Search

An efficient expression system for the previously only weakly expressed thermophilic lipase BTL2 (Bacillus thermocatenulatus lipase 2) was developed for the production of large amounts of lipase in Escherichia coli. Therefore, the gene was subcloned in the pCYT-EXP1 (pT1) expression vector downstream of the temperature-inducible lambda promoter PL. Three different expression vectors were constructed: (i) pT1-BTL2 containing the mature lipase gene, (ii) pT1-preBTL2 containing the prelipase gene and (iii) pT1-OmpABTL2 containing the mature lipase gene fused to the signal peptide of the OmpA protein, the major outer membrane protein of E. coli. With pT1-BTL2 and pT1-preBTL2, comparable expression levels of 7000-9000 U/g cells were obtained independently of the E. coli host. In contrast, with E. coli JM105 harbouring pT1-OmpABTL2, 660,000 soluble lipase U/g cells was produced, whereas, with E. coli DH5 alpha and BL321, production levels of 30,000 U/g cells were achieved. However, most of the lipase remained insoluble but active after cell breakage because of the unprocessed OmpA signal peptide. A simple cholate extraction followed by proteinase K cleavage and ultrafiltration allowed the isolation of 1.15 x 10(6) units of 90% pure mature lipase/wet cells.

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 lambda pL promoter and was 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 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.

An expression library was generated by partial Sau3A digestion of genomic DNA from the thermophile Bacillus thermocatenulatus and cloning of DNA fragments in pUC18 in Escherichia coli DH5alpha. Screening for lipase activity identified a 4.5 kb insert in pUC18 which directed the production of lipase in E. coli DH5alpha. A subclone with a 2.2 kb insert was sequenced. The lipase gene codes for a mature lipase of 388 amino acid residues, corresponding to a molecular weight of 43 kDa. As in other Bacillus lipases, an Ala replaces the first Gly in the conserved pentapeptide Gly-X-Ser-X-Gly found in most lipases. The region upstream of the lipase gene contains a Bacillus promoter which directs the expression of lipase in E. coli DH5alpha. The expressed lipase was isolated and purified 312-fold to homogeneity. N-terminal sequencing of the purified lipase revealed a correct cleavage of the preprotein in E. coli DH5alpha. Maximum activity was found at pH 8.0-9.0 with tributyrin and olive oil as substrates and at 60-70 degrees C with p-NPP and olive oil as substrates. The lipase showed high stability at pH 9.0-11.0 and towards various detergents and organic solvents.