Liao H, McKenzie T, Hageman R
Proc Natl Acad Sci U S A. 1986 Feb;83(3):576-80. doi: 10.1073/pnas.83.3.576.
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.
我们开发了一种快速生成热稳定酶变体的方法。我们的策略是将来自嗜温生物的特定酶的编码基因导入嗜热菌嗜热脂肪芽孢杆菌中。然后选择在嗜热菌较高生长温度下仍保留酶活性的变体。该策略应用于卡那霉素核苷酸转移酶，它赋予对抗生素卡那霉素的抗性。携带野生型酶的嗜热脂肪芽孢杆菌在47摄氏度时对抗生素有抗性，但在55摄氏度及以上则没有。通过选择自发突变体、使穿梭质粒通过大肠杆菌mutD5诱变菌株并通过转化引入嗜热脂肪芽孢杆菌以及在恒化器中培养嗜热菌，获得了在63摄氏度时对卡那霉素有抗性的变体。从这些变体中纯化的卡那霉素核苷酸转移酶都比野生型酶更耐不可逆热失活，并且都具有相同的单氨基酸替换，即第80位的天冬氨酸替换为酪氨酸。通过在70摄氏度下选择卡那霉素抗性，从第一个变体衍生出更耐热的突变体，这些突变体在第130位还带有苏氨酸到赖氨酸的额外变化。该策略适用于在嗜热菌中可选择或可通过平板测定法筛选的其他酶活性。
