0.6mmol/L copper ions,1g/L glucose as carbon source,15g/L tryptone and2g/L ammol/Lonium sulfate as nitrogen source, inoculum concentration3%, temperature37℃and shaking speed180r/min. IPTG was added to the culture at1.0mmol/L when the OD600nm of culture reached1.0. After12h cultivation at25℃,the highest laccase yield was obtained. The laccase activity of the extraction of the fermentation broth after optimization (3526U/mL) was2.96times than that before optimization (only1190U/mL).(2) CotA laccase was separated and purified from recombinant Escherichia coli BL21(DE3) by a series of operating approachs. The purified recombinant laccase were detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The purified CotA laccase was a monomer, and the molecular weight was67.5kDa. The recombinant laccase was obtained with an overall yield of27.83%and purification fold was5.26. The CotA laccase was stained red by syringaldazine after Native-PAGE.(3) The purified enzyme showed a similar behaviour to the native laccase produced by Bacillus subtilis WD23. Using syringaldazine as the substrate to determine the CotA laccase activity, the optimum temperature was45℃and the optimum pH was7.2. The temperature half-life of the CotA laccases was0.5h at80℃. The pH half-life was8h at pH9.0. Ca2+and Cu2+stimulate the laccase activity, while The CotA laccase was strongly inhibited by EDTA and Zn2+, and K+、Na+and Fe2+make seldom difference of it.(4) When the substrate of CotA is syringaldazine, the Km is1.10mmol/L.The laccase CotA could efficiently decolorize anthraquinone and azo dyes. CotA laccase could decolorized different dyes (Remazol brilliant blue R、Congo red、Isatin and Crystal Violet) within12h, and the decolorizing rate were93.15%,90.03%,59.92%and55.14%, respectively. This degradation potential increased the applicability of this engineering bacteria for the dye removal.