Energy consumption and its impact on the environment is one of the greatest human challenges and the production of bioethanol or other chemicals by cellulose enzyme hydrolysis can have an important effect in replacing fossil fuels and reducing of CO₂ production in the atmosphere. The Cel6B enzyme from Thermobifidia fusca, a thermal actinomist, a CBHII, belongs to the family of cellulases B, which is highly resistant to heat. Due to the low level of production of this enzyme in the primary host for industrial use, it must be optimized in systems such as bacteria. In this research, specific primers were used to confirm the presence of Cel6B gene in PSZ143 vector. Then, the PSZ143 vector containing the Cel6B gene was transferred by heat shock to E. coli BL21 (DE3) competent cells. After protein extraction, expression of a protein with a molecular weight of 59.6 KDa was confirmed using SDS-PAGE. Optimization results of protein production showed that the best time and best concentration of IPTG for induction and expression of the protein in E. coli was obtained 6 hours after induction and 0.6 mM, respectively. Also, cellobiohydrolase (CBH) activity on Phosphoric acid pretreated cotton (PC) substrate in lysed bacteria, was investigated by DNS method. The enzyme activity of cel6B in cellulose hydrolysis, in bacterial lysis and its culture media was obtained after IPTG induction of 0.8 mM, 1.21 and 0.07 U(mmol/min)/ml, respectively. Finally It has been shown that the expression of the cel6B protein is low due to the low CAI (Codon Adaptation Index) and the difference in codons preferred by E. coli. Also, by checking the native signal sequence of the cel6B gene in the Signal P4.1 server, the inability to secrete this protein in E. coli was proven.

Keywords: Bacteria, Cellobiohydrolase (CBH), Induction of protein expression, Cellulose

Full-Text [PDF 804 kb]   (222 Downloads)