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 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 Cu 2+. Copper concentration was limited up to 10 µM CuSO 4 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. (Journal of Biomolecular Screening 2005:624-631)

Heterologous laccase production and its role in industrial applications

“…Directed evolution followed by saturation mutagenesis rendered laccases able to tolerate high concentration of organic cosolvents. [68][69][70][71][72] A higher laccase production and an increased thermal stability have been obtained in P. pastoris through mutagenesis with low-energy nitrogen ion implantation. 101 Random mutagenesis through ethyl methane sulfonate-based (EMS) technique has improved laccase production up to 144 mg l -1 in P. pastoris.…”

Laccases and Their Applications: A Patent Review

“…In a recent investigation directed molecular evolution of laccases was carried out for organic cosolvents resistance [333]. The M. thermophila laccase expressed in S.…”

Nanobiocatalysts for biofuel cells and biosensor systems

“…It has been successfully expressed in yeasts for directed evolution [42]. Using in vivo the recombination approach, the thermostability of versatile (VL) and high redox potential laccases (HRPL) was improved [39].…”