Isolation of Thermostable Enzyme Mutants by Cloning and Selection in ThermophilicBacteria

摘要

The objective of this project was to isolate thermostable variants of enzymes andto understand the molecular basis for their increased stability. Such variants may be isolated by introducing the gene encoding the enzyme into a thermophile and selecting for enzymatic activity at the high growth temperatures of these organisms. Thermostable mutants of kanamycin nucleotidyltransferase (KNTase) were obtained in this way and found to be also more resistant to chemical denaturants, to protease, and to inclusion body formation during their overexpression in Escherichia coli. The tools for obtaining mutants at up to 80 deg C by cloning in Thermus thermophilus, including a plasmid that transforms and expresses heterologous genes in this thermophile, were constructed, and more thermostable mutants of KNTase were isolated. Crystals of a thermostable KNTase were obtained and the structure was solved to 2.7A; comparison with the wildtype structure will allow correlation of the known genetic change with the structural basis for enhanced stability. Work was also begun on isolating thermostable mutants of another enzyme, chloramphenicol acetyltransferase. Thermostability, Thermophiles, Kanamycin nucleotidyltransferase.