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Rapid method for screening enoate reductase activity in yeasts.

Stefano Raimondi¹, Lucia Roncaglia, Alberto Amaretti

¹Department of Chemistry, University of Modena and Reggio Emilia, Modena, Italy.

Journal of Microbiological Methods

|September 21, 2010

Summary

A new method screens yeasts for high Old Yellow Enzyme (OYE) activity by measuring their resistance to acrolein. This rapid technique identifies strains with significant enoate reductase capabilities for biocatalysis.

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Area of Science:

Biochemistry
Biotechnology
Enzymology

Background:

Old Yellow Enzymes (OYEs) are flavin-dependent oxidoreductases catalyzing alkene reduction.
OYEs play a role in detoxifying harmful compounds like acrolein.

Purpose of the Study:

To develop a high-throughput screening method for identifying yeasts with high OYE (enoate reductase) activity.
To correlate yeast growth resistance to acrolein with OYE activity.

Main Methods:

A screening method measuring yeast growth in acrolein-supplemented 96-well microtiter plates.
Development of an Acrolein Resistance Factor (ARF) to quantify growth inhibition.
Correlation of ARF with ketoisophorone (KIP) bioconversion efficiency across 30 yeast strains.

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Main Results:

The Acrolein Resistance Factor (ARF) effectively quantified yeast growth response to acrolein.
High ARF values strongly correlated with high OYE activity (measured by KIP bioconversion).
OYE gene presence was confirmed in active strains via PCR.

Conclusions:

The acrolein resistance screening method is a fast and effective way to select yeasts with high OYE activity.
This method facilitates the isolation and biocatalytic application of novel OYEs from yeast.
The approach supports strain selection for efficient whole-cell bioconversions.