Escherichia coli BL321 was transformed with the expression plasmid pCYTEXP1 carrying the BTL2 gene from Bacillus thermocatenulatus under the control of the strong temperature-inducible λpL promoter and was cultivated in a 100 1 bioreactor. The mature lipase was produced in large quantities (54000 U g-1 wet cells) and further purified to homogeneity by a two-step purification protocol (hydrophobic chromatography and gel filtration chromatography). The pure enzyme was characterized and its physicochemical properties compared to those of the BTL2 lipase which had previously been weakly expressed in E. coli under the control of its native promoter on pUC18, yielding 600 U g-1 wet cells. The specific activity of the overexpressed enzyme was approx. 5-fold higher than that of the weakly expressed enzyme. The two proteins showed the same pI and N-terminal sequence and had very similar thermostability, pH stability, optimum pH and temperature activity, and substrate specificity. Both enzymes were extremely stable in the presence of several organic solvents and detergents. With trioleylglycerol as a substrate, the overexpressed lipase cleaves each of the three ester bonds. The purified BTL2 lipase shows a strong tendency to aggregate. Direct evidence for changes in the aggregation state was obtained by gel filtration chromatography. The effect of aggregation on lipase activity was strongly dependent on both substrate and temperature during the assay. Under certain conditions, a direct relationship was found between the molecular mass of the lipase aggregates and the increase in activity upon the addition of 1% (w/v) sodium cholate.
Bibliographical noteFunding Information:
Dr M. Luisa Rúa gratefully acknowledges a scholarship from the Spanish Ministry of Education and Science.
- Bacillus thermocatenulatus