Gene Expression and Metabolome Analysis Reveals Anti-Inflammatory Impacts of 11,17diHDoPE on PM10-Induced Mouse Lung Inflammation.

Oxylipins, the metabolites of polyunsaturated fatty acids, are vital in regulating cell proliferation and inflammation. Among these oxylipins, specialized pro-resolving mediators notably contribute to inflammation resolution. Previously, we showed that the specialized pro-resolving mediators isomer 11,17dihydroxy docosapentaenoic acid (11,17diHDoPE) can be synthesized in bacterial cells and exhibits anti-inflammatory effects in mammalian cells. This study investigates the in vivo impact of 11,17diHDoPE in mice exposed to particulate matter 10 (PM10). Our results indicate that 11,17diHDoPE significantly mitigates PM10-induced lung inflammation in mice, as evidenced by reduced pro-inflammatory cytokines and pulmonary inflammation-related gene expression. Metabolomic analysis reveals that 11,17diHDoPE modulates inflammation-related metabolites such as threonine, 2-keto gluconic acid, butanoic acid, and methyl oleate in lung tissues. In addition, 11,17diHDoPE upregulates the LA-derived oxylipin pathway and downregulates arachidonic acid- and docosahexaenoic acid-derived oxylipin pathways in serum. Correlation analyses between gene expression and metabolite changes suggest that 11,17diHDoPE alleviates inflammation by interfering with macrophage differentiation. These findings underscore the in vivo role of 11,17diHDoPE in reducing pulmonary inflammation, highlighting its potential as a therapeutic agent for respiratory diseases.

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.

Production of C20 9S- and C22 11S-hydroxy fatty acids by cells expressing Shewanella hanedai arachidonate 9S-lipoxygenase.

The putative lipoxygenase (LOX) from the proteobacterium Shewanella hanedai was determined to be an 82 kDa monomeric enzyme by SDS-PAGE and gel filtration chromatography analysis. LOX was identified as a single-dioxygenating arachidonate (ARA) 9S-LOX by analyzing ARA-derived bioconversion products using high-performance liquid chromatography with reverse-, normal-, and chiral-phase columns and evaluating kinetic parameters for C20- and C22-polyunsaturated fatty acids (PUFAs). The catalytic efficiency (kcat/Km) values of 9S-LOX from S. hanedai for ARA, eicosapentaenoic acid, and docosahexaenoic acid were 3.1-, 4.1-, and 2.5-fold higher, respectively, than those only reported 9S-LOX from Sphingopyxis macrogoltabida with double-dioxygenating activity. To promote the production of C20 9S- and C22 11S-hydroxy fatty acids (HFAs) using Escherichia coli expressing 9S-LOX from S. hanedai, bioconversion conditions, including temperature, pH, solvent type and its concentration, concentrations of cells, and substrate, were optimized to 25 °C, pH 8.5, 6% (v/v) dimethyl sulfoxide, 0.2 g/l cells, and 7 mM ARA as substrate in a 500 ml-Erlenmeyer baffled flask with 50 ml reaction solution with agitation at 200 rpm in the presence of 10 mM cysteine as a reduction agent, respectively. Under these conditions, 6.4 mM 9S-hydroxyeicosatetraenoic acid, 6.2 mM 9S-hydroxyeicosapentaenoic acid, and 5.9 mM 11S-hydroxydocosahexaenoic acid were produced in 30 min, 40 min, and 60 min with specific productivities of 1067 µmol/min/g, 775 µmol/min/g, and 492 µmol/min/g, volumetric productivities of 213 µM/min, 155 µM/min, and 98 µM/min, and conversion yields of 91.4%, 88.6%, and 84.3%, respectively. To date, these are the highest specific productivities reported for the bioconversion of C20- and C22-PUFAs into HFAs. KEY POINTS: • Lipoxygenase from Shewanella hanedai was identified as arachidonate 9S-lipoxygenase • Optimization led to increased production of C20 9S- and C22 11S-hydroxy fatty acids • We reported the highest specific productivities of C20- and C22-hydroxy fatty acids.

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.

The DPA-derivative 11S, 17S-dihydroxy 7,9,13,15,19 (Z,E,Z,E,Z)-docosapentaenoic acid inhibits IL-6 production by inhibiting ROS production and ERK/NF-κB pathway in keratinocytes HaCaT stimulated with a fine dust PM10.

