Engineering thermostability in subtilisin BPN' by in vitro mutagenesis

A procedure has been developed for the isolation and identification of mutants of the bacterial serine protease, subtilisin, which exhibit enhanced thermostability. The cloned subtilisin BPN' gene from Bacillus amyloliquefaciens was treated with a variety of chemical mutagens to introduce random mutations in the coding sequence. Strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermostability were selected by a simple plate assay procedure, which screens for esterase activity on nitrocellulose filters after preincubation at elevated temperatures. The identification and characterization of eight different stabilizing mutations are described. Several mutants containing various combinations of these stabilizing mutations were constructed by oligonucleotide-directed mutagenesis. Combining independent, stabilizing mutations in the same subtilisin molecule has resulted in an approximate multiplicative decrease in the rate of thermal inactivation. In this way, a variant of subtilisin has been constructed which is about 12-fold more stable than wild-type subtilisin, with no radical changes in the tertiary protein structure but rather minor, independent alterations in amino acid sequence. The ultimate goal in these studies is to be able to accurately predict where stabilizing changes can be made in a protein.