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PubMed2001

Production, purification, characterization, and applications of lipases.

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. The many applications of lipases include speciality organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, resolution of racemic mixtures, and chemical analyses. This article discusses the production, recovery, and use of microbial lipases. Issues of enzyme kinetics, thermostability, and bioactivity are addressed. Production of recombinant lipases is detailed. Immobilized preparations of lipases are discussed. In view of the increasing understanding of lipases and their many applications in high-value syntheses and as bulk enzymes, these enzymes are having an increasing impact on bioprocessing.

Journal Article↗
PubMed2003

Identification of Propionibacterium acnes by polymerase chain reaction for amplification of 16S ribosomal RNA and lipase genes.

Propionibacterium acnes belongs to the cutaneous flora and is present in sebaceous follicles. The fatty acids that are released from sebum triglycerides by the action of this bacterial lipase play an important role in the pathogenesis of acne vulgaris. P. acnes is also involved in postoperative disorders and opportunistic infections in immunosuppressed hosts. Recently, it has been proposed that P. acnes causes sarcoidosis. Therefore, rapid isolation and identification of P. acnes is important. This study evaluated the polymerase chain reaction (PCR) for the detection of the 16S rRNA and lipase genes of P. acnes. The PCR used to detect the 16S rRNA gene could amplify the gene of P. acnes, but not the genes of the other tested strains of P. avidum, P. granulosum, P. lymphophilum, P. jensenii, P. acidipropionici and P. thoenii. The PCR to detect the lipase gene of P. acnes, however, could amplify not only the gene of P. acnes but also that of P. avidum. The PCR product of this lipase gene was not found in the strains of the other species tested. Therefore, the organism that has both the 16S rRNA gene and lipase gene was identified as P. acnes, while the strain with the lipase gene but not the 16S rRNA gene of P. acnes was characterized as P. avidum. These findings were confirmed by the conventional biochemical tests including lipase activity. Furthermore, out of the seven clinical isolates from acne vulgaris, four were identified as P. acnes and three as P. avidum by the PCR method and biochemical tests. The combination of two PCR, one for the detection of the 16S rRNA and the other of lipase genes was shown to be an easier, faster and more accurate method to identify P. acnes and P. avidum than conventional methods.

Journal Article↗
PubMed1981

Hormone-sensitive lipase in differentiated 3T3-L1 cells and its activation by cyclic AMP-dependent protein kinase.

Differentiation of 3T3-L1 fibroblasts to adipocyte-like cells was accompanied by a 19-fold increase in neutral triglyceride lipase activity, a 12-fold increase in diglyceride lipase activity, a 10-fold increase in monoglyceride lipase activity, and a 280-fold increase in cholesterol esterase activity. In contrast, acid acylhydrolase activities did not increase during differentiation. The rate of glycerol release from unstimulated intact cells increased by more than 1 order of magnitude upon differentiation. Isoproterenol (1 microM) and 1-methyl-3-isobutylxanthine (0.1 mM) further stimulated this rate of glycerol release 3-fold. The neutral triglyceride lipase activity in cell-free preparations of differentiated cells was activated 105% by cyclic AMP-dependent protein kinase. Neutral cholesterol esterase, diglyceride lipase, and monoglyceride lipase were also activated (117%, 10%, and 37+, respectively) by cyclic AMP-dependent protein kinase. In contrast, protein kinase had no effect on any of the four lysosomal acid acylhydrolase activities. Thus, hormone-sensitive lipase, the most characteristic and functionally important enzyme of adipose tissue, has been characterized in differentiated 3T3-L1 cells. The 3T3-L1 cell should be a valuable model system in which to study regulation of hormone-sensitive lipase, particularly its long-term regulation.

Adipose Tissue↗
PubMed1998

Digestion of ceramide by human milk bile salt-stimulated lipase.