11 S, 17S-dihydroxy 7,9,13,15,19 (Z,E,Z,E,Z)-docosapentaenoic acid (DoPE) is a derivative of docosapentaenoic acid, a specialized pro-resolving mediator of inflammation such as lipoxins, resolvins, maresins, and protectins. PM10 is a fine dust particle that induces oxidative stress, DNA damage, inflammation, aging, and cancer. The anti-inflammatory mechanism of DoPE, however, has not yet been elucidated. In these studies, we investigated whether DoPE has anti-inflammatory effects in human keratinocyte HaCaT cells. We demonstrated that DoPE suppressed PM10-induced expressions of IL-6 mRNA and protein in human HaCaT keratinocytes. We also investigated the modulating effects of DoPE on reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK). ROS production, extracellular signal regulated kinase (ERK) phosphorylation, and translocation of nuclear factor-kappa B (NF-kB) p65 and NF-kB activity were suppressed by DoPE in PM10-stimulated HaCaT cells. Collectively, our results demonstrated that DoPE inhibited IL-6 expression by reducing ROS generation, suppressing ERK phosphorylation, and inhibiting translocation of NF-kB p65 and NF-kB activity in PM10-stimulated HaCaT cells, suggesting that DoPE can be useful for the resolution of the inflammation caused by IL-6.

Biocatalytic synthesis of dihydroxy fatty acids as lipid mediators from polyunsaturated fatty acids by double dioxygenation of the microbial 12S-lipoxygenase.

Leukotrienes (LTs) and maresins (MaRs) are human lipid mediators (LMs) involved in immune response and anti-inflammation, respectively. These compounds and their isomers are generated in trace amounts by lipoxygenases (LOXs) in human macrophages and neutrophils. These LMs have been synthesized using nonenvironmentally benign synthetic protocols, which are expensive. 8S- and 15S-LOXs with double dioxygenating activities have previously been reported, whereas 12S-LOX with double dioxygenating activity have not been reported to date. Here, we discovered a wild-type 12S-LOX with double dioxygenating activity from the bacterium Endozoicomonas numazuensis, which produced dihydroxy fatty acids (DiHFAs) as LMs from polyunsaturated fatty acids via double dioxygenation. The enzyme activity for producing DiHFA was approximately 550-fold higher than that of mammalian LOX with double dioxygenating activity. The microbial 12S-LOX converted 3.00 mM of arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid to 2.37 mM (797 mg/L) 6-trans-8-cis-12S-epimer of LTB4, 1.59 mM (532 mg/L) 6-trans-8-cis-12S-epimer of LTB5, 1.35 mM (498 mg/L) 10-cis-12-trans-7S-epimer of MaR1n-3 DPA , and 1.54 mM (555 mg/L) 10-cis-12-trans-7S-epimer of MaR1 within 2 h, which were 5.3-, 7.6-, 3.1-, and 5.5-fold higher than those biosynthesized by the previously reported microbial engineered 12S-LOX with double dioxygenating activity, respectively. These findings contribute to the efficient and environmentally friendly biosynthesis of LMs and stimulate physiological study on LMs.

An integrative approach to improving the biocatalytic reactions of whole cells expressing recombinant enzymes.

Biotransformation is a selective, stereospecific, efficient, and environment friendly method, compared to chemical synthesis, and a feasible tool for industrial and pharmaceutical applications. The design of biocatalysts using enzyme engineering and metabolic engineering tools has been widely reviewed. However, less importance has been given to the biocatalytic reaction of whole cells expressing recombinant enzymes. Along with the remarkable development of biotechnology tools, a variety of techniques have been applied to improve the biocatalytic reaction of whole cell biotransformation. In this review, techniques related to the biocatalytic reaction are examined, reorganized, and summarized via an integrative approach. Moreover, equilibrium-shifted biotransformation is reviewed for the first time.

Construction of an engineered biocatalyst system for the production of medium-chain α,ω-dicarboxylic acids from medium-chain ω-hydroxycarboxylic acids.

Medium-chain α,ω-dicarboxylic acids produced from renewable long-chain fatty acids are valuable as precursors in the chemical industry. However, they are difficult to produce biologically at high concentrations. Although improved biocatalyst systems consisting of engineering of Baeyer-Villiger monooxygenases are used in the production of ω-hydroxycarboxylic acids from long-chain fatty acids, the engineering of biocatalysts involved in the production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids has been rarely attempted. Here, we used highly active bacterial enzymes, Micrococcus luteus alcohol dehydrogenase and Archangium violaceum aldehyde dehydrogenase, for the efficient production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids and constructed a biocatalyst with cofactor regeneration system by introducing NAD(P)H flavin oxidoreductase as the NAD(P)H oxidase. The inhibition of the biocatalyst by hydrophobic substrates was attenuated by engineering a biocatalyst system with an adsorbent resin, which allowed us to obtain 196 mM decanedioic, 145 mM undecanedioic, and 114 mM dodecanedioic acid from 200 mM of C10, C11, and C12 hydroxyl saturated carboxylic acids, respectively, and 141 mM undecanedioic acid from 150 mM C11 unsaturated carboxylic acids, with molar conversions of 98%, 97%, 95%, and 94%, respectively. The concentration of undecanedioic acid obtained was approximately 40-fold higher than that in the previously highest results. Our results from this study can be applied for the industrial production of medium-chain α,ω-dicarboxylic acids from renewable long-chain fatty acids.

