In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor

• The 'detergent lipase' Lipolasel((R)), from Thermomyces lanuginosa was subjected to a combinatorial protein engineering/phage display approach with the aim of identifying new enzyme variants with improved characteristics in the presence of detergents. First it was demonstrated that wild-type Lipolase((R)) could be produced in Escherichia coli retaining full activity and be displayed as an active enzyme fused to coat protein 3 on E. coli phage M13. A phagemid library designed to result in approximately two to three mutations per lipase gene was then constructed. Nine amino acids located in two regions close to the active site were targeted for randomization. Selections using a mechanism-based biotinylated inhibitor showed that phages displaying Lipolase((R)) could be specifically enriched from a population of control phages. Selections on a library phage stock in the presence of inhibitor and a commercial powder detergent resulted in a step-wise increase in the proportion of active clones. Analysis of 84 active clones revealed that they all expressed lipase activity, but with lower activities than that of a wild-type Lipolase((R))-producing clone. In six of the seven most active clones a wild-type serine at position 83 had been replaced by threonine, a substitution known to alter the substrate chain length preference of Lipolase((R)) variants. Furthermore, the selection had enriched enzyme variants with a high degree of conservatism in one of the variegated regions, suggesting that this region is important for enzymatic activity and that the designed selection procedure was relevant. The selected variants contained primarily basic amino acid residues within the other variegated region. Taken together, the described results show that selection protocols based on enzymatic activity can be designed for this enzyme class which should be of importance for future protein engineering attempts.

Nyckelord

phage display

directed evolution

library

lipolase

affinity fusion

Escherichia coli enzyme

Thermomyces lanuginosa

catalytic antibodies

lanuginosa lipase

display

bacteriophage

proteins

sites

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