Evolutionary molecular engineering by random elongation mutagenesis

We describe a new method of random mutagenesis that employs the addition of peptide tails with random sequences to the C-terminal of enzyme molecules. A mutant population of catalase I from Bacillus stearothermophilus prepare d by this method has a diversity in thermostability and enzyme activity equ al to that obtained after random point mutagenesis. When a triple mutant of catalase (1108T/D130N/I222T)-the thermostability of which is much lower th an that of the wild type-was subjected to random elongation mutagenesis, we generated a mutant population containing only mutants with higher thermost ability than the triple mutant. Some had an even higher stability than the wild-type enzyme, whose thermostability is considered to be optimized. Thes e results indicate that peptide addition expands the protein sequence space resulting in a new fitness landscape. The enzyme can then move along the r outes of the new landscape until it reaches a new optimum. The combination of random elongation mutagenesis with random point mutagenesis should be a useful approach to the in vitro evolution of proteins with new properties.