Details
| Original language | English |
|---|---|
| Pages (from-to) | 189-195 |
| Number of pages | 7 |
| Journal | Journal of Molecular Catalysis B: Enzymatic |
| Volume | 97 |
| Publication status | Published - 2013 |
Abstract
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 ÌŠC. 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.
Keywords
- (+)-Valencene, Basidiomycete, Heterologous expression, Lipoxygenase, Pleurotus
ASJC Scopus subject areas
- Chemical Engineering(all)
- Catalysis
- Chemical Engineering(all)
- Bioengineering
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Chemical Engineering(all)
- Process Chemistry and Technology
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In: Journal of Molecular Catalysis B: Enzymatic, Vol. 97, 2013, p. 189-195.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Orthologous lipoxygenases of Pleurotus spp. - A comparison of substrate specificity and sequence homology
AU - Leonhardt, Robin Hagen
AU - Plagemann, Ina
AU - Linke, Diana
AU - Zelena, Katerina
AU - Berger, Ralf G.
N1 - Funding information: Support of the work by the BMBF cluster Biokatalyse2021 (FKZ0315172B) is gratefully acknowledged.
PY - 2013
Y1 - 2013
N2 - 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 ÌŠC. 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.
AB - 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 ÌŠC. 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.
KW - (+)-Valencene
KW - Basidiomycete
KW - Heterologous expression
KW - Lipoxygenase
KW - Pleurotus
UR - http://www.scopus.com/inward/record.url?scp=84883863387&partnerID=8YFLogxK
U2 - 10.1016/j.molcatb.2013.08.014
DO - 10.1016/j.molcatb.2013.08.014
M3 - Article
AN - SCOPUS:84883863387
VL - 97
SP - 189
EP - 195
JO - Journal of Molecular Catalysis B: Enzymatic
JF - Journal of Molecular Catalysis B: Enzymatic
SN - 1381-1177
ER -