Glycerol Ester Hydrolase Research Articles

GehB Inactivates Lipoproteins to Delay the Healing of Acute Wounds Infected with Staphylococcus aureus.

Staphylococcus aureus is one of the most prevalent bacteria found in acute wounds. S. aureus produces many virulence factors and extracellular enzymes that contribute to bacterial survival, dissemination, and pathogenicity. Lipase GehB is a glycerol ester hydrolase that hydrolyzes triglycerides to facilitate the evasion of S. aureus from host immune recognition. However, the role and mechanism of lipase GehB in skin acute wound healing after S. aureus infection remain unclear. In this study, we found that the gehB gene deletion mutant (USA300ΔgehB) stimulated significantly higher levels of pro-inflammatory cytokines in RAW264.7 and Toll-like receptor 2 (TLR2)-transfected HEK293 cells than the wild-type USA300 strain did. Recombinant GehB-His treated lipoprotein (Lpp) reduced stimulation of TLR2-dependent TNF-α production by RAW264.7 macrophages. GehB delayed the skin acute wound healing in BALB/c mice infected with S. aureus, while wound healing was similar in C57BL/6 TLR2-/- mice infected with either wild-type USA300 or USA300ΔgehB. In BALB/c mice,we also observed more bacterial survival, less leukocyte recruitment, lower IL-8 production, and adipocyte differentiation in USA300-infected skin acute wound tissues than those in USA300ΔgehB-challenged ones. Our data indicated that GehB inactivates lipoproteins to shield S. aureus from innate immune killing, resulting in delayed the healing of skin acute wounds infected with S. aureus.

Elucidating the impact of bacterial lipases, human serum albumin, and FASII inhibition on the utilization of exogenous fatty acids by Staphylococcus aureus.

Incorporation of host-derived exogenous fatty acids (eFAs), particularly unsaturated fatty acids (UFAs), by Staphylococcus aureus could affect the bacterial membrane fluidity and susceptibility to antimicrobials. In this work, we found that glycerol ester hydrolase (Geh) is the primary lipase hydrolyzing cholesteryl esters and, to a lesser extent, triglycerides and that human serum albumin (HSA) could serve as a buffer of eFAs, where low levels of HSA facilitate the utilization of eFAs but high levels of HSA inhibit it. The fact that the type II fatty acid synthesis (FASII) inhibitor, AFN-1252, leads to an increase in UFA content even in the absence of eFA suggests that membrane property modulation is part of its mechanism of action. Thus, Geh and/or the FASII system look to be promising targets to enhance S. aureus killing in a host environment by restricting eFA utilization or modulating membrane properties, respectively.

The Phospholipase A1 Activity of Glycerol Ester Hydrolase (Geh) Is Responsible for Extracellular 2-12(S)-Methyltetradecanoyl-Lysophosphatidylglycerol Production in Staphylococcus aureus.

