Determination Of Lipase Activity Research Articles

The lipase/lipoxygenase bienzyme system in AOT reversed micelles in octane

In this work it is shown that the bienzyme lipase/lipoxygenase system can function in reversed micelles of bis(2-ethyl)hexyl sulfosuccinate (AOT) in octane. As a lipase substrate, a fish fat preparation (fat of sea mammals) with a high content of polyunsaturated fatty acids was used. It was demonstrated that the bienzyme reaction proceeded in a stationary mode and had a rate-limiting step catalyzed by lipase. Under optimal conditions, the efficacy of functioning of the bienzyme system was by an order of magnitude higher than that in water. The lipase/lipoxygenase bienzyme system can be used as a new method of spectrophotometric determination of lipase activity. The English version of the paper.

Validation and Comparison of Analytical Methods Based on the Release of p-Nitrophenol to Determine Lipase Activity in Milk

Three methods of determining lipase activity were optimized, validated, and compared using skim and whole milk. A chromogenic ester (p-nitrophenyl caprylate) was used in all to quantify the enzyme activity through the release of p-nitrophenol. It was measured colorimetrically (method A) or spectrophotometrically (methods B and C) with a clarifying reagent to render the samples measurable. Methods B and C differed because an inhibiting mixture was used in the latter method to better stop the enzymatic reaction. All the methods were reliable; they were linear in the range of 0 to 300 mU/ml of the enzyme, and the least detection and quantification limits were 9.31 and 31.03 mU/ml of lipase, respectively. Precision, measured as relative standard deviation, was between 1.52 and 4.94%, and mean recoveries ranged between 81 and 90%.Linearity, sensitivity, and accuracy were significantly different among the methods. Methods B and C had better linearity and sensitivity than method A, and the most accurate results were obtained with methods A and B in skim milk. Sensitivity was influenced by the fat content of the samples. On the other hand, the content of lipase did not influence the reliability of any method. Although, all of the methods were useful for routine analysis of quality control of milk, method B was most reliable. Moreover, it would be the method of choice because it was easier and less costly than the other methods.

Electrochemical determination of lipase activity in pharmaceutical preparations

Flow injection analysis with electrochemical detection was used for lipase assay in pharmaceutical preparations. The method is based on the fact that the current of voltammetric prepeak, accompanied by the reduction of 2-methyl-1,4-naphthoquinone (vitamin K 3, VK 3) due to the effects of fatty acids, is proportional to the acid concentration. A sample solution containing lipase was incubated with olive oil (substrate) at 37°C with stirring for 15 min, and the released fatty acids were extracted by ether. No emulsifying agent was needed in the incubation of the enzyme reaction mixture. An acid test solution prepared by dissolving the extracts in ethanol containing 3 mM VK 3 and 38 mM LiClO 4 (the same composition as the carrier solution) was injected into the flow injection system. The flow signal was linear to the lipase activity ranging from 10 to 1500 U/l for sample solutions obtained from the digestive preparations. By this method, analysis of the fat digestive power of the commercially available preparations could be accomplished with good results, thus showing this method to be practically useful.

An esterification method for determination of lipase activity

The choice of a lipase for an esterification reaction can be determined from the esterification reaction between butyric acid (0.16 M) and butanol (0.33 M) at 50 °C and agitated with 10 mg lipase. The decrease in butyric acid is measured by titration and 1 unit of lipase activity is defined as 1 μmol butyric acid consumed per min per mg lipase.

Lipases in autolysed cultures of filamentous fungi.

Fifty-one fungi from different genera and strains were checked in plate to determine lipase activity in protein precipitates from their autolysed cultures. Each of them was then analysed at 3.5, 6.5 and 9.2 pH units and, as a consequence, basic lipases with high activity at 9.2 pH were found after 1 h of incubation. Only 25% of the studied fungi showed this lipase activity, among them the best producers were fungi from genus Fusarium (47% of fungi had lipase activity). In addition to lipase activity, Fusaria showed a low hydrolytic activity on cutin and suberin. The genus Aspergillus produced lipase and cutinase activity to a similar extent. Aspergillus nidulans 2544 also showed suberinase activity in a considerable amount. Penicillium species had very low activities. Other species and strains from genus Trichoderma, order Mucorales and class Basidiomycetes, did not show lipase activity in their degradative processes.

