Rational design of trypsin variants with improved synthetic properties

摘要

The narrow substrate specificity of proteases generally limits the choice of amino acids between which a peptide bond can be synthesized.While for the carboxyl components the restricted enzyme specificity can be overcome by the use of substrate mimetics,no universal approach exists for broadening the specificity of the biocatalyst towards the amino component.In the present contribution we describe the suitability of site-directed mutagenesis to improve the binding of originally less specific amino components to the enzyme's active site,which was found to be crucial for efficient synthesis.Starting from the trypsin variant K60E,D189K,which is already known as a promising catalyst with reduced proteolytic activity,further mutations were introduced forming an artificial metal binding site within the S' subsite of the biocatalyst.The resulting enzyme variant shows a significantly altered specificity towards the amino component.Especially peptide sequences for which the wild-type enzyme is originally rather non-specific are efficiently recognized by the optimized trypsin variant ester hydrolysis studies using a library of substrate mimetics could further prove a high activity of the variant towards peptide 4-guanidinophenyl esters enabling its use as coupling reagent for peptide synthesis.Model peptide ligation reactions finally verified the improved acceptance of originally non-efficient peptide sequences resulting in an increase of product yields from about 20%(wild-type enzyme)up to nearly 100%(enzyme variant).