THERMOSTABILIZATION OF FIREFLY LUCIFERASE BY A SINGLE AMINO-ACID SUBSTITUTION AT POSITION-217
THERMOSTABILIZATION OF FIREFLY LUCIFERASE BY A SINGLE AMINO-ACID SUBSTITUTION AT POSITION-217
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DOI：
10.1021/bi00213a007
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发表时间：
1993-12-21
期刊：
影响因子：
2.9
通讯作者：
NAKANO, E
中科院分区：
文献类型：
--
作者：
KAJIYAMA, N;NAKANO, E
通过羟胺诱导来自“源氏”萤火虫（Luciola cruciata）的荧光素酶 cDNA 的随机诱变，试图分离耐热突变体。分离出三个突变体，并对编码这些蛋白质的 cDNA 进行了测序。所有突变体 cDNA 均携带相同的 C 到 T 转换突变，该突变导致第 217 位的 Thr 氨基酸被 Ile 取代。将野生型荧光素酶和热稳定变体 (Thr217Ile) 纯化至均质，并测定了它们的酶性质。 Thr2l7Ile 在热稳定性和 pH 稳定性方面优于野生型。此外，Thr217Ile突变体的比活性增加至野生型的130%。为了考察217位氨基酸残基取代对荧光素酶热稳定性的影响，我们通过定点诱变将217位的Thr残基替换为所有其余可能的氨基酸残基。这些取代突变体的热稳定性似乎与取代的氨基酸残基的疏水性相关。
Random mutagenesis of the luciferase cDNA from ''Genji'' firefly, Luciola cruciata, was induced by hydroxylamine in an attempt to isolate thermostable mutants. Three mutants were isolated, and the cDNAs encoding these proteins were sequenced. All mutant cDNAs carried the same C to T transition mutation that conferred an amino acid substitution of Thr by Ile at position 217. The wild-type luciferase and the thermostable variant (Thr2l7Ile) were purified to homogeneity, and their enzymatic properties were determined. Thr2l7Ile was superior to wild-type in thermal and pH stability. Furthermore, the specific activity of the Thr2l7Ile mutant was increased to 130% of that of the wild-type. In order to examine the effect of amino acid residue substitution at position 217 on the thermostability of luciferase, we replaced the Thr residue at position 217 with all of the rest of the possible amino acid residues by site-directed mutagenesis. The thermostability of these substitution mutants seemed to correlate with the hydrophobicity of the substituted amino acid residue.