Biotransformation of docosahexaenoic acid into 10R,17S-dihydroxydocosahexaenoic acid as protectin DX 10-epimer by serial reactions of arachidonate 8R- and 15S-lipoxygenases.

Protectins, 10,17-dihydroxydocosahexaenoic acids (10,17-DiHDHAs), are belonged to specialized pro-resolving mediators (SPMs). Protectins are generated by polymorphonuclear leukocytes in humans and resolve inflammation and infection in trace amounts. However, the quantitative production of protectin DX 10-epimer (10-epi-PDX, 10R,17S-4Z,7Z,11E,13Z,15E,19Z-DiHDHA) has been not attempted to date. In this study, 10-epi-PDX was quantitatively produced from docosahexaenoic acid (DHA) by serial whole-cell biotransformation of Escherichia coli expressing arachidonate (ARA) 8R-lipoxygenase (8R-LOX) from the coral Plexaura homomalla and E. coli expressing ARA 15S-LOX from the bacterium Archangium violaceum. The optimal bioconversion conditions to produce 10R-hydroxydocosahexaenoic acid (10R-HDHA) and 10-epi-PDX were pH 8.0, 30 °C, 2.0 mM DHA, and 4.0 g/L cells; and pH 8.5, 20 °C, 1.4 mM 10R-HDHA, and 1.0 g/L cells, respectively. Under these optimized conditions, 2.0 mM (657 mg/L) DHA was converted into 1.2 mM (433 mg/L) 10-epi-PDX via 1.4 mM (482 mg/L) 10R-HDHA by the serial whole-cell biotransformation within 90 min, with a molar conversion of 60% and volumetric productivity of 0.8 mM/h (288 mg/L/h). To the best of our knowledge, this is the first quantitative production of 10-epi-PDX. Our results contribute to the efficient biocatalytic synthesis of SPMs.

Biotransformation of Polyunsaturated Fatty Acids to Trioxilins by Lipoxygenase from Pleurotus sajor-caju.

A lipoxygenase from Pleurotus sajor-caju (PsLOX) was cloned, expressed in Escherichia coli, and purified as a soluble protein with a specific activity of 629 µmol/min/mg for arachidonic acid (AA). The native PsLOX exhibited a molecular mass of 146 kDa, including a 73-kDa homodimer, as estimated by gel-filtration chromatography. The major products converted from polyunsaturated fatty acids (PUFAs), including AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were identified as trioxilins (TrXs), namely 13,14,15-TrXB3 , 13,14,15-TrXB4 , and 15,16,17-TrXB5 , respectively, through high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The enzyme displayed its maximum activity at pH 8.0 and 20 °C. Under these conditions, the specific activity and catalytic efficiency of PsLOX for PUFAs exhibited the following order: AA>EPA>DHA. Based on HPLC analysis and substrate specificity, PsLOX was identified as an arachidonate 15-LOX. PsLOX efficiently converted 10 mM of AA, EPA, and DHA to 8.7 mM of 13,14,15-TrXB3 (conversion rate: 87 %), 7.9 mM of 13,14,15-TrXB4 (79 %), and 7.2 mM of 15,16,17-TrXB5 (72 %) in 15, 20, and 20 min, respectively, marking the highest conversion rates reported to date. Collectively, our results demonstrate that PsLOX is an efficient TrXs-producing enzyme.

Biotransformation of C20- and C22-polyunsaturated fatty acids to 11S- and 13S-hydroxy fatty acids by Escherichia coli expressing 11S-lipoxygenase from Enhygromyxa salina.

PURPOSE: Peroxidation and reduction of 11S- and 13S-positions on C20 and C22 polyunsaturated fatty acids (PUFAs) by Escherichia coli expressing highly active arachidonate (ARA) 11S-lipoxygenase (11S-LOX) from Enhygromyxa salina with the reducing agent cysteine. RESULTS: The specific activity and catalytic efficiency of ARA 11S-LOX from E. salina were 4.1- and 91-fold higher than those of only reported ARA 11S-LOX from Myxococcus xanthus, respectively. The hydroxy fatty acids (HFAs) obtained by the biotransformation of ARA, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexanoic acid (DHA) by Escherichia coli expressing 11S-LOX from E. salina in the presence of cysteine were identified as 11S-hydroxyeicosatetraenoic acid (11S-HETE), 11S-hydroxyeicosapentaenoic acid (11S-HEPE), 13S-hydroxydocosapentaenoic acid (13S-HDPA), and 13S-hydroxydocosahexaenoic acid (13S-HDHA), respectively. The recombinant cells converted 3 mM of ARA, EPA, DPA, and DHA into 2.9 mM of 11S-HETE, 2.4 mM 11S-HEPE, 1. 9 mM 13S-HDPA, and 2.2 mM 13S-HDHA in 60, 80, 120, and 120 min, corresponding to productivities of 72.5, 40.4, 18.5, and 22.4 µM min-1 and conversion yields of 96.7, 80.0, 62.3, and 74.6%, respectively. CONCLUSIONS: We report the highest concentrations, conversion yields, and productivities of 11S- and 13S-hydroxy fatty acids from C20- and C22-PUFAs achieved via E. coli expressing highly active E. salina 11S-LOX.

Phorbal-12-mysristate-13-acetate-induced inflammation is restored by protectin DX through PPARγ in human promonocytic U937 cells.

