ABSTRACT
We report a novel method for the rapid, sensitive, and quantitative detection of alkanes, alcohols, and aldehydes that relies on the reaction of bacterial luciferase with an aldehyde, resulting in the emission of light. Primary alcohols with corresponding aldehydes that are within the substrate range of the particular luciferase are detected after conversion to the aldehyde by an alcohol dehydrogenase. In addition, alkanes themselves may be detected by conversion to primary alcohols by an alkane hydroxylase, followed by conversion to the aldehyde by alcohol dehydrogenase. We developed a rapid bioluminescent method by genetically engineering the genes encoding bacterial luciferase, alcohol dehydrogenase, and alkane hydroxylase into a plasmid for simultaneous expression in an E. coli host cell line. Alkanes, alcohols, or aldehydes were detected within seconds, with sensitivity in the micromolar range, by measuring the resulting light emission with a microplate reader. We demonstrate the application of this method for the detection of alkanes, alcohols, and aldehydes and for the detection of alkane hydroxylase and alcohol dehydrogenase activity in vivo. This method is amenable to the high-throughput screening needs required for the identification of novel catalysts.
Subject(s)
Luciferases, Bacterial/metabolism , Luminescent Measurements/methods , Organic Chemicals/analysis , 1-Octanol/analysis , 1-Octanol/metabolism , Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/metabolism , Alcohols/analysis , Alcohols/metabolism , Aldehydes/analysis , Aldehydes/metabolism , Alkanes/analysis , Alkanes/metabolism , Cytochrome P-450 CYP4A/genetics , Cytochrome P-450 CYP4A/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fatty Alcohols/analysis , Fatty Alcohols/metabolism , Gene Expression , Genetic Vectors/genetics , Luciferases, Bacterial/genetics , Octanes/analysis , Octanes/metabolism , Organic Chemicals/metabolism , Plasmids/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , Pseudomonas putida/enzymology , Pseudomonas putida/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transformation, Bacterial , Vibrio/enzymology , Vibrio/geneticsABSTRACT
Biodesulfurization of fuel oils is a two-phase (oil/water) process which may offer an interesting alternative to conventional hydrodesulfurization due to the mild operating conditions and reaction specificity afforded by the biocatalyst. For biodesulfurization to realize commercial success, a variety of process considerations must be addressed including reaction rate, emulsion formation and breakage, biocatalyst recovery, and both gas and liquid mass transport. This study evaluates emulsion formation and breakage using two biocatalysts with differing hydrophobic characteristics. A Gram-positive (Rhodococcus erythropolis) biocatalyst, expressing the complete 4S desulfurization pathway, and a Gram-negative biocatalyst (Escherichia coli), expressing only the gene for conversion of dibenzothiophene (DBT) to DBT sulfone, are compared relative to their ability to convert DBT and the ease of phase separation as well as biocatalyst recovery following desulfurization.
