Esterolytic activities of rat intestinal mucosa. 1. Characterization, cellular distribution and subcellular localization of a glycerol-ester hydrolase.

The preferential cellular distribution in the villus tip and the subcellular localization in the endoplasmic reticulum of an intestinal glycerol-ester hydrolase from rat mucosa are described. The enzyme is shown not to be from either pancreatic or bacterial origin; it catalyzes the hydrolysis of short- and medium chain triglycerides and of p-nitrophenylacetate. Contrarily to the specificity found for the pig intestinal lipase (Serrero, Négrel and Ailhaud, 1975), no activity is detectable against acylCoA; a thiolester hydrolase different from the glycerol-ester hydrolase was demonstrated after differential solubilization and chromatographic separation. A high proportion of glycerol-ester hydrolase is present in the intestinal lumen; its possible complementary role in lipid degradation is discussed.     

A specific histochemical method for the demonstration of the activity of pancreatic lipase

Synopsis Emulsified long-chain triglyceride, a specific substrate for the enzyme pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3), has been used in a modification of the Gomori technique for the demonstration of lipase. In the range of tissues examined (pancreas, testis, cardiac stomach and liver), true pancreatic lipase activity was revealed only in pancreatic tissue, by contrast with results obtained with less specific methods.     

Structure-function relationships in the carboxylic-ester-hydrolase superfamily. Disulfide bridge arrangement in porcine intestinal glycerol-ester hydrolase.

CNBr fragments from porcine intestinal glycerol-ester hydrolase were separated by SDS/PAGE under reducing and nonreducing conditions, and their amino-acid sequences were analysed. Two intra-chain disulfide bridges were identified, namely Cys70-Cys99 (loop A) and Cys256-Cys267 (loop B). As the Cys71 sulfhydryl group could not be alkylated with iodoacetamide, it is suggested that the residue is blocked rather than being present in the free form. The two disulfide bridges of intestinal glycerol-ester hydrolase are present in the cholinesterase family, although the enzyme showed only about 35% identity with these proteins. Furthermore, the finding that glycerol-ester hydrolase was partly inactivated under reducing conditions suggests that one or both disulfide bridges are important for the enzyme conformation. Lastly, glycerol-ester hydrolase was also found to hydrolyse cholinergic substrates, although residues Trp86 and Asp74 which are considered to be the main constituents of the 'anionic' subsite responsible for substrate binding in cholinesterases were absent from loop A. Other amino-acid residues in the glycerol-ester hydrolase may therefore be responsible for the binding of cholinergic substrates to the enzyme.     

Carboxylic ester hydrolases of rat pancreatic juice

An attempt was made to establish the number and characteristics of the enzymes in pancreatic juice that hydrolyze nitrogen- and phosphorus-free esters of fatty acids. For this purpose model compounds were hydrolyzed by lyophilized rat pancreatic juice under conditions that accelerated or inhibited the reactions. Although it is not established with certainty, it is suggested that three enzymes are responsible for the hydrolysis of fatty acid esters. The first enzyme is glycerol-ester hydrolase (EC 3.1.1.3) or lipase. This enzyme hydrolyzes water-insoluble esters of primary alcohols. The reaction occurs at an oil/water interface and is inhibited by bile salts at pH 8. The enzyme is relatively stable at pH 9, but unstable at pH 4. It has a broad pH optimum between 7.5 and 9.5. The second enzyme hydrolyzes esters of secondary alcohols and of other alcohols as well. It has an absolute requirement for bile salts and has a pH optimum at about 8. The enzyme is unstable in pancreatic juice when maintained at pH 9, probably due to the action of trypsin. It may be identical with sterol-ester hydrolase (EC 3.1.1.13). The third enzyme hydrolyzes water-soluble esters. It too has an absolute requirement for bile salts, although a smaller amount is necessary for maximum activity. This enzyme also is unstable at pH 9, but can be differentiated from the preceding enzyme by its stability at pH 4 and its pH optimum of 9.0. Carboxylic-ester hydrolase (EC 3.1.1.1) is not found in pancreatic juice, although it is present in pancreatic tissue.     

Relative rates of hydrolysis by rat pancreatic lipase of esters of C2-C18 fatty acids with C1-C18 primary n-alcohols

The rate at which rat pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3) hydrolyzes the esters of primary n-alcohols containing from 1 to 18 carbon atoms with fatty acids containing from 2 to 18 carbon atoms was determined. The speed of hydrolysis was influenced, apparently independently, by both the acyl and the alkyl chains. With respect to the fatty acid moiety, the esters of dodecanoic acid were usually split at the most rapid rate. Esters of butyric acid were the next most susceptible. In the case of the alcohol moiety, esters of heptyl alcohol were hydrolyzed most rapidly. On the basis of the pattern of the relative rates of hydrolysis, it is proposed that the influence of the alcohol component is a result of its orienting the ester molecule at the oil/water interface. The fatty acid effect is attributed to enzyme-substrate specificity.     

