Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagen
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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis Hui Ma 1 & Kai-Zhong Xu 1 & Ya-Jing Wang 1 & Na Yan 1 & Xiang-Ru Liao 1 & Zheng-Bing Guan 1 Received: 23 June 2020 / Revised: 2 September 2020 / Accepted: 7 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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 costcompetitive 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. Keywords CotA-laccase . Bacillus pumilus . Site-saturation mutagenesis . Reactive Black 5 . Dye decolorization
Introduction Azo dyes (–N=N–) are synthetic organic compounds which were widely used in textile dyeing, paper making, and food manufacturing (Gao et al. 2015). Since synthetic dyes are designed to have photolytic stability and resistance to major oxidants, their removal from wastewater is rather difficult (Wang Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10897-1) contains supplementary material, which is available to authorized users. * Zheng-Bing Guan [email protected] 1
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People’s Republic of China
et al. 2017). In addition, a large number of azo dyes and their decomposition products are toxic and/or mutagenic. It will cause
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