期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1986
卷号：83
期号：3
页码：576-580
DOI：10.1073/pnas.83.3.576
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We developed a method for rapidly generating thermostable enzyme variants. Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus. Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected. This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the antibiotic kanamycin. B. stearothermophilus carrying the wild-type enzyme is resistant to the antibiotic at 47 degrees C but not at 55 degrees C and above. Variants that were kanamycin resistant at 63 degrees C were obtained by selection of spontaneous mutants, by passage of a shuttle plasmid through the Escherichia coli mutD5 mutator strain and introduction into B. stearothermophilus by transformation, and by growing the thermophile in a chemostat. The kanamycin nucleotidyltransferases purified from these variants were all more resistant to irreversible thermal inactivation than is the wild-type enzyme, and all have the same single amino acid replacement, aspartate to tyrosine at position 80. Mutants that are even more heat stable were derived from the first variant by selecting for kanamycin resistance at 70 degrees C, and these carry the additional change of threonine to lysine at position 130. This strategy is applicable to other enzymatic activities that are selectable in thermophiles or that can be screened for by plate assays.
