Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution

We attempted to apply the directed evolution approach to enhancing enzyme p roperties in the presence of organic solvents, in which enzyme stability an d activity were often drastically reduced. Stability and catalytic activity of phospholipase A(1) in the presence of an organic solvent were enhanced by error-prone polymerase chain reaction (PCR) and DNA shuffling followed b y a filter-based visual screening. Three mutants (SA8, SA17 and SA20) were isolated on indicator plates (i.e., 1% phosphatidylcholine gels containing 30% dimethyl sulfoxide (DMSO)) after a second mutant library was treated in 50% DMSO for 36 h. The half-life values of the three mutants exhibited an approximately 4-fold increase. The three mutants also exhibited increased s tability in all organic solvents tested compared with the wild-type enzyme. Thus, an enzyme variant having superior catalytic efficiency in most of th e organic solvents could be obtained by using any solvent suitable for desi gning the efficient screening system, regardless of the properties of the p articular solvent. (C) 2001 Elsevier Science B.V. All rights reserved.