title: Screening mutant libraries of fungal laccases in the presence of organic solvents.
abstract: Reliable screening methods are being demanded by biocatalysts' engineers, especially when some features such as activity or stability are targets to improve under non-natural conditions (i.e., in the presence of organic solvents). The current work describes a protocol for the design of a fungal laccase-expressed in Saccharomyces cerevisiae-highly active in organic cosolvents. A high-throughput screening assay based on ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) oxidation was validated. The stability of the ABTS radical cation was not significantly altered in the presence of acetonitrile, ethanol, or DMSO. With a coefficient of variance below 10% and a sensitivity limit of 15 pg laccase/microL, the assay was reproducible and sensitive. The expression system of Myceliophthora thermophila laccase variant T2 in S. cerevisiae was highly dependent on the presence of Cu2+. Copper concentration was limited up to 10 microM CuSO4 where expression levels (approximately 14-18 mg/L) were acceptable without compromising the reliability of the assay. A mutant library was created by error-prone PCR with 1.1 to 3.5 mutations per kb. After only 1 generation of directed evolution, mutant 6C9 displayed about 3.5-fold higher activities than parent type in the presence of 20% acetonitrile or 30% ethanol. The method provided here should be generally useful to improve the activity of other redox enzymes in mixtures of water/cosolvents.
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain. malcalde@icp.csic.es
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
authors.forename: Thomas
authors.initials: T
authors.lastname: Bulter
authors.forename: Miren
authors.initials: M
authors.lastname: Zumárraga
authors.forename: Humberto
authors.initials: H
authors.lastname: García-Arellano
authors.forename: Mario
authors.initials: M
authors.lastname: Mencía
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Plou
authors.forename: Antonio
authors.initials: A
authors.lastname: Ballesteros
mesh_terms: D000097:Acetonitriles / Q000494:pharmacology; D052160:Benzothiazoles; D002412:Cations / Q000737:chemistry; D002625:Chemistry, Organic / Q000379:methods; D002626:Chemistry, Pharmaceutical; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D019020:Directed Molecular Evolution; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D000431:Ethanol / Q000737:chemistry / Q000494:pharmacology; D015723:Gene Library; D005800:Genes, Fungal; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012997:Solvents / Q000737:chemistry*; D013451:Sulfonic Acids / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D052160:Benzothiazoles; D002412:Cations; D012997:Solvents; D013451:Sulfonic Acids; D014867:Water; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D000431:Ethanol; D042845:Laccase; D004121:Dimethyl Sulfoxide; C032159:acetonitrile