New fluorescent glycerolipids for a dual wavelength assay of lipase activity and stereoselectivity

A new type of fluorogenic alkyldiacyl glycerols was synthesized and used as fluorogenic substrates for the analysis of lipase activities and stereoselectivities. These compounds contain pyrene or perylene as a fluorophore and the trinitrophenyl-amino- (TNP) residue as a quencher. Both substituents are covalently bound to the ω-ends of the sn-2 and sn-1(3) acyl chains, respectively. Upon glycerolipid hydrolysis, the residues are separated from each other thus allowing determination of lipase activity from the continuous increase in fluorescence intensity which is caused by dequenching. Mixtures of enantiomeric fluorogenic alkyldiacyl glycerols, selectively labeled with pyrene or perylene as fluorophores, can be used for a dual-wavelength ‘stereoassay’ of lipases. Since absorption and emission maxima of both labels are clearly separated, hydrolysis of the respective enantiomeric substrates can be distinguished simultaneously, and the difference in their rates of hydrolysis can be taken as a parameter for the stereopreference of a lipase.

Determination of Lipase Activity by High Performance Liquid Chromatography

Lipase unit is defined as that amount of enzyme needed to produce 1 μmol non-esterified fatty acid in one minute under specified conditions. Non-esterified fatty acid is traditionally measured by acid-base titration. This was done in the present study in place of High-performance liquid chromatography (HPLC) method. HPLC with a light scattering detector was conducted to measure non-esterified fatty acids to determine the activity of lipase from Candida cylindracea. HPLC result was essentially the same as that obtained by titration, the former method being more sensitive.

A Semiautomated Reflectance Colorimetric Method for the Determination of Lipase Activity in Milk

Measurement of heat-stable lipase activity in dairy products relies on methods that are slow or that cannot be used in turbid solutions, which limits their industrial value. A need exists for a rapid, simple, informative assay to detect lipase activity in dairy products. In this study, we observed that hydrolysis of p-nitrophenyl esters, monitored by reflectance colorimetry, was linearly correlated to spectrophotometry (R2=0.93) and release of titratable FFA (R2=0.92 to 0.97), indicating that chromogenic substrates were useful in determining lipase activity. However, at the concentrations reported in milk, FFA inhibited p-nitrophenyl caprylate hydrolysis, which led to an underestimation of lipase activity in milk that had previously undergone lipolysis. Milk fat also significantly reduced hydrolysis of the chromogenic substrates tested but could be accounted for by a correction equation. To demonstrate the use of the assay, lipase activity in UHT skim milk inoculated with Pseudomonas fluorescens AFT36 was followed using reflectance colorimetry and tributyrin agar. Lipase activity increased as cell numbers increased during 106h of incubation. Extracellular lipase activity was detected after 10h of incubation using reflectance colorimetry, but 28h were required using tributyrin agar. Reflectance colorimetry and chromogenic substrates allowed a rapid, sensitive, and meaningful detection of esterase and lipase activity in milk.

Titrimetric test for lipase activity using stabilized triolein emulsions

A common assay system for lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) activity is based on the measurement of free fatty acids, liberated from triacylglycerols, such as triolein, by enzymatic hydrolysis. This enzymatic reaction involves, in addition to substrate concentration, the size of the surface area of the oil-water interface in the assay system. Only sufficiently dispersed and stabilized oil-water emulsions are suitable for reproducible determination of lipase activity, e.g., in subcellular fractions of the oil storing cotyledons from rape seedlings. Stabilization of the emulsified substrate by both gum arabic (Acacia) and the detergent, desoxycholate, was found to be crucial to the measurement of the lipase activity in a pH-stat. Furthermore, complete ionization of free fatty acids is achieved at pH 9.0 and, consequently, highest lipase activity was found in a test medium adjusted to this pH value. Inclusion of CaCl 2 into the assay medium leads to a decrease in lipase activity, addition of NaCl to an increase. Preincubation of enzyme preparations with different detergents activates the lipolytic breakdown of triolein emulsions. Producing a large surface area in the triolein emulsion and stabilizing it for hours, increases the sensitivity of the titrimetric assay method. Using such conditions it was possible to determine, for the first time, the low lipase activity in non-fatty tissues, such as hypocotyls of rape seedlings.