AIMS: Protectin DX (PDX), a specialized pro-resolving mediator, is an important pharmaceutical compound with potential antioxidant and inflammation-resolving effects. However, the fundamental mechanism by which PDX's ameliorate chronic inflammatory diseases has not yet been elucidated. This study aims to evaluate the anti-inflammatory properties and PPARγ-mediated mechanisms of PDX in phorbal-12-mysristate-13-acetate (PMA)-stimulated human promonocytic U937 cells. MAIN METHODS: We confirmed the effects of PDX on expressions of pro-inflammatory cytokines, mediators, and CD14 using conventional PCR, RT-qPCR, ELISA, and flow cytometry. Using western blotting, immunofluorescence, and reactive oxygen species (ROS) determination, we observed that PDX regulated PMA-induced signaling cascades. Molecular docking analysis and a cellular thermal shift assay were conducted to verify the interaction between PDX and the proliferator-activated receptor-γ (PPARγ) ligand binding domain. Western blotting was then employed to explore the alterations in PPARγ expression levels and validate PDX as a PPARγ full agonist. KEY FINDINGS: PDX attenuated protein and mRNA expression levels of interleukin-6, tumor necrosis factor-α, and cyclooxygenase-2 in PMA-treated U937 cells. PDX acts as a PPARγ agonist, exerting a modulating effect on the ROS/JNK/c-Fos signaling pathways. Furthermore, PDX reduced human monocyte differentiation antigen CD14 expression levels. SIGNIFICANCE: PPARγ exhibits pro-resolving effects to regulate the excessive inflammation. These results suggest that PDX demonstrates the resolution of inflammation, indicating the potential for therapeutic targeting of chronic inflammatory diseases.

Production of Bioactive Deapiosylated Platycosides from Glycosylated Platycosides in Balloon Flower Root Using the Crude Enzyme from the Food-Available Fungus Rhizopus oryzae.

Extract from balloon flower root (Platycodi radix) containing platycosides as saponins is a beneficial food additive and is used for their savory taste and the alleviation of respiratory diseases. Deglycosylated platycosides show greater pharmacological effects than glycosylated platycosides. However, there are no reports on the conversion of glycosylated platycosides into deapiosylated platycosides. In this study, we showed that the crude enzyme from Rhizopus oryzae, a generally recognized as safe (GRAS) fungus isolated from meju (fermented soybean brick), completely converted glycosylated platycosides in Platycodi radix extract into deapiosylated platycosides: deapiosylated platycodin D (deapi-PD), deapiosylated platycodin A (deapi-PA), deapiosylated polygalacin D (deapi-PGD), and deapiosylated platyconic acid A (deapi-PCA). Among these, deapi-PA and deapi-PCA were first identified using liquid chromatography/mass spectrometry. The anti-inflammatory and antioxidant effects of deapiosylated platycosides were greater than those of the precursor glycosylated platycosides. These deapiosylated platycosides could improve the properties of functional food additives.

Improved Bioactivity of 3-O-ß-D-Glucopyranosyl Platycosides in Biotransformed Platycodon grandiflorum Root Extract by Pectinase from Aspergillus aculeatus.

Platycodon grandiflorum (balloon flower) root (Platycodi radix, PR) is used as a health supplement owing to its beneficial bioactive properties. In the present study, the anti-inflammatory, antioxidant, and whitening effects of deglycosylated platycosides (saponins) from PR biotransformed by pectinase from Aspergillus aculeatus were investigated. The bioactivities of the platycosides improved when the number of sugar moieties attached to the aglycone platycosides was decreased. The deglycosylated saponins exhibited higher lipoxygenase inhibitory activities (anti-inflammatory activities) than the precursor platycosides and the anti-inflammatory compound baicalein. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the pectinase-treated PR extract was higher than that of the non-treated PR extract. The trolox-equivalent antioxidant capacity (TEAC) assay showed improved values as the saponins were hydrolyzed. The tyrosinase inhibitory activities (whitening effects) of deglycosylated platycosides were higher than those of the precursor platycosides. Furthermore, 3-O-ß-D-glucopyranosyl platycosides showed higher anti-inflammatory, antioxidant, and whitening activities than their precursor glycosylated platycosides. Therefore, 3-O-ß-D-glucopyranosyl platycosides may improve the beneficial effects of nutritional supplements and cosmetic products.

Biotransformation of Fructose to Allose by a One-Pot Reaction Using Flavonifractor plautiiD-Allulose 3-Epimerase and Clostridium thermocellum Ribose 5-Phosphate Isomerase.

D-Allose is a potential medical sugar because it has anticancer, antihypertensive, anti-inflammatory, antioxidative, and immunosuppressant activities. Allose production from fructose as a cheap substrate was performed by a one-pot reaction using Flavonifractor plautiiD-allulose 3-epimerase (FP-DAE) and Clostridium thermocellum ribose 5-phosphate isomerase (CT-RPI). The optimal reaction conditions for allose production were pH 7.5, 60°C, 0.1 g/l FP-DAE, 12 g/l CT-RPI, and 600 g/l fructose in the presence of 1 mM Co2+. Under these optimized conditions, FP-DAE and CT-RPI produced 79 g/l allose for 2 h, with a conversion yield of 13%. This is the first biotransformation of fructose to allose by a two-enzyme system. The production of allose by a one-pot reaction using FP-DAE and CT-RPI was 1.3-fold higher than that by a two-step reaction using the two enzymes.