Methods for identifying lipoxygenase producing microorganisms on agar plates

Plate assays for lipoxygenase producing microorganisms on agar plates have been developed. Both potassium iodide-starch and indamine dye formation methods were effective for detecting soybean lipoxygenase activity on agar plates. A positive result was also achieved using the β-carotene bleaching method, but the sensitivity of this method was lower than the other two methods. The potassium iodide-starch and indamine dye formation methods were also applied for detecting lipoxygenase production by Trichoderma reesei and Pichia pastoris transformants expressing the lipoxygenase gene of the fungus Gaeumannomyces graminis. In both cases lipoxygenase production in the transformants could be identified. For detection of the G. graminis lipoxygenase produced by Aspergillus nidulans the potassium iodide-starch method was successful. When Escherichia coli was grown on agar and soybean lipoxygenase was applied on the culture lipoxygenase activity could clearly be detected by the indamine dye formation method. This suggests that the method has potential for screening of metagenomic libraries in E. coli for lipoxygenase activity

Hydroperoxide production from linoleic acid by heterologous Gaeumannomyces graminis tritici lipoxygenase: Optimization and scale-up

Linoleic acid was converted into hydroperoxides by a Gaeumannomyces graminis tritici lipoxygenase produced recombinantly in Trichoderma reesei. Hydroperoxide production was optimized using a face-centred experimental design in order to study the effects of pH, temperature and time on the conversion of linoleic acid into four regioisomeric hydroperoxyoctadecadienoic acids (HPODE): 13-(Z,E)-, 9-(E,Z)-, 13-(E,E)-, 9-(E,E)-HPODE. Fitting equations described satisfactorily the system behavior and showed that reaction time was the most influencing independent variable. A set of independent variables (pH = 6.7, temperature = 23.9 degrees C and time = 18 h) allowed to obtain high yields of hydroperoxides (88.0%) with a good selectivity for the 13-(Z,E)-HPODE isomer (47.4%) when the initial substrate concentration was 10 g/L. The production was further investigated using industrially relevant linoleic acid concentrations (100-300 g/L) leading to HPODE yields of similar to 40% and the volumetric productivity 3.6 g/(L h), and a selectivity for 13-(Z,E)-HPODE of around 74%. (c) 2012 Elsevier B.V. All rights reserved