Isolation of a Bacillus stearothermophilus mutant exhibiting increased thermostability in its restriction endonuclease.
A procedure was developed for the selection of spontaneous mutants of Bacillus stearothermophilus NUB31 that are more efficient than the wild type in the restriction of phage at elevated temperatures. Inactivation studies revealed that two mutants contained a more thermostable restriction enzyme and one mutant contained three times more enzyme than the wild type. The restriction endonucleases from the wild type and one of the mutants were purified to apparent homogeneity. The mutant enzyme was more thermostable than the wild-type enzyme. The subunit molecular weight, amino acid composition, N-terminal and C-terminal amino acid residues, tryptic peptide map, and catalytic properties of the two enzymes were determined. The two enzymes have similar catalytic properties, but the molecular size of the mutant enzyme is approximately 6 to 7 kilodaltons larger than that of the wild-type enzyme. The mutant enzyme contains 54 additional amino acid residues, of which 26 to 28 are aspartate/asparagine, 8 to 15 are glutamate/glutamine, and 8 to 9 are tyrosine residues. The two enzymes contained similar amounts of the other amino acids, identical N-terminal residues, and different C-terminal residues. Tryptic peptide analyses revealed a high degree of homology between the two enzymes. The increased thermostability observed in the mutant enzyme appears to have been achieved by a mutation that resulted in the addition of amino acid residues to the wild-type enzyme. A number of mechanisms are discussed that could account for the observed difference between the mutant and wild-type enzymes.
Hendrix
J D
JD
Welker
N E
NE
D004262:DNA Restriction Enzymes / Q000235:genetics*; D001411:Geobacillus stearothermophilus / Q000235:genetics*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis
D010446:Peptide Fragments; D004262:DNA Restriction Enzymes