Carboxylester hydrolases, commonly named esterases, consist of a large spectrum of enzymes defined by their ability to catalyze the hydrolysis of carboxylic ester bonds and are widely distributed ...among animals, plants, and microorganisms. Lipases are lipolytic enzymes which constitute a special class of carboxylic esterases capable of releasing long-chain fatty acids from natural water-insoluble carboxylic esters. However, up to now, several unsuccessful attempts aimed at differentiating “lipases” from “esterases” by using various criteria. These criteria were based on the first substrate used chronologically, primary sequence comparisons, some kinetic parameters, or some structural features.
Lipids are biological compounds which, by definition, are insoluble in water. Taking into account this basic physico-chemical criterion, we primarily distinguish lipolytic esterases (L, acting on lipids) from nonlipolytic esterases (NL, not acting on lipids). In view of the biochemical data accumulated up to now, we proposed a new classification of esterases based on various criteria of physico-chemical, chemical, anatomical, or cellular nature. We believe that the present attempt matters scientifically for several reasons: (1) to help newcomers in the field, performing a few key experiments to figure out if a newly isolated esterase is lipolytic or not; (2) to clarify a debate between scientists in the field; and (3) to formulate questions which are relevant to the still unsolved problem of the structure–function relationships of esterases.
Excessive accumulation of triacylglycerol in peripheral tissues is tightly associated with obesity and has been identified as an independent risk factor for insulin resistance, type 2 diabetes, and ...cardiovascular complications. Here we show that ablation of carboxylesterase 3 (Ces3)/triacylglycerol hydrolase (TGH) expression in mice (
Tgh
−/−) results in decreased plasma triacylglycerol, apolipoprotein B, and fatty acid levels in both fasted and fed states. Despite the attenuation of very low-density lipoprotein secretion, TGH deficiency does not increase hepatic triacylglycerol levels.
Tgh
−/− mice exhibit increased food intake, respiratory quotient, and energy expenditure without change in body weight. These metabolic changes are accompanied by improved insulin sensitivity and glucose tolerance.
Tgh
−/− mice have smaller sized pancreatic islets but maintain normal glucose-stimulated insulin secretion. These studies demonstrate the potential of TGH as a therapeutic target for lowering blood lipid levels.
► Triacylglycerol hydrolase participates in adipose and liver lipid metabolism ► Loss of TGH lowers plasma fatty acid and triglyceride levels in mice ► Loss of TGH increases energy expenditure in mice ► Loss of TGH results in improved glucose metabolism and β cell function
Rhizopus oryzae lipase (ROL) was immobilized by physical adsorption onto silica aerogels. The functional properties of immobilized lipase were determined and compared to the soluble lipase ones. The ...optimum temperature for both free and immobilized lipase activities was 37°C. We found that the immobilization of R. oryzae lipase onto silica aerogels increased remarkably its stability at high temperatures and within a wade pH range. Besides the immobilized enzyme exhibited a high tolerance to apolar solvent and retained its fully activity in suspension after 4 months of storage at 4°C. This immobilized biocatalyst is applied in n-butyl oleate synthesis by esterification of oleic acid with n-butanol, using hexane as an organic solvent. The best conversion yield of the ester butyl oleate was obtained with the immobilized lipase (80% versus 35% with the free lipase). This catalytic esterification has been carried on the presence of hexane at 37°C with oleic acid to butanol molar ratio of 1:1 and 450IU of immobilized lipase. Furthermore, the reuse of the lipase immobilized by adsorption allowed us to observe that its can achieved 12 successive cycles, without a significant loss of its catalytic activity. Such results revealed good potential for recycling under non-aqueous system.
We have isolated a lipolytic halotolerant bacterium, designated as CJ3, that was identified as a Staphylococcus sp. Culture conditions were optimized and the highest extracellular lipase production ...amounting to 5U/ml was achieved after 24h of cultivation. The extracellular lipase was purified 24-fold by ammonium sulfate precipitation and a Sephacryl S-200 chromatography, and its molecular mass was found to be around 38kDa, as revealed by SDS-PAGE and gel filtration. The lipase substrate specificity was investigated using short (tributyrin) and long (olive oil) chain triglyceride substrates. The lipase was inhibited by submicellar concentrations of Triton X-100, and maximum specific activities were found to be 802U/mg on tributyrin and 260U/mg on olive oil at pH 8.0 and 45°C. The lipase was fairly stable in the pH range from 6.0 to 9.0, and about 69% of its activity was retained after incubation at 45°C for 60min. The enzyme showed a high tolerance to a wide range of salt concentration and a good stability in organic solvents, especially in long-chain alcohols.
In the present work we determined the total phenolic content of Aloe vera leaf skin (AVLS) extracts by using various solvents (hexane, chloroform-ethanol (1/1), ethyl acetate, butanol and water). We ...have also evaluated the antioxidant and the anti-PLA2 properties of these extracts by measuring their inhibition potency on the human pro-inflammatory phospholipase A2 (group IIA).
The water extract exhibits the highest inhibitory effect with an IC50 = 0.22 mg/ml and interestingly no effect was observed on the digestive phospholipase A2 (group IB) even at a concentration of 5 mg/ml. Antioxidant activities were also analyzed and the most active extracts were observed when using chloroform ethanol (1/1) and ethyl acetate (IC50 = 0.274 and 0.326 mg/ml, respectively). Analysis of the total phenolic content reveals that the water extract, with the best anti-PLA2 effect, was poor in phenolic molecules (2 mg GAE/g). This latter value has to be compared with the chloroform-ethanol and the ethyl acetate extracts (40 and 23.8 mg GAE/g, respectively), mostly responsible for the antioxidant activity.
