title: 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.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Vânia
authors.initials: V
authors.lastname: Brissos
authors.forename: Luciana
authors.initials: L
authors.lastname: Pereira
authors.forename: Florentina-Daniela
authors.initials: FD
authors.lastname: Munteanu
authors.forename: Artur
authors.initials: A
authors.lastname: Cavaco-Paulo
authors.forename: Lígia O
authors.initials: LO
authors.lastname: Martins
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D000880:Anthraquinones / Q000737:chemistry / Q000378:metabolism; D001391:Azo Compounds / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D052160:Benzothiazoles / Q000737:chemistry / Q000378:metabolism; D004396:Coloring Agents / Q000737:chemistry / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D007700:Kinetics; D042845:Laccase / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation; D010084:Oxidation-Reduction; D013451:Sulfonic Acids / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
chemical_list: D000880:Anthraquinones; D001391:Azo Compounds; D001426:Bacterial Proteins; D052160:Benzothiazoles; D004396:Coloring Agents; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C533003:CotA protein, Bacillus licheniformis; D042845:Laccase