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![Absorbance variations during -carotene cooxidation by the filtrate of a tangential ultrafiltration device separating soybean lipoxygenase-1 (retentate) from free substrate, product and derived species (filtrate) during the course of the dioxygenation reaction (see "Experimental Procedures"). Reaction carried out under N 2 (l) or air (E) bubbling. [LA] initial 1 mM; [-carotene] initial 3 M; [soybean lipoxygenase-1] 7.5 nM.](publication/247482911/figure/fig5/AS:1018110651932673@1619747903247/Absorbance-variations-during-carotene-cooxidation-by-the-filtrate-of-a-tangential.png)
Absorbance variations during -carotene cooxidation by the filtrate of a tangential ultrafiltration device separating soybean lipoxygenase-1 (retentate) from free substrate, product and derived species (filtrate) during the course of the dioxygenation reaction (see "Experimental Procedures"). Reaction carried out under N 2 (l) or air (E) bubbling. [LA] initial 1 mM; [-carotene] initial 3 M; [soybean lipoxygenase-1] 7.5 nM.
Source publication
The effect of oxygen concentration on the regiospecificity of the soybean lipoxygenase-1 dioxygenation reaction was studied.
At low oxygen concentrations (<5 μm), a dramatic change in the regiospecificity of the enzyme was observed with the hydroperoxy-octadecadienoic acid (HPOD) 13:9
ratio closer to 50:50 instead of the generally reported 95:5. Th...
Context in source publication
Context 1
... product, and derived species) was directly flowed into a -carotene solution circulating as a closed circuit into a spectrophotometer. Thus lipoxygenase is never in contact with the pigment, the bleaching of which can only be ascribed to the presence of free radicals not bound to the enzyme (see "Experimental Procedures"). Under these conditions, Fig. 7 shows that at low oxygen concentrations (N 2 bubbling), the absorbance variation is lower than that caused by -carotene dilution, indicating the release by the enzyme of free radical species not detectable under higher oxygen concentrations (air bubbling) nor in the absence of enzyme at low oxygen concentrations (data not ...
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Citations
The neural cell death in cerebral infarction is suggested to be ferroptosis-like cell death, involving the participation of 15-lipoxygenase. Ferroptosis is induced by lipid radical species generated through the one-electron reduction of lipid hydroperoxides, and it has been shown to propagate intracellularly and intercellularly. At lower oxygen concentration, it appeared that both regiospecificity and stereospecificity of conjugated diene moiety in lipoxygenase-catalysed lipid hydroperoxidation are drastically lost. As a result, in the reaction with linoleic acid, the linoleate 9-peroxyl radical-ferrous lipoxygenase complex dissolves into the linoleate 9-peroxyl radical and ferrous 15-lipoxygenase. Subsequently, the ferrous 15-lipoxygenase then undergoes one-electron reduction of 13-hydroperoxy octadecadienoic acid, generating an alkoxyl radical (Pseudoperoxidase reaction). A part of the produced lipid alkoxyl radicals undergoes cleavage of C-C bonds, liberating small molecular hydrocarbon radicals. Particularly, in ω-3 polyunsaturated fatty acids, which are abundant in the vascular and nervous systems, the liberation of small molecular hydrocarbon radicals was more pronounced compared to ω-6 polyunsaturated fatty acids. The involvement of these small molecular hydrocarbon radicals in the propagation of membrane lipid damage is suggested.