Conversion of Glycosylated Platycoside E to Deapiose-Xylosylated Platycodin D by Cytolase PCL5.

Platycosides, the saponins abundant in Platycodi radix (the root of Platycodon grandiflorum), have diverse pharmacological activities and have been used as food supplements. Since deglycosylated saponins exhibit higher biological activity than glycosylated saponins, efforts are on to enzymatically convert glycosylated platycosides to deglycosylated platycosides; however, the lack of diversity and specificities of these enzymes has limited the kinds of platycosides that can be deglycosylated. In the present study, we examined the enzymatic conversion of platycosides and showed that Cytolase PCL5 completely converted platycoside E and polygalacin D3 into deapiose-xylosylated platycodin D and deapiose-xylosylated polygalacin D, respectively, which were identified by LC-MS analysis. The platycoside substrates were hydrolyzed through the following novel hydrolytic pathways: platycoside E → platycodin D3 → platycodin D → deapiosylated platycodin D → deapiose-xylosylated platycodin D; and polygalacin D3 → polygalacin D → deapiosylated polygalacin D → deapiose-xylosylated polygalacin D. Our results show that cytolast PCL5 may have a potential role in the development of biologically active platycosides that may be used for their diverse pharmacological activities.

Whole-Cell Photoenzymatic Cascades to Synthesize Long-Chain Aliphatic Amines and Esters from Renewable Fatty Acids.

Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer-Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g-1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.

Production of 8S- and 10S-hydroxy polyunsaturated fatty acids by recombinant Escherichia coli cells expressing mouse arachidonate 8S-lipoxygenase.

OBJECTIVE: To quantitatively hydroxylate 8S- and 10S-positions on polyunsaturated fatty acids by recombinant Escherichia coli cells expressing mouse arachidonate 8S-lipoxygenase (8S-LOX). RESULTS: Hydroxylated products gained from the conversion of arachidonic acid (20:4Δ5Z,8Z,11Z,14Z, AA), eicosapentanoic acid (20:5Δ5Z,8Z,11Z,14Z,17Z, EPA), and (22:6Δ4Z,7Z,10Z,13Z,16Z,19Z, DHA) by recombinant E. coli cells containing 8S-LOX from mouse were identified as 8S-hydroxy-5,9,11,14(Z,E,Z,Z)-eicosatetranoic acid (8S-HETE), 8S-hydroxy-5,9,11,14,17(Z,E,Z,Z,Z)-eicosapentanoic acid (8S-HEPE), and 10S-hydroxy-4,8,12,14,16,19(Z,E,Z,Z,Z,Z)-docosahexaenoic acid (10S-HDoHE), respectively. Under the optimal hydroxylation conditions of pH 7.5, 30 °C, 5% (v/v) ethanol, 15 g cells l-1, and 5 mM substrate, AA, EPA, and DHA were hydroxylated into 4.37 mM 8S-HETE, 3.77 mM 8S-HEPE, and 3.13 mM 10S-HDoHE for 60, 90, and 60 min, with 87, 75, and 63% molar conversions, respectively. CONCLUSION: To the best of our knowledge, this is the first quantitatively biotechnological production of 8S-HETE, 8S-HEPE, and 10S-HDoHE.

Cloning and characterization of α-L-rhamnosidase from Chloroflexus aurantiacus and its application in the production of isoquercitrin from rutin.

OBJECTIVE: This study was conducted to characterize recombinant α-L-rhamnosidase from Chloroflexus aurantiacus and apply the enzyme in the production of isoquercitrin from rutin. RESULTS: The α-L-rhamnosidase from C. aurantiacus was cloned and expressed in Escherichia coli BL21 and purified as a soluble enzyme. α-L-rhamnosidase purified from C. aurantiacus has a molecular mass of approximately 105 kDa and is predicted to exist as a homodimer with a native enzyme of 200 kDa. The purified enzyme exhibited the highest specific activity for rutin among the reported isoquercitrin producing α-L-rhamnosidases and was applied in the production of isoquercitrin from rutin. Under the optimised conditions of pH 6.0, 50 °C, 0.6 U mL-1 α-L-rhamnosidase, and 30 mM rutin, α-L-rhamnosidase from C. aurantiacus produced 30 mM isoquercitrin after 2 h with a 100% conversion yield and productivity of 15 mM h-1. CONCLUSIONS: We achieved a high productivity of isoquercitrin from rutin. Moreover, these results suggest that α-L-rhamnosidase from C. aurantiacus is an effective isoquercitrin producer.