Phosphatidylglycerol (PG) is the major membrane phospholipid of Staphylococcus aureus and predominately consists of molecular species with ≥16-carbon acyl chains in the 1-position and anteiso 12(S)-methyltetradecaonate (a15) esterified at the 2-position. The analysis of the growth media for PG-derived products shows S. aureus releases essentially pure 2-12(S)-methyltetradecanoyl-sn-glycero-3-phospho-1'-sn-glycerol (a15:0-LPG) derived from the hydrolysis of the 1-position of PG into the environment. The cellular lysophosphatidylglycerol (LPG) pool is dominated by a15-LPG but also consists of ≥16-LPG species arising from the removal of the 2-position. Mass tracing experiments confirmed a15-LPG was derived from isoleucine metabolism. A screen of candidate secreted lipase knockout strains pinpointed glycerol ester hydrolase (geh) as the gene required for generating extracellular a15-LPG, and complementation of a Δgeh strain with a Geh expression plasmid restored extracellular a15-LPG formation. Orlistat, a covalent inhibitor of Geh, also attenuated extracellular a15-LPG accumulation. Purified Geh hydrolyzed the 1-position acyl chain of PG and generated only a15-LPG from a S. aureus lipid mixture. The Geh product was 2-a15-LPG, which spontaneously isomerizes with time to a mixture of 1- and 2-a15-LPG. Docking PG in the Geh active site provides a structural rationale for the positional specificity of Geh. These data demonstrate a physiological role for Geh phospholipase A1 activity in S. aureus membrane phospholipid turnover. IMPORTANCE Glycerol ester hydrolase, Geh, is an abundant secreted lipase whose expression is controlled by the accessory gene regulator (Agr) quorum-sensing signal transduction pathway. Geh is thought to have a role in virulence based on its ability to hydrolyze host lipids at the infection site to provide fatty acids for membrane biogenesis and substrates for oleate hydratase, and Geh inhibits immune cell activation by hydrolyzing lipoprotein glycerol esters. The discovery that Geh is the major contributor to the formation and release of a15-LPG reveals an unappreciated physiological role for Geh acting as a phospholipase A1 in the degradation of S. aureus membrane phosphatidylglycerol. The role(s) for extracellular a15-LPG in S. aureus biology remain to be elucidated.

Biological Evaluation of the Antibacterial Retinoid CD437 in Cutibacterium acnes Infection.

Acne vulgaris is a complex skin disease involving infection by Cutibacterium acnes, inflammation, and hyperkeratinization. We evaluated the activity of the retinoid 6-[3-(adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and 16 other retinoid analogs as potential anti-C. acnes compounds and found that CD437 displayed the highest antimicrobial activity with an MIC against C. acnes (ATCC 6919 and HM-513) of 1 μg/mL. CD437 demonstrated an MBC of 2 μg/mL compared to up to 64 μg/mL for the retinoid adapalene and up to 16 μg/mL for tetracycline, which are commonly used clinically to treat acne. Membrane permeability assays demonstrated that exposure of C. acnes ATCC 6919 to CD437 damaged the integrity of C. acnes ATCC 6919 bacterial membranes, and this finding was confirmed with scanning electron microscopy. Additionally, CD437 downregulated the expression of C. acnes ATCC 6919 virulence factors, including the genes encoding Christie-Atkins-Munch-Petersen factor 1 (CAMP1), CAMP2, glycerol-ester hydrolase B (GehB), sialidase B, and neuraminidase. In a mouse skin infection model of C. acnes ATCC 6919, topical treatment with CD437 ameliorated skin lesions and reduced the bacterial burden in situ (P < 0.001). In human NHEK primary cells, CD437 reduced the transcriptional levels of the coding genes for inflammatory cytokines (interleukin-1α, ~10-fold; interleukin-6, ~20-fold; interleukin-8, ~30-fold; and tumor necrosis factor-alpha, ~6-fold) and downregulated the transcriptional levels of KRT10 (~10-fold), FLG (~4-fold), and TGM1 (~2-fold), indicating that CD437 can diminish inflammation and hyperkeratinization. In summary, CD437 deserves further attention for its dual function as a potential acne therapeutic that potentially acts on both the pathogen and the host.

Screening, lipA Gene Amplification and Molecular Identification of Lipolytic Fungi from Contaminated Soils

