A 4.7-kb DNA PstI partially restricted insert encoding a secretory cellobiase (Cba) was isolated from Cellulomonas biazotea and cloned into Escherichia coli using an excretion vector, pM, forming a recombinant construct, pBZ4.7. Using an E. coli JMlO 1 (pBZ4.7) transformant, extracellular production of the recombinant Cba. of which part the activity was detected in periplasm and part even in supernatant was demonstrated. Previous results from the purification of the excreted Cba revealed that the Cba had a molecular mass of about 86 kDa, suggesting the size of the encoding gene, designated cba, would be about 2.7 kb in length....[ Read more ]

A 4.7-kb DNA PstI partially restricted insert encoding a secretory cellobiase (Cba) was isolated from Cellulomonas biazotea and cloned into Escherichia coli using an excretion vector, pM, forming a recombinant construct, pBZ4.7. Using an E. coli JMlO 1 (pBZ4.7) transformant, extracellular production of the recombinant Cba. of which part the activity was detected in periplasm and part even in supernatant was demonstrated. Previous results from the purification of the excreted Cba revealed that the Cba had a molecular mass of about 86 kDa, suggesting the size of the encoding gene, designated cba, would be about 2.7 kb in length.

DNA sequencing revealed that the cba gene contained an "ATG" start codon 327 nucleotides away from the first PstI site of the insert and an open-reading frame of 2,484 bp, which was terminated by "TGA" stop codon identified at 2814 nucleotide. The gene has a high G + C content of 76.4%. A putative ribosomal-binding site is identified a few bases upstream of the start codon. Three pairs of potential transcriptional termination signals are situated downstream of the stop codon. Comparison of the deduced protein sequence with other β-glucosidases revealed that there was a consensus region which might be the potential active site. The Asp²²⁸ is found to be conserved in all the β-glucosidases compared.

The cba gene is fused precisely to the ompA leader sequence in the pM vector under the influence of the tat promoter and IPTG induction to see whether the excretion of Cba could be enhanced. As a result of the lethal effect probably caused by both IPTG induction and the large size of the protein, the findings showed that there was an increase in the levels of excreted Cba at or before 2 hours but not at the later time points after induction.

The cba gene was cloned into Saccharomyces cerevisiae using the yeast expression vector, pMV2Adel to form a recombinant construct p43. Results from the expression of the yeast transformant harbouring p43 showed that the recombinant Cba was intracellularly borne. The finding could result from the presence of the homologous signal peptide of the Cba, which interfered the secretion of the Cba mediated by the signal peptide of the yeast α-galactosidase enzyme.