Enzymatic Biotransformation of Balloon Flower Root Saponins into Bioactive Platycodin D by Deglucosylation with Caldicellulosiruptor bescii ß-Glucosidase.

Platycodin D (PD), a major saponin (platycoside) in Platycodi radix (balloon flower root), has higher pharmacological activity than the other major platycosides; however, its content in the plant root is only approximately 10% (w/w) and the productivities of PD by several enzymes are still too low for industrial applications. To rapidly increase the total PD content, the ß-glucosidase from Caldicellulosiruptor bescii was used for the deglucosylation of the PD precursors platycoside E (PE) and platycodin D3 (PD3) in the root extract into PD. Under the optimized reaction conditions, the enzyme completely converted the PD precursors into PD with the highest productivity reported so far, increasing the total PD content to 48% (w/w). In the biotransformation process, the platycosides in Platycodi radix were hydrolyzed by four pathways: deapiosylated (deapi)-PE → deapi-PD3 → deapi-PD, PE → PD3 → PD, polygalacin D3 → polygalacin D, and 3″-O-acetyl polygalacin D3 → 3″-O-acetyl polygalacin D.

Stabilization and improved activity of arachidonate 11S-lipoxygenase from proteobacterium Myxococcus xanthus.

Lipoxygenases (LOXs) catalyze the dioxygenation of PUFAs to produce regio- and stereospecific oxygenated fatty acids. The identification of regio- and stereospecific LOXs is important because their specific products are involved in different physiological activities in various organisms. Bacterial LOXs are found only in some proteobacteria and cyanobacteria, and they are not stable in vitro. Here, we used C20 and C22 PUFAs such as arachidonic acid (ARA), eicosapentaenoic acid, and docosahexaenoic acid to identify an 11S-specific LOX from the proteobacterium Myxococcus xanthus and explore its in vitro stability and activity. The activity and stability of M. xanthus ARA 11S-LOX as well as the production of 11S-hydroxyeicosatetraenoic acid from ARA were significantly increased by the addition of phosphatidylcholine, Ca2+, and coactosin-like protein (newly identified in the yeast Rhodosporidium toluroides) as stimulatory factors; in fact, LOX activity in the presence of all three factors increased approximately 3-fold. Our results indicate that these stimulatory factors can be used to increase the activity and stability of bacterial LOX and the production of bioactive hydroxy fatty acids, which can contribute to new academic research.

Regiospecificity of a novel bacterial lipoxygenase from Myxococcus xanthus for polyunsaturated fatty acids.

Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific­oxygenated signaling compounds.

PKCι is a target of 7,8,4'-trihydroxyisoflavone for the suppression of UVB-induced MMP-1 expression.

The soy isoflavone daidzein is bioconverted to 7,8,4'-trihydroxyisoflavone (7,8,4'-THIF) by microorganisms. Here, we investigated the matrix metalloproteinase (MMP)-1 inhibitory properties of 7,8,4'-THIF that arise through the suppression of UVB-induced MMP-1 expression. 7,8,4'-THIF reduced UVB-induced MMP-1 expression at the transcriptional level in primary human dermal fibroblasts and inhibited UVB-induced transcriptional activity of AP-1, a major activator of MMP-1 expression. Additionally, it was observed that the mitogen-activated protein kinase (MAPK) pathway, a crucial signalling cascade for MMP-1 expression, was suppressed by 7,8,4'-THIF. Protein kinase C iota (PKCι) was suspected to be a direct target of 7,8,4'-THIF. The direct interaction between 7,8,4'-THIF and PKCι was confirmed using pull-down assays and immobilized metal ion affinity-based fluorescence polarization assays. Finally, we observed that 7,8,4'-THIF inhibited UVB-induced MMP-1 expression in a human skin equivalent model. Taken together, these results suggest that 7,8,4'-THIF, a bioconversion product of daidzein, suppresses UVB-induced MMP-1 expression.

Characterization of L-rhamnose isomerase from Clostridium stercorarium and its application to the production of D-allose from D-allulose (D-psicose).