Lipase is a glycerol ester hydrolase that hydrolyzes esters of triglycerides. This study aimed to isolate, screen and characterize lipolytic fungi with lipA gene from soil environments. Fungi [molds and yeasts] were isolated from contaminated soil samples randomly collected from Palm oil mills, Abattoirs and Automobile servicing workshops in Eleme and Obio Akpor local governments in Rivers State, Nigeria using standard microbiological techniques. Qualitative Lipase screening assay was carried out on the fungal isolates through gel diffusion assay using Tributryin, Tween 80 and Olive oil respectively. Isolates with maximum zones of clearance were selected and screened for Lipase gene for hydrolysis of triacylglyceride [lipA gene] through Lipase gene amplification and were identified by Internal Transcribed Spacer characterization.&#x0D; A total of 96 fungal isolates were obtained, lipolytic screening showed 57 lipolytic strains in which 21 lipolytic fungal strains were screened from contaminated soil from palm oil mills, 17 lipolytic strains from abattoirs and 19 lipolytic strains from automobile workshops respectively. Seven isolates with maximum zones of clearance showed lipA gene in their genomic DNA and were identified as Candida orthopsilosis PMS3 [Accession No: OL546803], Fusarium proliferatum PMS4 [Accession No: OL546804], Saccharomyces cerevisiae PMS8 [Accession No: OL546805], Penicillium paxilli ABS8 [Accession No: OL546799], Pichia kudriavzevii ENS1 [Accession No: OL546800] Aspergillus niger ENS3 [Accession No: OL546801] and Trichoderma viride ENS5 [Accession No: OL546802].&#x0D; This study showed that soil fungi are potential producers of biotechnologically important lipase with diverse industrial applications.

Triterpenoid acids isolated from Schinus terebinthifolia fruits reduce Staphylococcus aureus virulence and abate dermonecrosis

Staphylococcus aureus relies on quorum sensing to exert virulence to establish and maintain infection. Prior research demonstrated the potent quorum sensing inhibition effects of “430D-F5”, a refined extract derived from the fruits of Schinus terebinthifolia, a medicinal plant used for the traditional treatment of skin and soft tissue infections. We report the isolation and identification of three compounds from 430D-F5 that reduce virulence and abate dermonecrosis: 3-oxo-olean-12-en-28-oic acid (1), 3-oxotirucalla-7,24Z-dien-26-oic acid (2) and 3α-hydroxytirucalla-7,24 Z-dien-27-oic acid (3). Each compound inhibits all S. aureus accessory gene regulator (agr) alleles (IC50 2–70 μM). Dose-dependent responses were also observed in agr-regulated reporters for leucocidin A (lukA, IC50 0.4-25 μM) and glycerol ester hydrolase or lipase (gehB, IC50 1.5–25 μM). Surprisingly, dose-dependent activity against the nuclease reporter (nuc), which is under the control of the sae two-component system, was also observed (IC50 0.4–12.5 μM). Compounds 1-3 exhibited little to no effect on the agr-independent mgrA P2 reporter (a constitutive promoter from the mgrA two-component system) and the esxA reporter (under control of mgrA). Compounds 1-3 inhibited δ-toxin production in vitro and reduced dermonecrosis in a murine in vivo model. This is the first report of triterpenoid acids with potent anti-virulence effects against S. aureus.

Determination of Lipase Activities and Lipid Peroxidation Level of Fermented Oil Bean Seeds (Ugba, Pentaclethra macrophylla ), Castor Oil Seeds (Ogiri, Ricinus communis ) and Millet Seeds (Kunu, Eleusine coracana )

Lipases are glycerol ester hydrolases which hydrolyze esther linkages of glycerides at water-oil interface. The activities of lipase in fermented oil bean seeds (ugba), fermented castor oil seeds (ogiri) and kunu were determined with respect to the levels of free fatty acids produced from each sample. The samples were prepared by local adoption of processing oil bean and castor seeds. The seeds were cooked (100°C) in cooking pot for 4hr, chopped into pieces, washed with clean tap water and covered in a stainless pot and allowed to ferment at room temperature. The lipid peroxidation and lipase activity were also determined on the fermented products using standard methods. The total free fatty acid determined were 349± 7, 1026± 5 and 94± 5mg/ml for ugba, ogiri and kunu respectively. The level of lipid hydroperoxide concentrations of fermented ugba, ogiri and kunu were (2.5 ±0.3) ×10-4, (1.57 ±0.05) ×10-4 and (4.5 ±0.3) × 10-4 mg/ml malondialdehyde respectively. It was observed that the higher the peroxidation or concentration of malondialdehyde in a given sample, the least the lipase activity as determined by the level of free fatty acids. The findings of this research indicate that fermented ugba, ogiri and kunu could be good sources of lipase for industrial application.