BACKGROUND: There is a renewed interest in metabolism of sphingolipids because of their role in signal transduction. Sphingomyelin is the dominating phospholipid in human milk but its metabolism and possible function in the gastrointestinal tract of breast fed infants is unknown. We explored whether bile salt-stimulated milk lipase has a role in sphingolipid metabolism. METHODS: In vitro assays of sphingomyelinase and ceramidase activities, using radiolabeled substrates, human milk samples and purified native and recombinant variants of bile salt-stimulated milk lipase with or without known activators or inhibitors. RESULTS: Human whey and purified lipase catalysed hydrolysis of palmitoyl-labeled ceramide with the highest rate around pH 8.5-9.0. 1 mg of lipase hydrolysed 0.7 micromol ceramide in one hour at pH 8.5 in presence of 4 mM bile salt. The activity of whey was inhibited by antibodies towards human bile salt-stimulated milk lipase, indicating that this lipase accounted for virtually all ceramidase activity in the milk. In contrast, bile salt-stimulated milk lipase showed no activity against sphingomyelin. However we give evidence of a separate, hitherto unknown, acid sphingomyelinase in human milk. Under the used in vitro conditions this sphingomyelinase could account for hydrolysis of half of milk sphingomyelin in one hour. CONCLUSIONS: Human milk bile salt-stimulated milk lipase hydrolyses ceramide and may thus have a role in sphingomyelin digestion, but only after initial hydrolysis to ceramide and phosphorylcholine. Part of the latter could be carried out in the stomach by the acid milk sphingomyelinase now described. We speculate that these two milk enzymes may be of importance for optimal use of human milk sphingolipids.

Amidohydrolases↗
PubMed1984

Inhibitors of lipase activities in soybean and other oil seeds.

In the cotyledon extracts of seedlings of many oil seeds, including soybean, sunflower, cucumber, and peanut, the in vitro lipase activity was too low to account for the observed in vivo lipolysis. The low in vitro lipase activity was due to the presence of lipase inhibitors in the extracts. The inhibitors from soybean were characterized based on their effects on the hydrolysis of trilinolein by corn, pancreatic, and Rhizopus lipases. The inhibitors were not dialyzable and unaltered by RNase and beta-galactosidase treatment. However, they were sensitive to heating and protease digestion. The inhibitory effect of the inhibitors was expressed irrespective of the sequence of the addition of lipase, substrate, and inhibitors to the assay medium. The inhibitory effect was equally expressed when the inhibitors were added either before or after the lipase reaction had been in progress. The inhibitory effect of the inhibitors was independent of the amount of lipase present in the assay, but was dependent on the amount of substrate added. High substrate concentration eliminated totally the inhibitory effect of the inhibitors. Most of the inhibitors were recovered in the soluble fraction in subcellular fractionation. They were present in the 2-4S and not in the 7S, and 11S (storage proteins) protein fraction. There was a gradual decrease of the inhibitors in the cotyledons in the postgerminative growth. We suggest that the inhibitors are proteins which bind to the surface of the substrate micelles. The binding prevents the normal functioning of lipase which acts on the interfacial area between the aqueous medium and the micelle surface.

Journal Article↗
PubMed2001

Effect of hydrocarbon-water interfaces on synthetic and hydrolytic activities of lipases.

We have developed a new method of lipase activation for interesterification, in which lipase, in contact with a hydrocarbon-water interface, has been found to have high interesterification activity in an anhydrous solvent. We have applied this activation method to various lipase and obtained high synthetic activity in n-hexane, and have investigated the effect of various hydrocarbon-water interfaces on the synthetic and hydrolytic activities of lipases. The esterification and/or interesterification activity of lipases tested was improved by this activation method, using an n-tetradecane-water interface. From the initial group of lipases, three representative lipases (from Rhizopus japonicus, Chromobacterium viscosum and porcine pancreas) were selected for further study. The effect of various hydrocarbon-water interfaces on synthetic (interesterification or esterification) activity was studied. We demonstrated that the resulting synthetic activity was affected by the choice of hydrocarbon-water interface and that there were differences in the effects of interfaces on the synthetic activity of these lipases.

Journal Article↗
PubMed1995

The effects of O- and N-linked glycosylation on the secretion and bile salt-stimulation of pancreatic carboxyl ester lipase activity.

Pancreatic carboxyl ester lipase is a glycoprotein that requires millimolar concentrations of trihydroxy bile salts, such as cholate, for maximal catalytic activity against cholesteryl esters and triglycerides. Binding of cholate, with subsequent activation, has been proposed to occur in the carboxy-terminal region of carboxyl ester lipase, which contains multiple sites for O-linked glycosylation (1). To investigate the role of O- and N-linked glycosylation in the secretion of carboxyl ester lipase by cells and its activation by cholate, rat carboxyl ester lipase cDNA was transfected into the mutant chinese hamster ovary cell line, IdID, and the ability of the cells to modify the expressed carboxyl ester lipase by N- and O-linked glycosylation was modulated by using various incubation conditions and metabolic inhibitors. The results showed that, similar to other lipases, maximal secretion of carboxyl ester lipase activity required N-linked glycosylation. In contrast, O-linked glycosylation did not affect the secretion of carboxyl ester lipase activity. In addition, the cholate stimulation of hydrolysis was also independent of O-linked glycosylation.

Animals↗
PubMed2003

Purification and characterization of the lipase from Pseudomonas fluorescens HU380.