A significant correlation was established between the total phenolic content and the antioxidant capacity but not with the anti PLA2 activity. Results from phytochemical screening suggest that the anti PLA2 molecules were probably catechin tannins compounds.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In order to identify fish enzymes displaying novel biochemical properties, we have chosen the common stingray (Dasyatis pastinaca), one of the most primitive living jawed aquatic vertebrates as a ...starting biological material to purify a lipase. A stingray pancreatic lipase (SPL) was purified from delipidated pancreatic powder. The SPL molecular weight was around 55 kDa which is slightly higher than that of known classical pancreatic lipases (50 kDa). This increase in the molecular weight was due to glycosylation. Like classic pancreatic lipases, SPL was found to be much more active on short-chain triacylglycerols than on long-chain ones. Natural detergents act as inhibitors of the SPL activity. This inhibition can be reversed by the addition of stingray colipase. Starting from total pancreatic messenger RNAs (mRNAs), partial stingray pancreatic lipase complementary DNA (cDNA) was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR) and cloned into the PGEM-T vector. Partial amino acid sequence of the SPL was homologous to that of Japanese eel, porcine, and human pancreatic lipases. A 3D structure model of the sequenced part of SPL was built using the 3D structure of porcine pancreatic lipase as template, since both lipases shared an amino acid sequence identity of 60 %.
Crab digestive phospholipase (CDPL) was purified from the hepatopancreas of
Carcinus mediterraneus crabs. Homogeneous enzyme was obtained after two chromatography steps: anion exchange and size ...exclusion HPLC column. Homogeneous CDPL has a molecular mass of 14
kDa as determined by SDS/PAGE analysis. Unlike known digestive phospholipases like porcine PLA
2 (PPPL), CDPL displayed its maximal activity at 50
°C and not at 37
°C. A specific activity of 40
U/mg for the purified CDPL was measured using PC as substrate under optimal conditions (pH 8 and 50
°C) in the presence of 8
mM sodium deoxycholate (NaDC) and 10
mM CaCl
2. In contrast to PPPL, purified CDPL was completely inactivated at 60
°C. The N-terminal sequence was determined by automatic Edman degradation. No similarity between 12 N-terminal amino acid residues of CDPL was found with those of known digestive phospholipases. CDPL appears to be a new member of invertebrate phospholipases, and it is potentially useful for treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds which can be used in the food industry.
Hormone-sensitive lipase (HSL) plays an important role in the mobilization of free fatty acids (FFA) from adipocytes. The inhibition of HSL may offer a pharmacological approach to reduce FFA levels ...in plasma and diminish peripheral insulin resistance in type 2 diabetes. In this work, the inhibition of HSL by substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones has been studied in vitro. 5-methoxy-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one (compound 7600) and 5-methoxy-3-(3-methyl-4-phenylacetamidophenyl)-1,3,4-oxadiazol-2(3H)-one (compound 9368) were selected as the most potent HSL inhibitors. HSL is inhibited after few minutes of incubation with compound 7600, at a molar excess of 20. This inhibition is reversed in the presence of an emulsion of lipid substrate. The reactivation phenomenon is hardly observed when incubating HSL with compound 9368. The molecular mechanism underlying the reversible inhibition of HSL by compound 7600 was investigated using high performance liquid chromatography and tandem mass spectrometry. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound per enzyme molecule. The inhibition by compound 7600 involves a nucleophilic attack by the hydroxy group of the catalytic Ser of the enzyme on the carbon atom of the carbonyl moiety of the oxadiazolone ring of the inhibitor, leading to the formation of covalent enzyme–inhibitor intermediate. This covalent intermediate is subsequently hydrolyzed, releasing an oxadiazolone decomposition product, carbon dioxide and the active HSL form. On the basis of this study, a kinetic model is proposed to describe the inhibition of HSL by compound 7600 in the aqueous phase as well as its partial reactivation at the lipid–water interface.
► The specific inhibition of hormone-sensitive lipase (HSL) by oxadiazolones was studied. ► The IC50 of compound 7600 and compound 9368 was found to be around 250 nM and 90 nM, respectively. ► The HSL inhibition is reversed in the presence of lipid substrate. ► The mechanism of the reversible inhibition of HSL by compound 7600 was investigated using LC-MS.
Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for ...discovering new lipolytic enzymes.
A marine stingray phospholipase A2 (SPLA2) was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C) in the presence of 4 mM NaTDC and 8 mM CaCl2 using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry.
SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nutritional properties of Diplotaxis simplex Spreng., Brassicaceae, an edible wild cruciferous largely distributed in North Africa, were investigated. Potassium (3690–3780mg/100g) and calcium ...(900–1170mg/100g) were the most concentrated minerals. Linoleinic acid was found to be the main fatty acid (25.4–27.7%), followed by palmitic acid (13.2–15.3%). Moreover, lipidic fraction of leaves was characterized by a relatively high rate of ethyl linoleate (14.4%) and phytol (17.6%). Ethyl acetate extract of D. simplex flowers showed concentration-dependent α-amylase (IC50 3.46mg/ml) and α-glucosidase (IC50 0.046mg/ml) inhibitory activities. The positive in vitro enzymes inhibition was confirmed by a maltose tolerance test, which showed that treatment with flowers extract significantly inhibited the rise in blood glucose levels of maltose-loaded mice comparable to the standard antihyperglycemic agent acarbose. From these results, it may be concluded that D. simplex flowers can be used effectively as a safer alternative therapy to control postprandial hyperglycemia.
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