Bacterial lipolysis of immune-activating ligands promotes evasion of innate defenses

Commensal and pathogenic bacteria hydrolyze host lipid substrates with secreted lipases and phospholipases for nutrient acquisition, colonization, and infection. Bacterial lipase activity on mammalian lipids and phospholipids can promote release of free fatty acids from lipid stores, detoxify antimicrobial lipids, and facilitate membrane dissolution. The gram-positive bacterium Staphylococcus aureus secretes at least two lipases, Sal1 and glycerol ester hydrolase (Geh), with specificities for short- and long-chain fatty acids, respectively, each with roles in the hydrolysis of environmental lipids. In a recent study from our group, we made the unexpected observation that Geh released by S. aureus inhibits activation of innate immune cells. Herein, we investigated the possibility that S. aureus lipases interface with the host immune system to blunt innate immune recognition of the microbe. We found that the Geh lipase, but not other S. aureus lipases, prevents activation of innate cells in culture. Mutation of geh leads to enhancement of proinflammatory cytokine production during infection, increased innate immune activity, and improved clearance of the bacterium in infected tissue. These in vitro and in vivo effects on innate immunity were not due to direct functions of the lipase on mammalian cells, but rather a result of inactivation of S. aureus lipoproteins, a major pathogen-associated molecular pattern (PAMP) of extracellular gram-positive bacteria, via ester hydrolysis. Altogether, these studies provide insight into an adaptive trait that masks microbial recognition by innate immune cells through targeted inactivation of a broadly conserved PAMP.

Identification and Characterization of Bacterial Lipase from Plateu Soil in West Java

Lipases are known as glycerol ester hydrolases that catalyze the hydrolysis of triglycerides into free fatty acids and glycerol. Lipases are found in human, animal, plant, and microorganisms. The aim of this research is to identify lipase producers and characterize bacterial lipase from West Java plateau soil. Plateau soil bacteria samples were isolated on lipase screening medium containing Rhodamine B. Olive oil was used as a substrate in screening and production medium bacterial lipases. From 16 bacterial isolate of lipase producers, 14 were identified as Bacillus sp. and the others were identified as Pseudomonas alcaligenes. All isolates were taken into production step to determine their lipase activities. Moreover, top 3 lipase activities out of 16 lipase activities were chosen to find the optimum pH and temperature. Both characterizations showed pH optimum and temperature optimum from each lipase. These optimum condition were used in heat stability characterization for each lipase samples. The result showed that lipase from isolate COK 2 in optimum pH 4 and temperature 50oC was the most stable lipase due to this sample has good and stable activity for 1 to 5 hours incubation time. Lipase sample from isolate COK 2 has good efficiency for lipase productivity in acid condition and high temperature. Results of this investigation could encourage utilization of these activity enhancers for various industrial applications.

English

Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Oil spilled soil samples collected from Dharmapuri and Salem districts were screened for a novel lipase producing bacteria by screening in Tributyrin agar plate and Rhodamine olive oil agar and lipase screening liquid medium. Among the two hundred isolates thirty two isolates exhibited high lipolytic activity on TBA plate assay and twenty isolate exhibited high lipolytic activity on ROA plate assay The isolate BLP 141 produces lipase more than 12 U/ml in the further lipase screening liquid medium was selected as a novel lipase producing bacteria and identified by morphological and biochemical characteristics as Pseudomonas. The 16S rRNA gene amplified by bacterial universal primer 27F and 1492R yielded 1478 bp was analysed by BLAST confirmed it as Pseudomonas gessardii and was submitted in GenBank with accession number KJ547711.

