The methods used in the current work for identification of lipoxygenase producing transformants on agar plates are all based on the detection of the linoleic acid hydroperoxides formed in the lipoxygenase catalyzed reaction. In the indamine dye formation method 3-methyl-2-benzothiazolinone (MBTH) is coupled oxidatively with 3-(dimethylamino) benzoic acid (DMAB) in a hematin catalyzed reaction, which results in the generation of the indamine dye (Anthon and Barrett 2001
). In the potassium iodide starch method the fatty acid hydroperoxides formed oxidize iodide to iodine, which in turn reacts with starch (Williams et al. 1986
). β-Carotene can be used for lipoxygenase detection, since it is bleached by the fatty acid hydroperoxides (Villafuerte Romero and Barrett 1997
The indamine dye formation and KI-starch methods appeared to be more sensitive than the β-carotene bleaching method for detection of lipoxygenase activity. The poor sensitivity of the β-carotene bleaching method has also been established in a previous report in which different liquid assays for detecting lipoxygenase activity from vegetable extracts were compared (Villafuerte Romero and Barrett 1997
The reaction products of lipoxygenase-catalyzed oxidation of linoleic acid appeared to be toxic for E. coli
cells. It has also been shown previously that fatty acid hydroperoxides and their degradation products (oxylipins) impair the growth of many microbial plant pathogens (Prost et al., 2005
). If linoleic acid is present in the growth medium from the onset of the cultivation, the lipoxygenase producers may thus not be able to survive. Linoleic acid can also be spontaneously degraded via
auto-oxidation during longer incubations (Seo et al. 1999
). For these reasons, we chose to omit linoleic acid from the agar medium. Lipoxygenase positive T. reesei
and P. pastoris
transformants expressing the G. graminis
lipoxygenase gene could be clearly identified by both KI-starch and indamine dye formation methods. These methods could also most likely be used for other microbial production hosts. For the A. nidulans
transformants only the KI-starch assay was successful. The indamine dye formation method did not show clear results for detection of lipoxygenase (data not shown).
A potential problem in screening E. coli
-hosted metagenomic libraries is that the cells are able to use the lipoxygenase substrate, linoleic acid, for growth. This problem can, however, most probably be overcome by disrupting genes encoding fatty acid transport and/or β-oxidation present in the fad
regulon (DiRusso and Nyström, 1998
). However, as stated above, adding linoleic acid to the medium may not be feasible, because positive transformants can produce toxic substances via
the lipoxygenase catalyzed oxidation of the substrate.
Preferably tens of thousands of metagenomic library colonies would have to be screened to find lipoxygenase activity. Furthermore, it is typical for metagenomic expression libraries that the activities are low and may be detectable only after prolonged cultivations (Uchiyama and Miyazaki 2009
). The use of the KI-starch method is problematic for the detection of lipoxygenase producing transformants of metagenomic libraries, since the color reaction takes place under very acidic conditions. Iodine is also a well-known microbicide (Vasudevan and Tandon 2010
). We chose therefore the indamine dye formation method for investigation whether lipoxygenase activity can be detected in the presence of E. coli
cells. If linoleic acid and the coloring reagents were applied as one layer on the E. coli
culture supplemented with soybean lipoxygenase, the violet-blue color was for unknown reasons only transiently visible. However, when the substrate agarose was applied first, followed by incubation and application of the coloring agarose, the soybean lipoxygenase catalyzed reaction was clearly detectable. EDTA intensified the signal, possibly because it chelates metal ions, which can catalyze hydroperoxide degradation.
It can be concluded that the KI-starch and the indamine dye formation method can both be used for detection of lipoxygenase producing transformants of P. pastoris and T. reesei, and possibly also of other microbial hosts. Furthermore, the indamine dye formation method holds promise for identification of lipoxygenase positive transformants of E. coli-hosted metagenomic libraries. However, at the present the detection of lipoxygenase production by natural isolates growing on agar remains a challenge.