Creation of the foldase independent lipase from pseudomonas cepacia using directed evolution

Abstract

A lipase gene, lipA, and a lipase-specific foldase gene, lipB of Pseudomonas cepacia KTCT 2966 (recently reclassified as Burkholderia cepacia) were cloned, sequenced and expressed in Escherichia coli. Enzymatically active lipase was produced only in the presence of the lipase-specific foldase gene. Because the lipase from Pseudomonas cepacia is widely used in industry as catalyst in organic synthesis, there are many attempts to increase lipase productivity. However, much difficulty has been encountered mainly due to its requirements for lipase-specific foldase. To obtain the lipase variants that do not require the lipase-specific foldase for correct folding, random mutations were introduced to the lipase gene by error-prone PCR, and three lipase variants were selected. Three lipase variants, M3, M5 and M7, showed lipase activities in the absence of the lipase-specific foldase, and all of them have a Y219F substitution seemed important for folding of the lipase. In the absence of the foldase, lipase variants, M3, M5 and M7, showed the specific activities of 0.05, 0.07, 0.16 U/mg respectively, while the LipA did not have lipase activity. Interestingly, M7 variant showed the highest lipase activity, however, when it was expressed with LipB, the lipase activity was not noticeably increased. Amino acid substitutions in M7 variant seemed to participate in the folding of lipase to make it active, and the foldase did not cause a significant change in enzyme activity in M7 variant. Therefore, the possibility of creating an active lipase which does not require the lipase-specific foldase is demonstrated.