Hydroperoxy‐9Z,11E‐octadecadienoic acid (13‐HPODE) can be obtained from safflower oil in an enzyme cascade utilizing lipase, lipoxygenase (LOX), and catalase for in situ oxygen generation. The application of immobilized enzymes may open a new path to a cost‐efficient production of 13‐HPODE, which is used for the synthesis of green note aroma hexanal. Ten immobilization supports are compared for immobilization of lipoxygenase‐1 from Glycine max (LOX‐1) and oxirane‐based Immobead 150 P proves to be best with a maximum LOX‐1 activity of 22 470 U g−1. The immobilizate is successfully recycled in eight consecutive batches and maintains full activity over a period of 16 h using a 3D‐printed column reactor. Catalase from Micrococcus lysodeikticus and LOX‐1 are co‐immobilized on Immobead 150 P allowing a constant production of 13‐HPODE for up to six cycles with a maximum product conversion of 45% and a 13‐regioselectivity of 83% . In this two‐enzyme system with H2O2‐dosage, foam generation is significantly reduced. Co‐immobilization of LOX‐1 and lipaseis possible; however, rapid lipase deactivation occurs. Therefore, a two‐reactor approach with oil hydrolysis in the first reactor is proposed. Immobilized lipases from C. rugosa are suitable for safflower oil hydrolysis and maintain full activity over ten hydrolysis cycles. Practical applications: Linoleic acid hydroperoxide (13‐HPODE) is the starting material for the synthesis of the green note aroma compound hexanal. The byproduct of the hydroperoxide splitting is ω‐oxododecenoic acid, which is currently not employed industrially. The bifunctional oxodocecenoic acid is interesting as precursor for the synthesis of polymer building blocks. Simple one‐step derivatization of the oxo‐group can yield suitable C12 monomers such as dicarboxylic acids, ω‐amino acids, or ω‐hydroxy acids. Cost‐efficiency is a key parameter to incorporate these new biobased building blocks for polymer applications. In this approach, immobilized enzymes are used for the synthesis of 13‐HPODE starting from safflower oil with in situ oxygen generation to prevent excessive foam formation. A two‐reactor concept is designed to circumvent hydroperoxide‐induced lipase deactivation. Direct comparison of both batch and continuous process is performed and provides information for the implementation of the enzyme cascade and the design of an optimized reactor system. The synthesis of linoleic acid based hydroperoxide 13‐HPODE is achieved with immobilized lipase and co‐immobilized lipoxygenase (LOX) and catalase starting from safflower oil in an organic solvent‐free system. A reactor cascade is needed to prevent lipase deactivation by the hydroperoxide. In situ oxygen supply for LOX is generated by hydrogen peroxide dosing and catalase activity.
Oxo-octadecadienoic acids (OxoODEs) act as peroxisome proliferator-activated receptor (PPAR) agonists biologically, and are known to be produced in the lipoxygenase/linoleate system. OxoODEs seem to originate from the linoleate alkoxyl radicals that are generated from (E/Z)-hydroperoxy octadecadienoic acids ((E/Z)-HpODEs) by a pseudoperoxidase reaction that is catalyzed by ferrous lipoxygenase. However, the mechanism underlying the conversion of alkoxyl radical into OxoODE remains obscure. In the present study, we confirmed that OxoODEs are produced in the lipoxygenase/linoleate system in an oxygen-dependent manner. Interestingly, we revealed a correlation between the (E/Z)-OxoODEs content and the (E/E)-HpODEs content in the system. (E/E)-HpODEs could have been derived from (E/E)-linoleate peroxyl radicals, which are generated by the reaction between a free linoleate allyl radical and an oxygen molecule. Notably, the ferrous lipoxygenase-linoleate allyl radical (LOx(Fe²⁺)-L·) complex, which is an intermediate in the lipoxygenase/linoleate system, tends to dissociate into LOx(Fe²⁺) and a linoleate allyl radical. Subsequently, LOx(Fe²⁺) converts (E/Z)-HpODEs to an (E/Z)-linoleate alkoxyl radical through one-electron reduction. Taken together, we propose that (E/Z)-OxoODEs and (E/E)-HpODEs are produced through radical–radical dismutation between (E/Z)-linoleate alkoxyl radical and (E/E)-linoleate peroxyl radical. Furthermore, the production of (E/Z)-OxoODEs and (E/E)-HpODEs was remarkably inhibited by a hydrophobic radical scavenger, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO). On the contrary, water-miscible radical scavengers, 4-hydroxyl-2,2,6,6-tetramethylpiperidine 1-oxyl (OH-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmΔP) only modestly or sparingly inhibited the production of (E/Z)-OxoODEs and (E/E)-HpODEs. These facts indicate that the radical–radical dismutation between linoleate alkoxyl radical and linoleate peroxyl radical proceeds in the interior of micelles.
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The positive effect of lipoxygenase, added as an enzyme-active soy flour, during the production of white bread is well established. In addition to increasing the mixing tolerance and overall dough rheology, lipoxygenase is also an effective bleaching agent. It is known that these effects are mediated by enzyme-coupled cooxidation of gluten proteins and carotenoids. However, the mechanism whereby these effects are achieved is not yet fully understood. In order to gain a better understanding into the reactions governing the beneficial effects of lipoxygenases in bread dough, an in-depth knowledge of the lipoxygenase catalytic mechanism is required. Until now no single review combining the molecular enzymology of lipoxygenase enzymes and their application in the baking industry has been presented. This review, therefore, focuses on the extraction and molecular characterization of lipoxygenases in addition to the work done on the application of lipoxygenases in the baking industry.
