Expression system of CotA‐laccase for directed evolution and high‐throughput screenings for the oxidation of high‐redox potential dyes

Abstract

Laccases are useful biocatalysts for many diverse biotechnological applications. In this study we have established efficient and reliable expression systems and high-throughput screenings for the recombinant CotA-laccase from Bacillus subtilis. The expression levels of cotA-laccase were compared in five different Escherichia coli host strains growing in 96-well microtiter plates under different culture conditions. Lower coefficients of variance (around 15%) were achieved using crude cell lysates of BL21 and KRX host strains growing under microaerobic conditions. Reproducible high-throughput screenings for the decolorization of high redox potential azo and anthraquinonic dyes were developed and optimized for identification of variants with increased redox potential. The enzymatic assays developed were tested for the screening of one mutant library from CotA-laccase created by error-prone PCR.

Features and applications of bilirubin oxidases

“…While the ability of different species of laccase to decolorize synthetic dyes is well documented, only a limited number of studies, in CotA BODs (Brissos et al 2009;Durand et al 2012b;) and in fungal BODs (Durand et al 2012a;Liu et al 2009), report the utilization of BODs for such applications. These papers demonstrate how BODs have a high efficiency of decolorizing compounds such as Trypan blue and Remazol brilliant blue R under mild pH conditions.…”

Heterologous laccase production and its role in industrial applications

“…The recombinant expression of Bacillus subtilis CotA in E. coli has allowed its deep characterization, structure solving, and functional evolution. 8,31,32 However, very often the production yield is low, and recombinant enzymes form aggregates difficult to purify. 33 On the other hand, recombinant production of Streptomyces coelicolor laccase (SLAC) in Streptomyces lividans has yielded considerable large amount of laccase (350 mg l -1 ) with high purity.…”

Hyper-Production of Laccase By Pseudomonas putida LUA15.1 through Mutagenesis

“…Thus, bacterial laccases have significantly more potential industrial applications, especially in decolorization of industrial textile dye effluents which constitute a major threat to soil and ground water reservoirs worldwide. Furthermore, bacterial systems are easier to handle than fungal ones [3]. Laccase activity has also been demonstated in a number of bacteria including Bacillus subtilis, Bacillus vallismortis, Bacillus pumilus, Bordetella compestris, Caulobacter crescentus, Escherichia coli, Mycobacterium tuberculosum, Pseudomonas putida, Pseudomonas syringae, Pseudomonas aeruginosa, Yersinia pestis and Geobacillus thermocatenulatus [4][5][6][7][8][9][10][11].…”