OBJECTIVE: To characterize L-rhamnose isomerase (L-RI) from the thermophilic bacterium Clostridium stercorarium and apply it to produce D-allose from D-allulose. RESULTS: A recombinant L-RI from C. stercorarium exhibited the highest specific activity and catalytic efficiency (k cat/K m) for L-rhamnose among the reported L-RIs. The L-RI was applied to the high-level production of D-allose from D-allulose. The isomerization activity for D-allulose was maximal at pH 7, 75 °C, and 1 mM Mn2+ over 10 min reaction time. The half-lives of the L-RI at 65, 70, 75, and 80 °C were 22.8, 9.5, 1.9, and 0.2 h, respectively. To ensure full stability during 2.5 h incubation, the optimal temperature was set at 70 °C. Under the optimized conditions of pH 7, 70 °C, 1 mM Mn2+, 27 U L-RI l-1, and 600 g D-allulose l-1, L-RI from C. stercorarium produced 199 g D-allose l-1 without by-products over 2.5 h, with a conversion yield of 33% and a productivity of 79.6 g l-1 h-1. CONCLUSION: To the best of our knowledge, this is the highest concentration and productivity of D-allose reported thus far.

The Ginsenoside Derivative 20(S)-Protopanaxadiol Inhibits Solar Ultraviolet Light-Induced Matrix Metalloproteinase-1 Expression.

Ginsenosides are major pharmacologically active compounds present in ginseng (Panax ginseng). Among the ginsenosides, 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol (GPPD) and ginsenoside Rb1 (Rb1) have previously been reported to exhibit anti-wrinkle effects. In this study, 20(S)-protopanaxadiol (20(S)-PPD), an aglycone derivative of the Rb1 metabolite was investigated for its anti-wrinkle benefit and compared to GPPD and Rb1. The anti-wrinkle effect of 20(S)-PPD during solar UV light was investigated using a human skin equivalent model and human keratinocytes. 20(S)-PPD attenuated solar UV-induced matrix metalloproteinase (MMP)-1 expression to a greater extent than GPPD and Rb1. 20(S)-PPD treatment modulated MMP-1 mRNA expression and the transcriptional activity of activator protein (AP)-1, a major transcription factor of MMP-1. Two upstream signaling pathways for AP-1, the MEK1/2-ERK1/2-p90RSK and MEK3/6-p38 pathways, were also suppressed. Taken together, these findings highlight the potential of 20(S)-PPD for further development as a preventative agent for sunlight-induced skin wrinkle. J. Cell. Biochem. 118: 3756-3764, 2017. © 2017 Wiley Periodicals, Inc.

Comparison of Biochemical Properties of the Original and Newly Identified Oleate Hydratases from Stenotrophomonas maltophilia.

Oleate hydratases (OhyAs) catalyze the conversion of unsaturated fatty acids to 10-hydroxy fatty acids, which are used as precursors of important industrial compounds, including lactones and ω-hydroxycarboxylic and α,ω-dicarboxylic acids. The genes encoding OhyA and a putative fatty acid hydratase in Stenotrophomonas maltophilia were identified by genomic analysis. The putative fatty acid hydratase was purified and identified as an oleate hydratase (OhyA2) based on its substrate specificity. The activity of OhyA2 as a holoenzyme was not affected by adding cofactors, whereas the activity of the original oleate hydratase (OhyA1) showed an increase. Thus, all characterized OhyAs were categorized as either OhyA1 or OhyA2 based on the activities of holoenzymes upon adding cofactors, which were determined by the type of the fourth conserved amino acid of flavin adenine dinucleotide (FAD)-binding motif. The hydration activities of S. maltophilia OhyA2 toward unsaturated fatty acids, including oleic acid, palmitoleic acid, linoleic acid, α-linolenic acid, and γ-linolenic acid, were greater than those of OhyA1. Moreover, the specific activity of S. maltophilia OhyA2 toward unsaturated fatty acids, with the exception of γ-linolenic acid, was the highest among all reported OhyAs.IMPORTANCE All characterized OhyAs were categorized as OhyA1s or OhyA2s based on the different properties of the reported and newly identified holo-OhyAs in S. maltophilia upon the addition of cofactors. OhyA2s showed higher activities toward polyunsaturated fatty acids (PUFAs), including linoleic acid, α-linolenic acid, and γ-linolenic acid, than those of OhyA1s. This suggests that OhyA2s can be used more effectively to convert plant oils to 10-hydroxy fatty acids because plant oils contain not only oleic acid but also PUFAs. The hydration activity of the newly identified OhyA2 from S. maltophilia toward oleic acid was the highest among the activity levels reported so far. Therefore, this enzyme is an efficient biocatalyst for the conversion of plant oils to 10-hydroxy fatty acids, which can be further converted to important industrial materials.