Pyrethroids are ester-containing synthetic compounds derived from natural pyrethrins. They are widely used as insecticides in agriculture, animal husbandry, and homes, but their persistence poses risks to non-target organisms and natural habitats [1;2]. Microbial lipolytic enzymes (e.g., carboxylesterases EC 3.1.1.1, lipases EC 3.1.1.3) offer potential for pyrethroid degradation due to their broad substrate specificity, thermo-, pH and other attractive characteristics can be a new and effective means of removing pyrethroids from the environment. Bacteria such as Bacillus sp., Pseudomonas sp., Serratia sp., Micrococcus sp., Sphingomonas sp., and Staphylococcus sp. produce lipolytic enzymes capable of breaking ester bonds in compounds like cypermethrin, permethrin, and deltamethrin [1].
In this study, plant growing substrate samples from three Lithuanian farmlands were investigated using conventional methods to identify pyrethroid-degrading bacteria. To isolate bacteria capable of degrading pyrethroids, an enrichment culture was used with permethrin as the sole carbon source. The selected bacterial isolates were identified through 16S rRNA gene sequencing and phylogenetic analysis. After identifying the target bacteria, 12 cultures from the genera Pseudomonas, Staphylococcus, Micrococcus, Bacillus, Peribacillus and Paenibacillus sp. were assessed based on the hydrolysis zones around their colonies in solid minimal salts media supplemented with permethrin. Further, target proteins were purified from both intracellular and extracellular fractions of the most active bacterial strains and analyzed using zymographic analysis. For the latter, tributyrin and permethrin were used as substrates. Lipolytic activity of partially purified enzymes was also tested using para-nitrophenyl butyrate (p-NPB) substrate.
In four bacterial strains, which according to the phylogenetic analysis most likely belong to Pseudomonas knackmussii, Staphylococcus warneri, Micrococcus aloeverae and Solibacillus sp., 55-70 kDa lipolytic enzymes active towards tributyrin and permethrin in the zymogram gels, were determined. In the partially purified fractions of other studied bacterial strains enzymes (20-100 kDa) active towards only the tributyrin were determined.