Purification of carboxyl ester lipase from human pancreas and the amino acid sequence of the N-terminal region

A procedure for the purification of carboxyl ester lipase from human pancreas has been developed. The determined N-terminal 10 amino acid residues of the purified enzyme, NH2-Ala-Lys-Leu-Gly-Ala-Val-Tyr-Thr-Glu-Gly, was identical to the terminal of human milk bile salt-activated lipase. The human pancreatic carboxyl ester lipase has an apparent molecular weight slightly smaller than that of human milk bile salt-activated lipase (105,000 vs 125,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, it is possible that the human pancreatic carboxyl ester lipase and human milk bile salt-activated lipase could be produced by the same gene by a different splice or post-translational modification. Alternatively, they could simply be the products of two closely related but separate genes.     

Modeling the catalytic activity and kinetics of lipase (glycerol-ester hydrolase)

In order to design industrial scale reactors and proceises for multi-phase biocatalytic reactions, it is essential to understand the mechanisms by which such systems operate. To illustrate how such mechanisms can be modeled, the hydrolysis of the primary ester groups of triglycerides to produce fatty acids and monoglycerides by lipase (glycerol-ester hydrolase) catalysis has been selected as an example of multiphase biocatalysis. Lipase is specific in its behavior such that it can act only on the hydrolyzed (or emulsified) part of the substrate. This follows because the active center of the enzyme is catalytically active only when the substrate contacts it in its hydrolyzed form. In other words, lipase acts only when it can shuttle back and forth between the emulsion phase and the water phase, presumably within an interphase or boundary layer between these two phases. In industrial applications lipase is employed as a fat splitting enzyme to remove fat stains from fabrics, in making cheese, to flavor milk products, and to degrade fats in waste products. Effective use of lipase in these processes requires a fundamental understanding of its kinetic behavior and interactions with substrates under various environmental conditions. Therefore, this study focuses on modeling and simulating the enzymatic activity of the lipase as a step towards the basic understanding of multi-phase biocatalysis processes.     

Purification from human plasma of a heparin-released lipase with activity against triglyceride and phospholipids.

A triglyceride lipase different from lipoprotein lipase, but measurable only after intravenous heparin injection, has been isolated from human plasma by sequential use of heparin-Sepharose and concanavalin A-Sepharose affinity chromatography. Using these procedures, phospholipase A1 activity was found to chromatograph identically with the triglyceride lipase. The constancy of the ratio of activities after isoelectric focusing (pI 4.1) and during thermal deactivation indicates that this enzyme has hydrolase activity against both triglycerides and phospholipids. This conclusion was supported further by the homogeneity of the protein as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

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 Chromobacterium viscosum 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.     

Glycerol Ester Hydrolase Activity of Microbacterium thermosphactum

Microbacterium thermosphactum possesses a significant glycerol ester hydrolase (lipase, EC 3.1.1.3) activity and a weak but definite carboxylic ester hydrolase (esterase, EC 3.1.1.1) activity. Harvested whole cell preparations contained 53 units of lipase activity with tripropionin as the substrate. This activity decreased with an increasing chain length of fatty acid in the triglyceride to 13 units with trilaurin as the substrate and no activity with tripalmitin. Maximum lipase activity was found at a temperature of 35 to 37 C and at a pH of 7.1 to 7.3. Lipase activity was associated with three different protein peaks when the protein of cell-free extract was fractionated by polyacrylamide gel electrophoresis.     

Purification of rat pancreatic lipase

A procedure for the isolation of lipase (glycerolester hydrolase, EC 3.1.1.3) from rat pancreas is described. The purification scheme includes homogenization of the pancreas, centrifugation at 3,000 rpm, centrifugation at 40,000 rpm, DEAE-cellulose chromatography, precipitation of amylase as the amylase-glycogen complex, gel filtration of the amylase-free proteins on Sephadex G-100, and chromatography on carboxymethyl-Sephadex C-50. The enzyme showed only one band on polyacrylamide gel electrophoresis and had a specific activity of 5330 +/- 80 units/mg of protein.     

Evidence that pancreatic lipase is responsible for the hydrolysis of cutin,a biopolyester present in mammalian diet,and the role of bile salt and colipase in this hydrolysis

The enzyme, which catalyzes hydrolysis of cutin, an insoluble biopolyester of hydroxy and epoxy fatty acids, was purified from porcine pancreas. With three different purification methods, previously used for the purification of pancreatic lipase, it is shown that cutin hydrolase is pancreatic lipase. This enzyme released oligomers and all types of monomers from the polymer with a pH optimum around 7.5. Taurodeoxycholate inhibited cutin hydrolysis by lipase and colipase reversed this inhibition. Evidence is presented which suggests that bile salt stabilizes the enzyme at the surface of the insoluble substrate and that the interaction of the polymer surface with the lipase-colipase-bile salt system is similar to that previously observed with triglycerides. Diethyl-p-nitrophenyl phosphate inhibited cutin hydrolysis by lipase but the hydrolysis was insensitive to diisopropyl fluorophosphate.     