Hydrolysis of p-nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: A simple test for the determination of lipase activity in organic media

The rate of p-nitrophenyl palmitate (pNPP) hydrolysis in n-heptane by a lipase preparation from Pseudomonas cepacia was studied. In organic media, the reaction rate could be easily measured by spectrophotometry after extraction of the liberated p-nitrophenol by an alkaline water phase. Optimal conditions for substrate hydrolysis were selected and proposed for the easy and fast assay of lipase activity in organic media. Agitation and the amount of added water showed little effect on reaction rates. A temperature of 37°C and substrate concentration of 50 m m were selected. The reaction rate in n-heptane was only 0.16% of that in water.

New fluorogenic triacylglycerol analogs as substrates for the determination and chiral discrimination of lipase activities

A new type of fluorogenic and isomerically pure 1(3)-O-alkyl-2,3 (3,2)-diacyl glycerols was synthesized that can be used as substrate for the determination of lipase activities. These compounds contain a fluorescent pyrene acyl chain and, as a potent quencher of pyrene fluorescence, a trinitrophenylamino acyl residue. In their intact form, the fluorogens show only low fluorescence intensity. Upon lipase-induced or chemical hydrolysis of the substrates, however, the fluorophore and quencher separate from each other. This leads to a gradual increase in pyrene fluorescence, reflecting the time-dependent progress of lipolysis and, under substrate saturation conditions, lipase activity. This lipase assay is continuous and does not require separation of substrate and reaction products. Short- and long-chain homologues as well as optical isomers of the fluorogenic alkyldiacyl glycerols were hydrolyzed by pancreatic lipase, hepatic lipase, and lipo-protein lipase at highly different rates depending on the substrate or enzyme preparation and source (e.g., postheparin plasma or cultured cells). It is proposed that a useful set of enantiomeric and/or homologous substrates in combination with appropriate reaction media might be applied to the selective determination of a lipase in a mixture of lipases, e.g., hepatic and lipoprotein lipase in PHP, for medical diagnostics.

Enantiomeric perylene-glycerolipids as fluorogenic substrates for a dual wavelength assay of lipase activity and stereoselectivity.

A new type of fluorogenic alkyldiacyl glycerols was synthesized and used as fluorogenic substrates for the analysis of lipase activities and stereoselectivities. These compounds contain perylene as a fluorophore and the trinitrophenylamino (TNP) residue as a quencher. Both substituents are covalently bound to the omega-ends of the sn-2 and sn-1 (3) acyl chains, respectively. Upon glycerolipid hydrolysis, the residues are separated from each other thus allowing determination of lipase activity by the continuous increase in fluorescence intensity which is caused by dequenching. Using enantiomeric pairs of these compounds, we were able to analyze lipase stereoselectivity depending on the reaction medium. Mixtures of enantiomeric fluorogenic alkyldiacyl glycerols, selectively labelled with pyrene or perylene as fluorophores, can be used for a dual-wavelength "stereoassay" of lipases. Since absorption and emission maxima of both labels are clearly separated, hydrolysis of the respective enantiomeric substrates can be determined simultaneously, and the difference in the rates of hydrolysis can be taken as a parameter for the stereopreference of a lipase. Hydrolysis rates measured with perylene-substituted lipids are generally lower than those obtained with the pyrene analogs. Thus, with a mixture of perylene and pyrene-substituted lipids, we observe a higher apparent stereoselectivity of lipases since we measure a combination of stereo- and substrate selectivity. In the presence of albumin, all microbial lipases tested so far exhibit stereopreference for the sn-1 glycerol position. In our assay, the apparent stereoselectivities are highest if in the presence of albumin, the sn-1 position carries pyrene and the sn-3 position is substituted with perylene. The lipase stereoselectivity assay described here requires the simultaneous measurement of the fluorescence intensities at two different wavelengths in a single cuvette and can thus be carried out using existing and cheap instrumentation that was developed for the fluorimetric analysis of Ca+2 concentrations.