A lipase, which markedly splits polyunsaturated fatty acid ester (PUFA) bonds, from newly isolated Pseudomonas fluorescens HU380 was purified. The purification procedure included Phenyl-Toyopearl fractionation, DEAE-Sepharose chromatography, and Superdex-200HR chromatography. The enzyme was purified 24.3-fold with a yield of 14% and a specific activity of 9854 U/mg. Its molecular weight was estimated on SDS-PAGE to be 64,000. The optimum pH and temperature were 8.5 and 45 degrees C, respectively. The lipase was stable over the pH range of 6.0-7.0 at 30 degrees C for 24 h, and up to 40 degrees C at pH 7.0 for 60 min, when 0.1% Triton X-100 was present. The lipase preferably acted on short to middle-chain fatty acid simple methyl-esters and triglycerides, and cleaved mainly 1,3-ester bonds and to a lesser extent the 2-position ester bond of triolein. The lipase was inhibited by Co2+, Ni2+, Fe3+, Fe2+, and EDTA, and activated by Ca2+. Its N-terminal amino acid sequence was determined to be GVYDYKNFGTADSKALFSDAMAITLY, which exhibited considerable similarity with those of the lipases from other P. fluorescens strains, but no significant homology with other lipases. This lipase was able to decompose fats and oils that contained eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) without significantly affecting the contents of these fatty acids. The results suggest that the lipase may be useful when applied to the processing of industrial fats and oils containing EPA and DHA, such as fish oil splitting.

Journal Article↗
PubMed1974

Reversible protein kinase activation of hormone-sensitive lipase from chicken adipose tissue.

Hormone-sensitive lipase partially purified from adipose tissue of laying hens was markedly activated by cyclic AMP-dependent protein kinase. Activation was approximately 4-fold (ranging up to as great as 10-fold) compared with the much lower degree of activation obtained with analogous preparations from rat and human adipose tissues (59 and 86%, respectively). The partially purified preparations contained adequate endogenous protein kinase activity to effect complete activation with addition of cyclic AMP, ATP, and Mg(2+). Activation was blocked by protein kinase inhibitor (from rabbit skeletal muscle) but could be restored fully by addition of excess exogenous protein kinase (from bovine skeletal muscle). The fully activated lipase was slowly deactivated by dialysis at 4 degrees C and then rapidly and almost fully reactivated by addition of cyclic AMP and ATP-Mg(2+). Reactivation was blocked by protein kinase inhibitor. This deactivation-reactivation cycle was rapid at 23 degrees C with dialysis against charcoal and could be demonstrated repeatedly using a single preparation. The reversible deactivation of protein kinase-activated enzyme is presumed to reflect the action of a lipase phosphatase. Lipase prepared from tissue previously exposed to glucagon yielded a much smaller degree of activation than lipase prepared from tissue not exposed to the lipolytic hormone, indicating that the physiological hormone-induced activation is probably similar to or identical with the protein kinase activation demonstrated in the cell-free preparations. Under the conditions of assay used, the partially purified lipase fraction contained diglyceride, monoglyceride, and lipoprotein lipase activities. However, treatment with cyclic AMP-dependent protein kinase had virtually no effect on these lipase activities.

Adenosine Triphosphate↗
PubMed1987

Lipases operative at the fat cell surface: attempt at an integrated approach.

Extracellular acylglycerols are hydrolysed by lipases active at the surface of intact fat cells isolated from rat or human adipose tissue. During short-term incubation, rat fat cells hydrolyse di-[3H]oleyl-[14C]glycerol at a rate of 70 +/- 7.7 mU/10(6) cells (mean +/- S.E.) versus 440 +/- 62 mU/10(6) cells for the hydrolysis of mono-[3H]oleylglycerol; these relatively high lipolytic potencies may serve, among other functions, to counteract the cytolytic effect of both esters. Reaction rates with both substrates are unchanged by addition of various apolipoproteins C and by the nutritional state of the animals. Fat cells incorporate 15-20 per cent of the total [3H]-oleic chains liberated by hydrolysis, with no correlation between uptake and hydrolysis rates. [3H]-oleic chains in cell lipids are found mainly as diacylglycerol (15 per cent) and triacylglycerol (80 per cent). Both lipolytic processes differ from the hydrolysis of trioleylglycerol by cell-bound lipoprotein lipase, which occurs at lower rates (6.5 +/- 0.6 mU/10(6) cells) and depends on apolipoprotein C-II and nutritional state of the animals. The results support the accepted view that lipoprotein lipase and monoacylglycerol lipase are distinct enzymes. Differences between lipoprotein lipase and diacylglycerol lipase activities raise the possibility of different catalytic entities. In conclusion, isolated fat cells in suspension hydrolyse and incorporate lipids. This model should approximate physiological conditions more closely than the use of lipases in the free state.

