Screening for thermostable mutant of kanamycin nucleotidyltransferase by the use of a transformation system for a thermophile, Bacillus stearothermophilus.
A structural gene of kanamycin nucleotidyltransferase cloned into a single-stranded bacteriophage M13 was subjected to mutagenesis with hydroxylamine. Having recloned the mutagenized gene of the enzyme in a vector plasmid pTB922, the recombinant plasmid was used to transform Bacillus stearothermophilus with a purpose of screening for the more thermostable enzyme than the wild type. Out of greater than 8 X 10(3) transformants, 12 clones that were suspected to harbor the mutant gene encoding the more thermostable enzyme were isolated by shifting from a permissive (55 degrees C) to a nonpermissive (61 degrees C) temperature that inactivates the wild-type enzyme. DNA sequence analysis of the mutant genes revealed two types of mutation of single base substitution and hence a single amino acid replacement. The first type was the replacement of an aspartate by a tyrosine at position 80 of the wild-type enzyme, while the second was that of a threonine by a lysine at position 130. Purified enzymes from the two mutant genes were confirmed to be substantially more thermostable than the wild type in vitro. The method of screening for a thermostable kanamycin nucleotidyltransferase presented here could be applied to any other enzyme, if a transformation system of a thermophile were available. Indeed, thermostable mutants with a subtle amino acid change would be of value for better understanding of forces and interactions that contribute to the stability of a protein.
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005796:Genes; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics*; D013696:Temperature; D014170:Transformation, Genetic
D006898:Hydroxylamines; D019811:Hydroxylamine; D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase