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

¹ Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Madrid, Spain.
² Department of Chemical Engineering, University of California, Los Angeles.
³ Centro Nacional de Biotecnologia CSIC-UAM, Madrid, Spain.

^* To whom correspondence should be addressed. E-mail: malcalde{at}icp.csic.es.

	Abstract

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

First published on August 15, 2005, doi:10.1177/1087057105277058

Journal of Biomolecular Screening 2005;10:624.

A more recent version of this article appeared on September 1, 2005


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