Characterization of two fungal lipoxygenases expressed in Aspergillus oryzae
Akiko Sugio1, Lars Henrik Østergaard2, Kenji Matsui3
1Novozymes Japan Ltd., CB-6 MTG, 1-3 Nakase, Mihama-ku, Chiba 261-8501, Japan.
Abstract:
Two fungal lipoxygenase genes were cloned from a rice pathogen, Magnaporthe salvinii, and the take-all fungus, Gaeumannomyces graminis var. tritici, and successfully expressed in Aspergillus oryzae in secreted form. The lipoxygenases expressed, termed MLOX and GLOX, were purified and characterized to evaluate suitability for industrial applications. Both enzymes were active broadly at pH 4-11 and had optimum temperatures around 60 °C, but they were largely different in substrate specificity. Where MLOX was active broadly on arachidonic acid, EPA and DHA, and even on derivatives of fatty acids, such as methyl linoleate or linoleoyl alcohol, GLOX was more specific to linoleic acid and linolenic acid. The most remarkable difference between the two fungal LOXs was the positional and stereo-specificity of oxygenation reactions on polyunsaturated fatty acids. When using linoleic acid as the substrate, the product of MLOX was 9S-hydroperoxy-(E,Z)-octadecadienoic acid (9S(E,Z)-HPODE), on the other hand, the product of GLOX was 13R(E,Z)-HPODE. The enzymes were evaluated for a couple of potential applications and found to be effective on bleaching colored compounds such as carotenoids.
Insights
Two fungal lipoxygenases (MLOX and GLOX) were cloned and expressed for industrial use. These enzymes show distinct substrate specificities and stereospecificity, demonstrating potential in applications like bleaching colored compounds.
Area of Science:
- Biotechnology
- Enzymology
- Fungal Genetics
Background:
- Lipoxygenases (LOXs) are crucial enzymes in fatty acid metabolism.
- Fungal LOXs offer potential for industrial biocatalysis due to their unique properties.
Purpose of the Study:
- To clone and express lipoxygenase genes from Magnaporthe salvinii (MLOX) and Gaeumannomyces graminis (GLOX).
- To characterize MLOX and GLOX for industrial applications, focusing on substrate specificity and stereospecificity.
Main Methods:
- Gene cloning and expression in Aspergillus oryzae.
- Enzyme purification and characterization (pH, temperature, substrate specificity).
- Analysis of oxygenation products using linoleic acid as substrate.
Main Results:
- MLOX and GLOX were successfully expressed and purified.
- Both enzymes exhibited broad pH activity (4-11) and optimal temperature around 60°C.
- MLOX showed broad substrate specificity (arachidonic acid, EPA, DHA), while GLOX was specific to linoleic and linolenic acids.
- MLOX produced 9S(E,Z)-HPODE from linoleic acid, whereas GLOX produced 13R(E,Z)-HPODE.
- Both enzymes effectively bleached colored compounds like carotenoids.
Conclusions:
- MLOX and GLOX are distinct fungal lipoxygenases with potential industrial value.
- Their differing specificities and stereospecificity open avenues for targeted biocatalytic applications.
- The enzymes' bleaching capability highlights their utility in decolorization processes.
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