A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation.
A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation.
复制标题
辅因子消耗筛选确定了有前景的 NfsB 家族硝基还原酶用于二硝基甲苯修复。
DOI：
10.1007/s10529-019-02716-z
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发表时间：
2019
影响因子：
2.7
通讯作者：
Williams EM
中科院分区：
文献类型：
--
作者：
Williams EM
ObjectivesTo survey a library of overexpressed nitroreductases to identify those most active with 2，4- and 2，6-dinitrotoluene substrates，as promising candidates for phytoremediation of soil and underground pollutenitol pollutants.ResultsTo indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to comparison relative rates of NADPH consumption for 58 nitroreductase candidates，在一个硝基还原酶缺失的大肠杆菌菌株中过表达。尽管筛选仅提供单一底物浓度下的活性数据，但通过改变底物浓度和孵育时间，我们表明我们可以首先区分活性较高和活性较低的酶，然后区分集合中最具活性的硝基还原酶所表现出的相对还原率。我们观察到NfsA和NfsB硝基还原酶家族的成员对2，4-二硝基甲苯最有活性，但只有NfsB家族的成员有效地还原2，6-二硝基甲苯。两个NfsB家族成员，来自枯草芽孢杆菌的YfkO和来自创伤弧菌的NfsB，似乎对这些底物特别有效。两种酶的纯化His 6标记的重组蛋白，使在体外测定的Michaelis-Menten动力学参数与每个二硝基甲苯substrate.ConclusionsVibrio创伤弧菌NfsB是一个特别有前途的候选人的生物修复应用，是ca。与基准E相比，对2，4-二硝基甲苯的催化效率高5倍，对2，6-二硝基甲苯的活性高2,6倍以上。大肠杆菌硝基还原酶NfsA和NfsB。
ObjectivesTo survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants.ResultsTo indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain ofEscherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO fromBacillus subtilisand NfsB fromVibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His6-tagged recombinant proteins enabled in vitro determination of Michaelis–Menten kinetic parameters with each dinitrotoluene substrate.ConclusionsVibrio vulnificusNfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmarkE. colinitroreductases NfsA and NfsB.