Orthologous lipoxygenases of Pleurotus spp. – A comparison of substrate specificity and sequence homology

• Published in: Journal of molecular catalysis. B, Enzymatic Enzymatic, 2013-12, Vol.97, p.189-195
• Main Authors: Leonhardt, Robin-Hagen, Plagemann, Ina, Linke, Diana, Zelena, Katerina, Berger, Ralf G.
• Format: Article
• Language: English
• Subjects: (+)-Valencene; Basidiomycete; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Heterologous expression; Lipoxygenase; Methods. Procedures. Technologies; Pleurotus
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2013.08.014

•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 30–35̊C.•Activities with linoleic acid as substrate ranged from 95U/mg to 118U/mg.•Valencene dioxygenation differed strongly from 101.5 to 357.5μg 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 95U/mg to 118U/mg, with Km values between 58 and 106μM. Optimum reaction conditions were pH 7 and 30–35̊C. 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.