This Article Full Text (PDF) All Versions of this Article: 1087057105277058v1 10/6/624    most recent References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Web of Science (10) Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Alcalde, M. Articles by Ballesteros, A. Search for Related Content PubMed PubMed Citation Articles by Alcalde, M. Articles by Ballesteros, A. Social Bookmarking                         What's this?

Screening Mutant Libraries of Fungal Laccases in the Presence of Organic Solvents

Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Madrid, Spain

Thomas Bulter

Department of Chemical Engineering, University of California, Los Angeles

Miren Zumárraga

Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Madrid, Spain

Humberto García-Arellano

Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Madrid, Spain

Mario Mencía

Centro Nacional de Biotecnologia CSIC-UAM, Madrid, Spain

Francisco J. Plou

Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Madrid, Spain

Antonio Ballesteros

Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Madrid, Spain

Reliable screening methods are being demanded by biocatalysts’ engineers, especially when some features such as activity or stability are targets to improve under nonnatural 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/µL, 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 µM CuSO₄ where expression levels (~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.

Key Words: laccase • directed molecular evolution • high-throughput screening • organic solvents • Saccharomyces cerevisiae

This version was published on September 1, 2005

Journal of Biomolecular Screening, Vol. 10, No. 6, 624-631 (2005)
DOI: 10.1177/1087057105277058


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?