Identification of the Major Prostaglandin Glycerol Ester Hydrolase in Human Cancer Cells

Prostaglandin glycerol esters (PG-Gs) are produced as a result of the oxygenation of the endocannabinoid, 2-arachidonoylglycerol, by cyclooxygenase 2. Understanding the role that PG-Gs play in a biological setting has been difficult because of their sensitivity to enzymatic hydrolysis. By comparing PG-G hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. The principal role played by LYPLA2 in PGE2-G hydrolysis was confirmed by siRNA knockdown. Purified recombinant LYPLA2 hydrolyzed PG-Gs in the following order of activity: PGE2-G > PGF2α-G > PGD2-G; LYPLA2 hydrolyzed 1- but not 2-arachidonoylglycerol or arachidonoylethanolamide. Chemical inhibition of LYPLA2 in the mouse macrophage-like cell line, RAW264.7, elicited an increase in PG-G production. Our data indicate that LYPLA2 serves as a major PG-G hydrolase in human cells. Perturbation of this enzyme should enable selective modulation of PG-Gs without alterations in endocannabinoids, thereby providing a means to decipher the unique functions of PG-Gs in biology and disease.

Identification and characterization of fatty acid alkyl esterases found in Staphylococcus aureus (1002.3)

Identification and Characterization of Fatty Acid Alkyl Esterases Found in Staphylococcus aureus Alternative energy is a major field of current research. Biodiesel, a mixture of fatty acid alkyl esters, is the most versatile biofuel in current use. This is due to the fact that it is similar enough to gasoline that it is compatible with the diesel engines found throughout the existing global infrastructure. Biodiesel precursor lipids are abundant in cultivated feedstock organisms such as algae and bacteria. However, the standard process for converting oil to biodiesel is heat‐intensive and requires complete removal of water, greatly reducing the net energy gained in its manufacture. Our work constitutes an attempt to explore enzymatic synthesis of biodiesel with lipids like those derived from emerging fuel crops.Previous literature describes fatty acid alkyl ester formation in Staphylococcal lesions, formed by partially characterized esterase activity from an unidentified source. We have identified the enzymes responsible for this activity by using a combination of size exclusion chromatography, gas chromatography‐mass spectrometry, and mass spectrometric protein sequencing. These two highly similar enzymes in the glycerol ester hydrolase (geh) family of proteins catalyze the synthesis of fatty acid alkyl esters in aqueous conditions at or near room temperature. We have demonstrated that other, similar lipases do not exhibit this behavior. We have expressed one of these Staphylococcal esterases in E. coli, and shown via gas chromatography that the expressed protein catalyzes the formation of fatty acid alkyl esters. Based on sequence similarity to homologous proteins that have already been crystallized, we have predicted a structure for these enzymes and intend to engineer mutants with higher rates of catalysis.Grant Funding Source: NSF

In-Vitro Effect of Iloprost on Lipase Activity in Serum

Lipid-metabolizing enzymes may be altered in cells by prostacyclin. Changes in enzyme activity are known to affect the metabolism of acylglycerols and that of cholesterol esters and phospholipids. The activity of the lipase glycerol ester hydrolase in acetone–butanol powders made from serum obtained from healthy blood donors and five patients with advanced atherosclerotic lower limb peripheral vascular disease who had been treated with iloprost was investigated. The protein extracts were preincubated with iloprost, the stable prostacyclin analogue. The enzyme was found to be inhibited at different concentrations of iloprost and the pI50 was 6.7 (−log M for 50% inhibition of the enzyme). The results indicate a possible importance of the enzyme response to prostacyclin in the metabolism of acylglycerols and to the changes in metabolism which occur in atherosclerosis.

A modified method based on p-nitrophenol assay to quantify hydrolysis activities of lipases in litters

Lipases are glycerol ester hydrolases (EC 3.1.1.3) produced by a wide range of microorganisms. They catalyse the hydrolysis of different esters depending on the water content of the reaction medium. Here, we developed a simple methodology to quantify lipase hydrolysis activities using two different litters: a litter of Quercus pubescens (QP) and a litter of both Q. pubescens and Q. ilex. Different p-nitrophenyl esters were used to test hydrolysis in a reaction medium with an organic solvent (heptane). We showed that these activities depended on the amount of litter, the incubation time and the substrate concentration and that they increased with temperature. Furthermore, the lipases from the studied litters were still active after 2 h at 70 °C. These activities showed common properties of lipases: the highest activities were obtained with a medium-acyl chain substrate, p-nitrophenyl laurate. Moreover abiotic hydrolysis with short-chain acyl substrates was observable. The following parameters are recommended to quantify hydrolysis activities of lipases in litters: 10 mM of p-nitrophenyl laurate in 2 ml of heptane, 1 g of litter, 2 ml of water incubated at 30 °C for 2 h.

