Modulator-mediated synthesis of active lipase of Pseudomonas sp 109 by Escherichia coli cell-free coupled transcription/translation system

Catalytically active lipase was synthesized using Escherichia coli S30 extr act from the signal-deleted lipL gene (rlipL) in the presence of its N-term inal hydrophobic fragment-truncated modulator (rLimL) that was purified fro m the overexpressing E. coli cells. The specific activity of the lipase thu s synthesized was 125 times higher than that of the purified one from Pseud omonas sp. 109. No lipase activity was detected in the absence of rLimL, ev en though the lipase protein itself was synthesized. Active lipase was also produced in vitro by coexpression of rlipL and the modulator gene (rlimL), although a much smaller amount of the lipase was formed. In the absence of rLimL, aggregates of the lipase were formed during its folding process. Th e addition of rLimL proportionally raised both lipase solubility and enzyme activity. An unstable but high activity peak of the lipase was found durin g its folding process.