Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case

Thermostability is a key feature for commercially attractive variants of th e fungal enzyme phytase. In an initial set of experiments, we restored ioni c interactions and hydrogen bonds on the surface of Aspergillus terreus phy tase, which are present in the homologous but more thermostable enzyme from A. niger. Since these mutations turned out to be neutral, we replaced - in the same region and based on the crystal structure of A. niger phytase - e ntire secondary structure elements. The replacement of one a-helix on the s urface of A. terreus phytase by the corresponding stretch of A. niger phyta se resulted in an enzyme with improved thermostability and unaltered enzyma tic activity. Surprisingly, the thermostability of this hybrid protein was very similar to that of A. niger phytase, although the fusion protein conta ined only a 31 amino acid stretch of the more stable parent enzyme. This re port provides evidence that structure-based chimeric enzymes can be used to exploit the evolutionary information within a sequence alignment. We propo se this method as an alternative to directed enzyme evolution if due to exp ression constraints the screening of large mutant populations is not feasib le. (C) 2001 Elsevier Science B.V. All rights reserved.