Cellobiase is one of the three key cellulases involved in the complete hydrolysis of cellulose residues, thereby resulting in fermentable glucose as the final product for further applications such as biofuel production. In production of cellulases by recombinant technology, an extracellular approach is generally more preferable than a cytoplasmic method. Our laboratory has worked on Cellulomonas biazotea for years and found that this microbe produces high levels of extracellular cellobiases. Among the five identified cellobiases (Cba, Cba2, Cba3, Cba4 and Cba5) of C. biazotea, Cba2 is shown to be the only enzyme possessing a secretion signal, and it has been found to be a major secretory cellobiase in C. biazotea. Therefore, Cba2 may play an important role in cellulose degradat...[ Read more ]

Cellobiase is one of the three key cellulases involved in the complete hydrolysis of cellulose residues, thereby resulting in fermentable glucose as the final product for further applications such as biofuel production. In production of cellulases by recombinant technology, an extracellular approach is generally more preferable than a cytoplasmic method. Our laboratory has worked on Cellulomonas biazotea for years and found that this microbe produces high levels of extracellular cellobiases. Among the five identified cellobiases (Cba, Cba2, Cba3, Cba4 and Cba5) of C. biazotea, Cba2 is shown to be the only enzyme possessing a secretion signal, and it has been found to be a major secretory cellobiase in C. biazotea. Therefore, Cba2 may play an important role in cellulose degradation in C. biazotea.

This thesis summarizes research findings on the cloning of cba2 and recombinant production of Cba2 in Escherichia coli. The cba2 gene was cloned from a genomic library of C. biazotea using a shotgun approach. Initially, neither culture media nor cell lysates of E. coli transformants harbouring the cba2 gene construct (pMcba2) showed to produce any detectable Cba2 activity. Cba2 expression in another host system, Bacillus subtilis encountered the same problem. Results from time-course experiments showed that transformants expressing Cba2 displayed normal cell growth and high plasmid stability. To facilitate immunogenic detection of Cba2, a 6× His-tag was fused at the C-terminus of Cba2. Western blot analysis revealed that Cba2 was expressed in insoluble form in the cytoplasm. Subsequently, chemical chaperone supplement in the culture medium and altered expression condition such as temperature and pH were applied attempting to enhance active Cba2 expression. Some Cba2 activity was detected in the cell lysate upon introduction of polyol osmolytes, particularly sorbitol, in the culture medium of a transformant expressing Cba2. The results obtained from the sorbitol experiment and cba2 cloning are elaborated herein. In addition, the transcriptional efficiency of cba2 is evaluated by northern blot analysis and the effect of low temperature and high pH in improving the soluble Cba2 level is also discussed.