High-Level Expression of Pseudomonas sp. LBC505 Endoglucanase Gene in Escherichia coli

Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUC19 to yield plasmid pLCl. The Pseudomonas sp. LBC505 endoglucanase gene was subcloned in a temperature-regulated Es-cherichia coli expression vector, pAS1, containing the leftward promoter $P_L$ of bacteriophage lambda. The level of gene expression was controlled by the thermal inactivation of the heat-sensitive lambda cI857 repressor. Best yield of endoglucanase was obtained by lowering the incubation temperature to $37^{\circ}C$ after induction at $42^{\circ}C$ for 1h. Under these conditions enzyme production continued for about 5h at a gradually decreasing rate. Ecoli harboring recombinant plasmid pASC10 expressed 4.3 times as much CMCase activity as E.coli containing pLCl. To enhance the expression level of endogl, ucanase gene, we have also changed the presumptive Shine-Dalgamo sequence (AGAGGT) of the gene to consensus sequence (AGGAGGT) by site-directed mutagenesis. The genes mutated were subcloned in pASl resulting in the formation of recombinant plasmid pASS50. E.coli harboring the plasmid pASS50 expressed 6.2-fold higher levels of CMCase activity than that of E.coli harboring pLC1.