A method to quantify transesterification activities of lipases in litters

Lipases are glycerol ester hydrolases (EC 3.1.1.3) produced by a wide range of microorganisms. They catalyse the hydrolysis of different esters but this reaction is reversible, depending on the water content of the reaction medium, via esterification and transesterification. The synthetic activity of lipases can be of major importance in natural ecosystems since it can be involved in carbon stockage in soils or litters. Here, the detection of transesterification activities of lipases in litter is reported for the first time. We used two different litters: litter of Quercus pubescens (QP) and litter of both Q. pubescens and Q. ilex. Different p-nitrophenyl esters and pentanol were used to test transesterification in a reaction medium with an organic solvent (heptane). We showed that these activities were proportional to the amount of litter, the incubation time and the substrate concentration and that they increased with temperature. Furthermore, the lipases from the litters studied were very thermostable since they were still active after 2 h at 70 °C. These activities showed common properties of lipases: the highest activities were obtained with a medium acyl-chain substrate p-nitrophenyl caprylate and transesterification activities were correlated to water activity, a w. The following parameters are recommended to quantify transesterification activities in litter: 10 mM of p-nitrophenyl caprylate, 1 g of litter, 500 µL of pentanol, q.s.p. 4 mL of heptane incubated at 30 °C for 2 h.

Properties and application of poly(methacrylic acid‐co‐dodecyl methacrylate‐cl‐N,N‐methylene bisacrylamide) hydrogel immobilized Bacillus cereus MTCC 8372 lipase for the synthesis of geranyl acetate

AbstractA range of fatty acid esters is now being produced commercially with immobilized microbial lipases (glycerol ester hydrolases; EC) in nonaqueous solvents. In this study, a synthetic hydrogel was prepared by the copolymerization of methacrylic acid and dodecyl methacrylate in the presence of a crosslinker, N,N‐methylene bisacrylamide. A purified alkaline thermotolerant bacterial lipase from Bacillus cereus MTCC 8372 was immobilized on a poly(methacrylic acid‐co‐dodecyl methacrylate‐cl‐N,N‐methylene bisacrylamide) hydrogel by an adsorption method. The hydrogel showed a 95% binding efficiency for the lipase. The bound lipase was evaluated for its hydrolytic potential toward various p‐nitrophenyl acyl esters with various C chain lengths. The bound lipase showed optimal hydrolytic activity toward p‐nitrophenyl palmitate at a pH of 8.5 and a temperature of 55°C. The hydrolytic activity of the hydrogel‐bound lipase was enhanced by Hg2+, Fe3+, and NH ions at a concentration of 1 mM. The hydrogel‐bound lipase was used to synthesize geranyl acetate from geraniol and acetic acid in n‐heptane. The optimization of the reaction conditions, such as catalyst loading, effect of substrate concentration, solvent (n‐pentane, n‐hexane, n‐heptane, n‐octane, and n‐nonane), reaction time, temperature, molecular sieve (3 Å × 1.5 mm) and scale up (at 50‐mL level), was studied. The immobilized lipase (25 mg/mL) was used to perform an esterification in n‐alkane(s) that resulted in the synthesis of approximately 82.8 mM geranyl acetate at 55°C in n‐heptane under continuous shaking (160 rpm) after 15 h when geraniol and acetic acid were used in a ratio of 100 : 100 mM. The addition of a molecular sieve (3 Å × 1.5 mm) to the reaction system at a concentration of 40 mg/mL in reaction volume (2 mL) resulted in an increase in the conversion of reactants into geranyl acetate (90.0 mM). During the repetitive esterification under optimum conditions, the hydrogel‐bound lipase produced ester (37.0 mM) after the eighth cycle of reuse. When the reaction volume was scaled up to 50 mL, the ester synthesized was 58.7 mM under optimized conditions. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Identification of catalytically active groups of wheat (Triticum aestivum) germ lipase