Determination of lipase activity in porcine pancreas and clinical analysis of lipase in human serum with surface acoustic wave enzyme sensor system

A new kind of surface acoustic wave (SAW) enzyme sensor system has been applied to the assay of extracted lipase activity based on triacetin solution as substrate. A linear relationship between the frequency change and the enzyme activity up to 500 U/L has been obtained, and the detection limit has been evaluated to 0.3 U/L. Kinetic parameters of the extracted lipase as well as that of the standard enzyme have been estimated. The effects of temperature, pH value and surfactants have been investigated. A comparison between SAW sensor system and the conductometric method has been carried out. The technique has been applied to the determination of lipase activity in serum samples.

Bacterial lipases

Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activitiy. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the α/β-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved β-ϵ-Ser-α motif. The active site is covered by a lid-like a-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate. This movable lid-like α-helix explains at a molecular level the lipase-specific phenomenon of interfacial activation. At least some of the pathogenic bacterial species produce a lipase which has been studied with respect to its role as a virulence factor. Lipases of Propionibacterium acnes and Staphylococcus epidermidis may be involved in colonization and persistence of these bacteria on the human skin. Lipases of S. aureus and P. aeruginosa are produced during the bacterial infection process and, at least in vitro, considerably impair the function of different cell types involved in the human immune response like macrophages or platelets. The present state of knowledge suggests to classify the lipases as important bacterial virulence factors which exert their harmful effects in combination with other bacterial enzymes, in particular the phospholipases C. Most of the steadily increasing interest in bacterial lipases is based on their biotechnological applications which are partly based on their potential to catalyze not only hydrolysis but also synthesis of a variety of industrially valuable products. Optically active compounds, various esters and lactones are among the substances synthesized using bacterial lipases. Recently, an important application emerged with the addition of bacterial lipases to household detergents in order to reduce or even replace synthetic detergent chemicals which pose considerable environmental problems. As a main conclusion, [ipases represent an extremely versatile group of bacterial extracellular enzymes that are capable of performing a variety of important reactions, thereby presenting a fascinating field tot future research.

Bacterial lipases

Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activity. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the alpha/beta-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved beta-epsilon Ser-alpha motif. The active site is covered by a lid-like alpha-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Stable expression of enzymatically active human pancreatic lipase in V79 cells: purification and characterization of the recombinant enzyme

The serum activity of human pancreatic lipase (HPL; EC 3.1.1.3), the main lipolytic enzyme secreted by the pancreas, is a valuable marker of pancreatic disorders. However, determining lipase activity in human serum is difficult because the substrates used vary in their lipase specificity. A lipase reference material is therefore needed. We describe the production of recombinant HPL (rHPL) in V79 Chinese hamster lung cells. A cDNA encoding the sequence of HPL was subcloned into the pcDNAI eukaryotic expression vector, which was then used to transfect V79 cells. The 50-kDa purified recombinant enzyme is fully active, is glycosylated, and has a pI of 7.0. The catalytic properties of rHPL, determined by using triolein as the substrate, were similar to those of the native enzyme. The V79rHPL cell line we developed might be useful for the production of rHPL suitable for the preparation of a reference material.

