| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 69607 | Journal of Molecular Catalysis B: Enzymatic | 2013 | 7 Pages |
•Lipoxygenase genes were amplified from five Pleurotus spp.•Sequences showed similarities >95% on the amino acid level.•Reaction optima of the heterologous enzymes were pH 7 and 30–35 ̊C.•Activities with linoleic acid as substrate ranged from 95 U/mg to 118 U/mg.•Valencene dioxygenation differed strongly from 101.5 to 357.5 μg product/mg enzyme.
A selection of Pleurotus spp. was screened for intracellular lipoxygenase activity, and strains with distinguished activities were chosen for a screening on the molecular level. Lipoxygenase genes from five different Pleurotus spp. were amplified from the corresponding cDNA, functionally expressed using a cold shock expression system in Escherichia coli BL21DE3 Star cells and characterized for specific activity and reaction optima. All lipoxygenase sequences coded for proteins of 643 amino acids, sharing similarities >95% among each other and to a previously characterized LOXPsa1 from Pleurotus sapidus. Lipoxygenase activities were quantified using linoleic acid as substrate and reached similar values ranging from 95 U/mg to 118 U/mg, with Km values between 58 and 106 μM. Optimum reaction conditions were pH 7 and 30–35 ̊C. However, the uncommon trait of accepting (+)-valencene, a sesquiterpene hydrocarbon substrate, differed strongly between two clusters of highly homologous sequences. No exchange of amino acids adjacent to the active site was found. Cloning and expression of a truncated LOXPsa1 sequence missing 144 amino acid residues of the N-terminal barrel domain yielded soluble protein but no measurable activity.
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