THERMOALKALOPHILIC LIPASE OF BACILLUS-THERMOCATENULATUS LARGE-SCALE PRODUCTION, PURIFICATION AND PROPERTIES - AGGREGATION BEHAVIOR AND ITS EFFECT ON ACTIVITY

Escherichia coli BL321 was transformed with the expression plasmid pCY TEXP1 carrying the BTL2 gene from Bacillus thermocatenulatus under the control of the strong temperature-inducible lambda pL promoter and wa s cultivated in a 100 1 bioreactor. The mature lipase was produced in large quantities (54 000 U g(-1) wet cells) and further purified to ho mogeneity by a two-step purification protocol (hydrophobic chromatogra phy and gel filtration chromatography). The pure enzyme was characteri zed and its physicochemical properties compared to those of the BTL2 l ipase which had previously been weakly expressed in E. coli under the control of its native promoter on pUC18, yielding 600 U g(-1) wet cell s. The specific activity of the overexpressed enzyme was approx. 5-fol d higher than that of the weakly expressed enzyme. The two proteins sh owed the same pI and N-terminal sequence and had very similar thermost ability, pH stability, optimum pH and temperature activity, and substr ate specificity. Both enzymes were extremely stable in the presence of several organic solvents and detergents. With trioleylglycerol as a s ubstrate, the overexpressed lipase cleaves each of the three ester bon ds. The purified BTL2 lipase shows a strong tendency to aggregate. Dir ect evidence for changes in the aggregation state was obtained by gel filtration chromatography. The effect of aggregation on lipase activit y was strongly dependent on both substrate and temperature during the assay. Under certain conditions, a direct relationship was found betwe en the molecular mass of the lipase aggregates and the increase in act ivity upon the addition of 1% (w/v) sodium cholate. (C) 1997 Elsevier Science B.V.