HIGH-LEVEL EXPRESSION OF THE THERMOALKALOPHILIC LIPASE FROM BACILLUS-THERMOCATENULATUS IN ESCHERICHIA-COLI

An efficient expression system for the previously only weakly expresse d thermophilic lipase BTL2 (Bacillus thermocatenulatus lipase 2) was d eveloped for the production of large amounts of lipase in Escherichia coli. Therefore, the gene was subcloned in the pCYTEXP1 (pT1) expressi on vector downstream of the temperature-inducible lambda promoter P-L. Three different expression vectors were constructed: (i) pT1-BTL2 con taining the mature lipase gene, (ii) pT1-preBTL2 containing the prelip ase gene and (iii) pT1-OmpABTL2 containing the mature lipase gene fuse d to the signal peptide of the OmpA protein, the major outer membrane protein of E. coli. With pT1-BTL2 and pT1-preBTL2, comparable expressi on levels of 7000-9000 U/g cells were obtained independently of the E. coli host. In contrast, with E. coli JM105 harbouring pT1-OmpABTL2, 6 60000 soluble lipase U/g cells was produced, whereas, with E. coli DH5 alpha and BL321, production levels of 30000 U/g cells were achieved. However, most of the lipase remained insoluble but active after cell b reakage because of the unprocessed OmpA signal peptide. A simple chola te extraction followed by proteinase K cleavage and ultrafiltration al lowed the isolation of 1.15 x 10(6) units of 90% pure mature lipase/we t cells.