Nonadditivity of mutational effects on the properties of catalase I and its application to efficient directed evolution
Nonadditivity of mutational effects on the properties of catalase I and its application to efficient directed evolution
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DOI：
10.1093/protein/11.9.789
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发表时间：
1998-09-01
期刊：
影响因子：
--
通讯作者：
Urabe, I
中科院分区：
文献类型：
--
作者：
Matsuura, T;Yomo, T;Urabe, I
嗜热脂肪芽孢杆菌过氧化氢酶Ⅰ具有高过氧化氢活性和低过氧化氢活性。对来自第一随机突变群体的突变体D130 N进行第二次随机诱变，观察反应特异性的变化，所述突变体D130 N具有比野生型酶表现出的更高的过氧化物活性和更低的过氧化氢活性。从第二个突变体群体中筛选出突变体I108 T/D130 N/I222 T，并对纯化酶的反应特异性进行了检测，结果表明，过氧化氢酶Ⅰ对过氧化物酶的特异性由原来的98%过氧化氢酶和2%过氧化物酶提高到58%。从两个随机突变群体的统计分析，平均程度的非加性的突变效应估计为0.13，而不管酶的性质。结果表明，第二个突变群体的属性的分布模式可以很好地预测从第一个突变群体的数据，考虑到非加性的程度。提出并讨论了在酶的定向进化中，通过预测合适的突变率和有效的样本量进行进一步突变和选择的策略，实现酶的高效自适应步行。
Catalase I of Bacillus stearothermophilus has high catalatic and low peroxidatic activities. The mutant from the first random mutant population, D130N, which has higher peroxidatic and lower catalatic activities than those exhibited by the wild-type enzyme, was subjected to second random mutagenesis in observance of the change in reaction specificity. From the second mutant population, the mutant I108T/D130N/I222T was selected and examined, The reaction specificity of the purified enzymes revealed that catalase I being originally 98% catalase and 2% peroxidase was brought to 58% specificity to peroxidase after two-step adaptive walks. From the statistical analysis of the two random mutant populations, the average degree of nonadditivity of the mutational effects was estimated to be 0.13 irrespective of the properties of the enzyme. It was demonstrated that the distribution pattern of a property of the second mutant population can be predicted well from the data of the first mutant population by taking into consideration the degree of nonadditivity. The strategy for an efficient adaptive walk in directed evolution of enzymes through the prediction of appropriate mutation rate and effective sample size for further mutation and selection was presented and discussed.