THERMOSTABLE MUTANTS OF KANAMYCIN NUCLEOTIDYLTRANSFERASE ARE ALSO MORE STABLE TO PROTEINASE-K, UREA, DETERGENTS, AND WATER-MISCIBLE ORGANIC-SOLVENTS
THERMOSTABLE MUTANTS OF KANAMYCIN NUCLEOTIDYLTRANSFERASE ARE ALSO MORE STABLE TO PROTEINASE-K, UREA, DETERGENTS, AND WATER-MISCIBLE ORGANIC-SOLVENTS
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DOI：
10.1016/0141-0229(93)90151-q
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发表时间：
1993-04-01
影响因子：
3.4
通讯作者：
LIAO, HH
中科院分区：
文献类型：
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作者：
LIAO, HH
先前通过克隆和选择嗜热脂肪芽孢杆菌中的酶活性来增强热稳定性，从而分离出一系列卡那霉素核苷酸转移酶 (KNTase) 变体，用于系统地测试热稳定性酶也能更耐受其他形式的蛋白质变性的假设。纯化的 KNTase 用蛋白酶 K 处理，或在尿素、N-月桂酰肌氨酸、Triton X-100、四氢呋喃、乙醇或二甲基甲酰胺存在下于 37 ℃ 进行测定。对于所有这些试剂，KNTase 按顺序显示出增强的变性抗性：野生型、突变体 TK9（具有 Thr130-->Lys 取代）、TK1（Asp80-->Tyr）和 TK101（两种取代）。这与它们的热稳定性增加的顺序相同，表明突变产生增强的热变性抗性的结构机制也产生对化学形式的酶失活的稳定性。这些结果表明，即使在非嗜热温度下，嗜热菌的选择也是获得具有更高整体稳定性的酶变体的有用方法。
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.