Understanding gas migration pathways is critical to unraveling structure-function relationships in enzymes that process gaseous
substrates such as O2, H2 and N2. This work investigates the role of a defined pathway for O2 in regulating the peroxidation
of linoleic acid by soybean lipoxygenase 1 (SLO-1). Computational and mutagenesis studies provide strong support for a dominant
delivery channel that shuttles molecular oxygen to a specific region of the active site, thereby ensuring the regio- and stereospecificity
of product. Analysis of reaction kinetics and product distribution in channel mutants also reveals a plasticity to the gas
migration pathway. The findings show that a single site mutation (Ile553Trp) limits oxygen accessibility to the active site,
greatly increasing the fraction of substrate that reacts with oxygen free in solution. They also show how a neighboring site
mutation (Leu496Trp) can result in a redirection of oxygen toward an alternate position of the substrate, changing the regio-
and stereospecificity of peroxidation. The present data indicate that modest changes in a protein scaffold may modulate the
access of small gaseous molecules to enzyme bound substrates.
Hydroxy fatty acids (HFAs) derived from omega-3 polyunsaturated fatty acids have been known as versatile bioactive molecules. However, its practical production from omega-3 or omega-3 rich oil has not been well established. In the present study, the stereo-selective enzymatic production of 9R-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9R-HOTE) from α-linolenic acid (ALA) in perilla seed oil (PO) hydrolyzate was achieved using purified recombinant 9R-lipoxygenase (9R-LOX) from Nostoc sp. SAG 25.82. The specific activity of the enzyme followed the order linoleic acid (LA) > ALA > γ-linolenic acid (GLA). A total of 75% fatty acids (ALA and LA) were used as a substrate for 9R-LOX from commercial PO by hydrolysis of Candida rugosa lipase. The optimal reaction conditions for the production of 9R-HOTE from ALA using 9R-LOX were pH 8.5, 15°C, 5% (v/v) acetone, 0.2% (w/v) Tween 80, 40 g/L ALA, and 1 g/L enzyme. Under these conditions, 9R-LOX produced 37.6 g/L 9R-HOTE from 40 g/L ALA for 1 h, with a conversion yield of 94% and a productivity of 37.6 g/L/h; and the enzyme produced 34 g/L 9R-HOTE from 40 g/L ALA in PO hydrolyzate for 1 h, with a conversion yields of 85% and a productivity of 34 g/L/h. The enzyme also converted 9R-hydroxy-10E,12Z-octadecadienoic acid (9R-HODE) from 40 g/L LA for 1.0 h, with a conversion yield of 95% and a productivity of 38.4 g/L. This is the highest productivity of HFA from both ALA and ALA-rich vegetable oil using LOX ever reported. Therefore, our result suggests an efficient method for the production of 9R-HFAs from LA and ALA in vegetable oil using recombinant LOX in biotechnology.
One of the most significant properties of lipoxygenase (LOX) as a biocatalyst is its stereo-selective oxygenation. In this study, the stereo-specific production of 9R-hydroxy-10E,12Z-octadecadienoic acid (9R-HODE) from linoleic acid was achieved using whole recombinant Escherichia coil cells expressing LOX from Nostoc sp. SAG 25.82. The optimal conditions for the production of 9R-HODE were pH 7.5, 25 degrees C, 40 gl(-1) cells, 15 gl(-1) linoleic acid, 2% (v/v) methanol, 1 working volume/oxygen volume/min (vvm) oxygenation rate, and 250 rpm agitation speed in 500 ml-baffled flask containing a working volume of 50 ml. Under these optimized conditions, whole recombinant cells expressing 9R-LOX protein produced 14.3 gl(-1) 9R-HODE for 1 h, with a conversion yield of 95% (w/w) and a productivity of 14.3 gl(-1) h(-1). The oxygen supply method significantly influenced stereo- and regio-selectivity of the oxygenation of linoleic acid. Among the oxygen supply methods tested, oxygenation (1 vvm) with agitation (250 rpm) resulted in the highest 9R/13S-HODE ratio of the products at 96:4. This is the first application using whole recombinant cells harboring R-specific LOX for the stereo-selective production of an R-specific hydroxy fatty acid.
