Cloning And Expression Of Cellulase (EGⅠ) From T. Knoningii In Escherichia Coli

Cellulase is the mainly enzyme to disaggregate cellulose from natrue.T. knoningii is one of microorganism which could produt cellulase,but fungi ferment take long time,and the yield and character of the cellulase are not easy to control.Thus we use gene engineering to cloning and expression of cellulase (EGâ… )cDNA from T.knoningii in E.coli.T.Knoningii need induce to produt cellulose,so by measuring activity of cellulase in medium with different carbon sources in certain time,the effects of different carbon sources on the cellulase production during the fermentation were researched.The results showed that,in a shake flask fermentation using Avicel as the carbon source,directly induce three days,gave the highest activity of cellulase(17.58U/mL with CMCase,11.39 U/mL with P-NPCase,1.68 U/mL with FPAase).The cellulase gene was amplified from total RNA of T.Knoningii which culture in the Avicel that directly inducted by RT-PCR technique.The PCR product was cloned into the expression Vector pET·His which is controlled by T7promoter,and the prokaryotic expression vector pET·His -EGâ… was thus constructed successfully.The reconstructed plasmid was transformed into E.coli BL21(DE3)plysS competent cell.The bacterium was induced by IPTG(0.4mmol/L)for 3 hours and analyzed by SDS-PAGE,approximately 45kDa exogenous protein was observed on the SDS-PAGE. Study on the impurified recombinant enzyme properties,the recombinant enzyme showed the optimal activity at pH 6.0 and 50℃.We also found that the recombinant enzyme was obviously activated by Mn²⁺ion,but was inhibited by Fe³⁺and Cu²⁺.The recombinant enzyme activity was measured to the highest when the consisitency of Mn²⁺was 2mmol/L.