Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents.
A series of variants of kanamycin nucleotidyltransferase (KNTase), isolated previously on the basis of enhanced thermostability by cloning and selection for enzymatic activity in the thermophile Bacillus stearothermophilus, was used to systematically test the hypothesis that thermostable enzymes would also be more resistant to other forms of protein denaturation. The purified KNTases were treated with proteinase K or assayed at 37 degrees C in the presence of urea, N-lauroylsarcosine, Triton X-100, tetrahydrofuran, ethanol, or dimethylformamide. With all these agents, the KNTases displayed increasing resistance to denaturation in the order: wild type, mutant TK9 (with a Thr130-->Lys substitution), TK1 (Asp80-->Tyr), and TK101 (both substitutions). This is the same order in which their thermostability increases, indicating that the structural mechanism(s) whereby the mutations yield enhanced resistance to heat denaturation also yield stabilization towards chemical forms of enzymatic inactivation. These results suggest that selection in thermophiles is a useful method to obtain enzyme variants with increased overall stability, even at nonthermophilic temperatures.
Liao
H H
HH
University of Wisconsin Biotechnology Center, Madison 53705.
D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019286:Endopeptidase K; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D003902:Detergents; D011994:Recombinant Proteins; D012997:Solvents; D014508:Urea; D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase; D012697:Serine Endopeptidases; D019286:Endopeptidase K