Expression of the Endoglucanase Gene in Escherichia coli for the Second-generation Bioethanol Production
Keywords:
Bioethanol, Biomass, Endoglucanase, CelluloseAbstract
The endoglucanase enzyme in Escherichia coli was expressed to improve enzymatic activities at low temperature and facilitate large-scale enzyme production. The endoglucanase gene (GenBank CAE51308.1) encoding endoglucanase enzyme (EC 3.2.1.4) was selected from Clostridium thermocellum, a major component in the cellulosome complex and effective in cellulose hydrolysis. The mature endoglucanase gene was cloned into pET28a (+) vector and expressed in E. coli BL21 (DE3) pLysS. SDS-PAGE revealed the molecular weight of recombinant endoglucanase enzyme at around 72 kDa. Carboxymethylcellulose (CMC) was used as a substrate to determine endoglucanase enzyme activity. Under various combinations of pH, temperature and time results showed that the recombinant enzyme reached maximum activity (3.05 U/mL) at pH 10, 40 °C for 10 min. Further study of large-scale enzyme production together with exoglucanase and β-glucosidase enzymes is planned for biomass hydrolysis in second-generation bioethanol production.
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