Sunday, April 29, 2007

A sure deductive method to improve the thermostability and activity of an enzyme

Zhizhuang Xiao, Hélène Bergeron, Stephan Grosse, and Peter C. K. Lau. Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, QC H4P 2R2, Canada

Engineering proteins to withstand a broad range of conditions is still a coveted objective, holding the potential to advance applications in both health and environment sectors. Rational protein engineering based on knowledge of the 3D structure has successfully increased thermostability of many enzymes. However, the often-disastrous effects of 'rationally introduced' mutations on the stability and activity of proteins has prompted Charles Craik to muse that 'protein terrorism' was a more suitable descriptor than protein engineering. Directed evolution uses irrational approaches such as random mutagenesis and DNA shuffling to arrive at a desired characteristic in the absence of knowledge about the 3D protein structure. However, directed evolution is limited by the number of isolates that can be analyzed, and how the selected property is quantified. We introduce a simple deductive method to rapidly predict the amino acids involved in protein thermostability based on sequence alignment. Pectate lyase is an enzyme that has been used in the production of high quality fibre for textile and paper-making industries. Analysis of molecular dissection indicated that 70% of the predicted residues in a Xanthomonas campestris pectate lyase notably affect its thermostability or catalytic activity. Replacement of the identified amino acids with those highly conserved in thermo-stable counterparts significantly improves the thermostability and activity of this pectate lyase. In terms of success rate in the number of clones that are needed to be screened, this new method appears to be a lot more efficient than other conventional methods.