Purification and partial characterization of feline classical pancreatic lipase

Classical pancreatic lipase has been purified and partially characterized in many species. The objective of this project was to purify feline classical pancreatic lipase (fPL) from pancreatic tissue and partially characterize this protein. Pancreata were collected from cats (Felis catus) euthanized for unrelated research projects. Fat was removed by trimming away grossly visible fat and by extraction in organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamidine and phenylmethylsulfonyl fluoride, followed by anion-exchange, size-exclusion, and cation-exchange chromatography. Feline pancreatic lipase was successfully purified from feline pancreatic tissue. The purified product showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of approximately 52.5 kDa. Exact molecular mass was determined by mass spectrometry as 52.4 kDa. Approximate specific absorbance at 280 nm of fPL was 1.18 for a 1 mg/ml solution. N-terminal amino acid sequence of the first 25 amino acid residues showed the sequence Lys-Glu-Ile-?-Phe-Pro-Arg-Leu-Gly-?-Phe-Ser-Asp-Asp-Ala-Pro-Trp-Ala-Gly-Ile-Ala-Gln-Arg-Pro-Leu. This sequence showed close homology with the amino acid sequence of classical pancreatic lipase in other species.     

Purification of classical pancreatic lipase from dog pancreas

The purification of canine classical pancreatic lipase from canine pancreatic juice, but not from pancreatic tissue, has been reported previously. Given the logistic difficulties associated with collection of pancreatic juice in dogs and efforts to minimize experiments in live animals the objective of this project was to purify canine classical pancreatic lipase from dog pancreas. Dog pancreata were collected from research dogs that had been sacrificed for unrelated research projects. Pancreatic tissue was delipidated using organic solvents. The delipidated pancreatic extract was further purified by extracting the enzymes in a Tris-buffer containing two different protease inhibitors, benzamindine and phenylmethylsulfonyl fluoride (PMSF), followed by anion exchange chromatography, gel-filtration, and cation exchange chromatography. The purified protein showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of approximately 50.7. Isoelectric focusing showed isoelectric points ranging from 6.0 to 6.2. N-terminal amino acid sequencing of the first 25 amino acid residues showed the sequence Lys-Glu-Val-X-Phe-Pro-Arg-Leu-Gly-X-Phe-Ser-Asp-Asp-Ser-Pro-Trp-Ala-Gly-Ile-Val-Glu-Arg-Pro-Leu. This sequence showed close homology with classical pancreatic lipase in pigs, horses, and human beings. We conclude that canine classical pancreatic lipase can be successfully purified from canine pancreatic tissue.     

Subcellular localization of cholesterol ester hydrolase in the human intestine

Immunocytochemistry and subcellular fractionation were used to localize the cholesterol ester hydrolase in the human small intestine. A positive immunoreaction, when using antibodies directed against pancreatic cholesterol ester hydrolase, was mainly found in endocytotic vesicles. Moreover, a label by gold particles was observed in intercellular spaces where lymphatic tissue merges. No specific immunoreactivity was obtained with the mucosa when sera directed against human pancreatic chymotrypsinogen and human pancreatic lipase were used. Conventional subcellular fractionation was performed after extensive washing of enterocytes to rule out any possible contamination by pancreatic enzymes. In these conditions a bile salt-dependent cholesterol ester hydrolase activity was detected in the soluble fraction of cells. Data agree with the concept that the intestinal cholesterol ester hydrolase may have a pancreatic origin. The absorption, if any, of this enzyme by enterocytes seems specific since other pancreatic (pro)enzymes tested (lipase, chymotrypsinogen) are not detected in these cells.     

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 (W₀), 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.     

Purification of extracellular lipase from Pseudomonas aeruginosa.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s.     

Calf-spleen nicotinamide--adenine dinucleotide glycohydrolase. Solubilization purification and properties of the enzyme.

NAD glycohydrolase of calf spleen was solubilized with pancreatic lipase and purified approximatively 800-fold to a specific activity of 7 units/mg of protein by successive DEAE-cellulose and carboxymethyl-cellulose chromatography. The purified enzyme has a molecular weight of 24,000 and is characterized by a double band on disc gel electrophoresis. Some kinetic properties of the NAD-glycohydrolase-catalyzed hydrolsis of NAD have been examined using a titrimetric assay for enzyme activity. The reaction is subject to inhibition be excess of substrate, which disappears at high ionic strength and low pH. At a pH below 5 the kinetic displays an apparent activation by substrate. The effects of pH (4.5-9.0) on the kinetic parameters do not reveal an essential ionizable group in the catalytic process.     

A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene

An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity. Keywords: Bacter     

Further characterization of intestinal lactase/phlorizin hydrolase

Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig enzyme were shown to have a considerable activity against cellotriose and cellotetraose, and a low but significant activity against cellulose. The lactase/phlorizin hydrolase isolated from pigs in which the pancreatic ducts had been disconnected 3 days before death and from Ca2+-precipitated enterocyte membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase.