Prolyl endopeptidase is the only endopeptidase that specifically cleaves peptides at proline residues. Although this unique specificity is advantageous for application in protein chemistry, the stability of the enzyme is lower than those of commonly used peptidases such as subtilisin and trypsin. Therefore, we attempted to apply a directed evolution system to improve the thermostability of the enzyme. First, an efficient expression system for the enzyme in Escherichia coli was established using the prolyl endopeptidase gene from Flavobacterium meningosepticum. Then, a method for screening thermostable variants was developed by combining heat treatment with active staining on membrane filters. Random mutagenesis by error-prone PCR and screening was repeated three times, and as a result the thermostability of the enzyme was increased step by step as the amino acid substitutions accumulated. The most thermostable mutant obtained after the third cycle, PEP-407, showed a half-life of 42 min at 60 degrees C, which was 60 times longer than that of the wild-type enzyme. The thermostable mutant was also more stable with a high concentration of glycerol, which is a necessary condition for in