ChemInform Abstract: On‐Line Determination of Lipase Activity and Enantioselectivity Using an Immobilized Enzyme Reactor Coupled to a Chiral Stationary Phase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa

Lipase from Pseudomonas aeruginosa is a M r, 29 kDa protein with a single functional disulfide bond as shown by a shift in electrophoretic mobility after treatment with dithiothreitol and iodoacetamide. Limited proteolysis of lipase with Staphylococcus aureus protease V8 resulted in cleavage after amino acid residues Asp 38 and Glu 46. Comparison of the lipase amino acid sequence with those of other hydrolases with known 3D structures indicated that the folding pattern might be compatible with the α/β hydrolase fold, thereby allowing us to construct a 3D model which fitted the biochemical properties. The model predicts a catalytic triad consisting of Ser 82, Asp 229 and His 251, and contains a disulfide bond connecting residues Cys 183 and Cys 235. Residues Asp 38 and Glu 46 are located at the surface of the enzyme, whereas the disulfide bond is rather inaccessible, which is in agreement with the finding that the protein needed to be partly unfolded before a reduction of the disulfide bond could take place. A striking prediction from the model was the lack of a lid-like α-helical loop structure covering the active site which confers to other well-characterized lipases a unique property known as interfacial activation. Experimental determination of lipase activity under conditions where the substrate existed either as monomeric solutions or aggregates confirmed the absence of interfacial activation.

Use of pyrenemethyl laurate for fluorescence-based determination of lipase activity in intact living lymphoblastoid cells and for the diagnosis of acid lipase deficiency

Pyrenemethyl laurate (PMLes), a fluorogenic substrate for determining in vitro lipase activity [Nègre, Salvayre, Dagan and Gatt (1989) Biochim. Biophys. Acta 1006, 84-88], has been administered to cultured lymphoblastoid cells from normal subjects and from a patient affected with Wolman disease, which is characterized by a deficiency of lysosomal acid lipase. The intracellular degradation of PMLes was dependent on the mode of administration of the substrate into the cells, and occurred by two separate pathways involving lysosomal and extra-lysosomal hydrolases. PMLes incorporated into LDL was taken up by normal lymphoblastoid cells through the apolipoprotein-B/E-receptor-mediated pathway and degraded in the lysosomal compartment, as suggested by the degradation block in Wolman cells. In contrast, when PMLes dissolved in 2% dimethyl sulphoxide was added directly to the culture medium, its hydrolysis was similar in lymphoblastoid cells from controls and from patients affected with Wolman disease, neutral lipid storage disease or familial hypercholesterolaemia. This suggested that the administered PMLes was degraded by a non-lysosomal enzyme which is not deficient in Wolman cells. This enzyme also differs from the neutral lipase system which is deficient in lymphoblastoid cells from patients with neutral lipid storage disease. When pyrenemethanol was administered directly to the cell culture, it was only poorly acylated and was rapidly released into the culture medium. These results and the fluorescence properties of PMLes ('monomeric' emission in a hydrophobic environment and 'excimeric' emission in a hydrophilic environment) and pyrenemethanol ('monomeric' emission in a hydrophilic environment) allowed us to design a 'direct reading' procedure by monitoring (without any lipid extraction) the fluorescence of intact living cells and that of the culture medium during pulse-chase experiments. This method allowed the direct evaluation of the time course of in situ degradation of PMLes. In pulse-chase experiments with LDL-PMLes, the fluorescence of normal cells decreased relatively rapidly with time whereas the fluorescence of the culture medium increased concomitantly. With Wolman cells, the cellular fluorescence decreased only very slightly, whereas that of the culture medium remained at the basal level; this demonstrates the catabolic block in intact living cells from patients with Wolman disease. In vitro degradation of PMLes indicated the existence of two PMLes-degrading enzymes in lymphoblastoid cell homogenates: one is the acid lipase which is involved in PMLes degradation in the lysosomal compartment (and is deficient in Wolman cells), while the second is a cytoplasmic enzyme (not deficient in Wolman cells).

On-line determination of lipase activity and enantioselectivity using an immobilized enzyme reactor coupled to a chiral stationary phase

Lipase has been immobilized on an artificial immobilized membrane HPLC phase and the enzyme reactor coupled to a HPLC chiral stationary phase. The resulting system was used for on-line determination of the enantioselectivity of the immobilized enzyme. Enantioselective hydrolyses using lipase from Candida cylindracea immobilized on an IAM HPLC support.