Abstract

Reactive Black 5 (RB5) is a typical refractory azo dye. Widespread utilization of RB5 has caused a variety of environmental and health problems. The enzymatic degradation of RB5 can be a promising solution due to its superiority as an eco-friendly and cost-competitive process. Bacterial CotA-laccase shows great application prospect to eliminate hazardous dyes from wastewater. However, efficient decolorization of RB5 CotA-laccase generally requires the participation of costly, toxic mediators. In the present study, we modified the amino acids Thr415 and Thr418 near the type 1 copper site and the amino acid Gln442 at the entrance of the substrate-binding pocket of Bacillus pumilus W3 CotA-laccase to boost its RB5 decolorization activity based on molecular docking analysis and site-saturation mutagenesis. Through the strategies, two double site mutants T415D/Q442A and T418K/Q442A obtained demonstrated 43.94 and 52.64% RB5 decolorization rates in the absence of a mediator at pH 10.0, respectively, which were about 3.70- and 4.43-fold higher compared with the wild-type CotA-laccase. Unexpectedly, the catalytic efficiency of the T418K/Q442A to ABTS was enhanced by 5.33-fold compared with the wild-type CotA-laccase. The mechanisms of conferring enhanced activity to the mutants were proposed by structural analysis. In summary, the mutants T415D/Q442A and T418K/Q442A have good application potentials for the biodegradation of RB5.

Key points

• Three amino acids of CotA-laccase were manipulated by site-saturation mutagenesis.

• Decolorization rate of two mutants to RB5 was enhanced 3.70- and 4.43-fold, respectively.

• The mechanisms of awarding enhanced activity to the mutants were supposed.

中文翻译：

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通过位点饱和诱变提高短小芽孢杆菌W3 CotA-漆酶对活性黑5的脱色活性。

摘要

活性黑5（RB5）是一种典型的难熔偶氮染料。RB5的广泛使用已引起各种环境和健康问题。RB5的酶促降解可能是一个有前途的解决方案，因为它具有环保和成本竞争性的优势。细菌CotA-漆酶在消除废水中有害染料方面具有广阔的应用前景。然而，RB5 CotA-漆酶的有效脱色通常需要昂贵的有毒介体的参与。在本研究中，我们修饰了1型铜位点附近的氨基酸Thr415和Thr418，以及短小芽孢杆菌的底物结合口袋入口处的氨基酸Gln442。基于分子对接分析和位点饱和诱变，W3 CotA-漆酶可增强其RB5脱色活性。通过该策略，获得的两个双位点突变体T415D / Q442A和T418K / Q442A分别在pH 10.0下不存在介体的情况下显示出43.94％和52.64％的RB5脱色率，分别比野生型高约3.70和4.43倍型CotA漆酶。出乎意料的是，与野生型CotA-漆酶相比，T418K / Q442A对ABTS的催化效率提高了5.33倍。通过结构分析提出了赋予突变体增强活性的机制。总之，突变体T415D / Q442A和T418K / Q442A对于RB5的生物降解具有良好的应用潜力。

关键点

•通过位点饱和诱变操作了CotA-漆酶的3个氨基酸。

•两个突变体对RB5的脱色率分别提高了3.70和4.43倍。

•假定了赋予突变体增强活性的机制。