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首页> 外文期刊>Journal of Biotechnology >Implication of a mutation in the flavin binding site on the specific activity and substrate specificity of glycine oxidase from Bacillus subtilis produced by directed evolution
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Implication of a mutation in the flavin binding site on the specific activity and substrate specificity of glycine oxidase from Bacillus subtilis produced by directed evolution

机译:黄素结合位点突变对定向进化产生的枯草芽孢杆菌甘氨酸氧化酶比活性和底物特异性的影响

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摘要

Directed evolution was used to expand the substrate specificity and functionality of glycine oxidase by using a high-throughput screening assay based on the 4-aminoantipyrine peroxidase system, with a coefficient of variance below 4%. After screening the library, one mutant with the desired changes was found. The mutant was purified and characterized, showing important changes compared to the wild-type, especially towards cyclic d-amino acids. Amino acid substitution of Ile15 for Val, where the consensus sequence for flavin binding site is placed, seems to be responsible for these changes in specific activity and substrate specificity. The effect of this mutation was explained by using a computer-based three-dimensional model.
机译:通过基于4-氨基安替比林过氧化物酶系统的高通量筛选测定,使用定向进化来扩展甘氨酸氧化酶的底物特异性和功能,方差系数低于4%。筛选文库后,发现了一个具有所需变化的突变体。对该突变体进行了纯化和鉴定,与野生型相比,显示出重要的变化,特别是对环状d-氨基酸。 Ile15被Val取代,在其中黄素结合位点的共有序列被氨基酸取代,这似乎是这些比活性和底物特异性变化的原因。通过使用基于计算机的三维模型解释了此突变的影响。

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