The active site of wheat germ lipase was studied by the Dixon method and chemical modification. The profile of curve logV = f(pH), pK and ionization heat values, lipase photoinactivation, and lipase inactivation with diethylpyrocarbonate and dicyclohexylcarbodiimide led us to assume that the active site of the enzyme comprises the carboxylic group of aspartic or glutamic acid and the imidazole group of histidine. Apparently, the OH-group of serine plays a key role in catalysis: as a result of incubation for 1 h in the presence of phenylmethylsulfonyl fluoride, the enzyme activity decreased by more than 70%. It is shown that ethylenediamine tetraacetate is a noncompetitive inhibitor of lipase. Wheat germs are very healthful because they are rich in vitamins, essential amino acids, and proteins. For this reason, wheat germs are widely used in food, medical, and feed mill industries [1-3]. However, their use is limited by instability during storage, which is largely determined by the effect of hydrolytic and redox enzymes. Representative enzymes of this group are lipase (glycerol ester hydrolase, EC 3.1.1.3), which hydrolyzes triglycerides of higher fatty acids, and lipoxygenase (EC 1.13.11.13), which oxidizes polyunsaturated higher fatty acids.

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w0 (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.

Effect of aprotic solvents on the enzymatic activity of lipase in AOT reverse micelles

The modification of reverse micellar systems composed of AOT, isooctane, water by the addition of aprotic solvents has been performed. The impact of this change on the activity, stability and kinetics of solubilized Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was investigated. Of seven aprotic solvents tested, dimethyl sulfoxide (DMSO) was found to be most effective. It was found that lipase activity was enhanced by optimizing some relevant parameters, such as water–AOT molar ratio ( W 0), buffer pH and surfactant concentration. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to deduce some kinetic parameters ( V max, K m and K ad), and the values of K m and K ad were significantly reduced by the presence of DMSO. Higher lipase stability was found in AOT reverse micelles with DMSO compared with that in simple AOT systems with half-life of 125 and 33 days, respectively. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were used to elucidate the effects of DMSO on the properties of AOT reverse micelles.

Propionibacterium acnes GehA lipase, an enzyme involved in acne development, can be successfully inhibited by defined natural substances

Propionibacterium acnes, a usual inhabitant of human skin, plays an important role in acne development, related to the production of numerous enzymatic activities involved in the degradation of host molecules. Among these enzymes, P. acnes lipase (GehA, glycerol-ester hydrolase A) has been recognized as one of the major factors in the pathogenesis of acne, being responsible for the hydrolysis of sebum and the release of inflammatory compounds. Anti-acne treatments are based on the use of retinoids or benzoyl peroxide, frequently in combination with antibiotics. However, the low effectiveness of such treatments and the increasing antibiotic resistance has led to the development of alternative therapies such as Kampo formulations, containing traditional herbal drugs. Search for new anti-acne treatments led us to perform the cloning, characterization and inhibition of P. acnes GehA, considered an interesting pharmaceutical target for anti-acne therapies. The genetic, molecular and biochemical properties of the cloned lipase were analysed, and several inhibitor agents were tested, including natural substances like saponins, alkaloids or flavonoids. Among these, the flavonoids (±)-catechin and kaempferol were the most promising candidates for acne treatment, whereas saponins like glycyrrhicic acid and digitonin produced a lower inhibition of the enzyme. No inhibition by alkaloids was found. Therefore, the inhibition caused by (±)-catechin and kaempferol on GehA, together with their wide anti-acne properties and low toxicity, make them very suitable candidates for the treatment of acne and other P. acnes-related diseases.