Rapid method for screening enoate reductase activity in yeasts.
Old Yellow Enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the asymmetric reduction of electron-poor alkenes (enoate reductase activity). Since OYEs are involved in detoxification of acrolein, a high-throughput method for selecting yeasts expressing high enoate reductase activity, based on their acrolein resistance, was developed. The screening method was based on the measurement of growth in acrolein-supplemented medium, in 96-well microtiter plate cultures. A quantitative descriptor (Acrolein Resistance Factor=ARF) was firstly designed for quantifying the influence of both acrolein concentration and time of exposure on yeast growth. Besides, the efficiency of bioconversion of ketoisophorone (KIP) was exploited to measure OYE activity. In order to validate the method, ARF was correlated with the bioconversion of KIP on thirty yeast strains, belonging to 7 genera. With only a few exceptions, all strains exhibiting the highest ARF also displayed the maximum OYE activity. The presence of OYE genes in the strains showing OYE activity was confirmed by PCR amplification. Based on the results herein reported, this method should be profitably used as a fast screening procedure aimed at selecting outstanding strains for whole-cell bioconversions and could open many possibilities for the isolation and the biocatalytic exploitation of new OYEs from yeasts.
Raimondi
Stefano
S
Department of Chemistry, University of Modena and Reggio Emilia, Modena, Italy.
Roncaglia
Lucia
L
Amaretti
Alberto
A
Leonardi
Alan
A
Buzzini
Pietro
P
Forti
Luca
L
Rossi
Maddalena
M
D000171:Acrolein / Q000378:metabolism / Q000633:toxicity; D002087:Butyrates / Q000378:metabolism* / Q000633:toxicity; D003470:Culture Media / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism*; D008403:Mass Screening / Q000379:methods*; D008828:Microbiological Techniques / Q000379:methods*; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D009574:Nitro Compounds / Q000378:metabolism* / Q000633:toxicity; D010084:Oxidation-Reduction; D015003:Yeasts / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*
C508635:6-(nitrooxy)hexyl-(2z)-4-(acetyloxy)-3-(4-(methylsulfonyl)phenyl)-2-phenylbut-2-enoate; D002087:Butyrates; D003470:Culture Media; D005656:Fungal Proteins; D009574:Nitro Compounds; D000171:Acrolein; D009252:NADPH Dehydrogenase