Adipose Tissue↗
PubMed1995

Lipase structures at the interface between chemistry and biochemistry.

In this chapter we review recent molecular knowledge on two structurally related mammalian triglyceride lipases which have evolved from a common ancestral gene. The common property of the lipase family members is that they interact with non-polar substances. Pancreatic lipase hydrolyzes triglycerides in the small intestine in the presence of many dietary components, other digestive enzymes and high concentrations of detergents (bile salts). Lipoprotein lipase acts at the vascular side of the blood vessels where it hydrolyses triglycerides and some phospholipids of the circulating plasma lipoproteins. A third member of the gene family, hepatic lipase, is found in the liver of mammals. Also, this lipase is involved in lipoprotein metabolism. The three lipases are distantly related to some non-catalytic yolk proteins from Drosophila (Persson et al., 1989; Kirchgessner et al., 1989; Hide et al., 1992) and to a phospholipase A1 from hornet venom (Soldatova et al., 1993).

Amino Acid Sequence↗
PubMed1979

Selective measurement of triacylglycerol lipase activities in pig post-heparin plasma.

Immunochemical methods for the selective measurement of pig post-heparin plasma lipoprotein lipase and hepatic lipase are described and validated. A simple two step purification method for porcine hepatic lipase from hepatic perfusate based on affinity chromatography and gel filtration is reported. The activity of the post-heparin plasma lipoprotein lipase and hepatic lipase in swine is reported. It is demonstrated that fasting decreases the activity of post-heparin plasma lipoprotein lipase activity more than two-fold while it does not affect the hepatic lipase activity significantly.

Animals↗
PubMed1986

Inhibition of human and rat lipoprotein lipase by high-density lipoprotein.

The hydrolysis in vitro of preactivated Intralipid (an artificial triacylglycerol-phospholipid emulsion) by rat adipose tissue lipoprotein lipase is inhibited by rat high-density lipoprotein (HDL). The aim of this work was to investigate whether human lipoprotein lipase was also inhibited, the mechanism of inhibition of the rat enzyme by HDL, and the role of the various individual apolipoproteins. Both human and rat lipoprotein lipase from post-heparin plasma are inhibited by HDL. This inhibition is considerably decreased if the HDL is first made 'apolipoprotein poor' by removal of some transferable apolipoproteins. In contrast, both native and apolipoprotein poor HDL inhibit the hydrolysis of Intralipid by rat hepatic lipase. Apolipoproteins C and E, either free in solution or attached to lipid vesicles, inhibit the hydrolysis of activated Intralipid by rat lipoprotein lipase to a maximum of 85% and 50%, respectively. Apolipoprotein A attached to vesicles gives little inhibition. HDL apolipoprotein and apolipoprotein C compete with the substrate for binding to lipoprotein lipase with apolipoprotein C having a higher affinity for the enzyme than HDL apolipoprotein. The inhibition of lipoprotein lipase by HDL can be explained by the association of the constituent apolipoproteins, in particular apolipoprotein C, with the enzyme so that there is less enzyme available to act on substrate.

Animals↗
PubMed1981

Ascites chylomicron: a poor substrate for hepatic triglyceride lipase.

A patient with nephrotic syndrome and morbus Kimura (eosinophilic granuloma) showed chylous ascites. Ascites chylomicrons were analyzed and used to study the substrate specificity of lipoprotein lipase and hepatic triglyceride lipase. Ascites triglyceride and cholesterol concentrations were 191 and 12 mg/dl, respectively. Both apo CII and apo CIII content in ascites were approximately one-third of those of plasma from normal subjects. Ascites chylomicrons were incubated with either lipoprotein lipase or hepatic triglyceride lipase, which were prepared from postheparin plasma using heparin-Sepharose affinity chromatography. Lipoprotein lipase hydrolyzed ascites chylomicrons, while hepatic triglyceride lipase did not. These results suggest different functions of these two lipases in chylomicron catabolism.

Ascites↗
PubMed1980

Immunochemical assay of rat postheparin plasma triacylglycerol lipases.

Immunochemical methods for selective measurement of lipoprotein lipase and hepatic lipase activities in rat postheparin plasma are described and validated. Lipoprotein lipase was measured using a substrate containing 10% serum and 0.1 M NaCl after inactivation of hepatic lipase with a specific antiserum. Hepatic lipase was measured at 1.0 M NaCl with a serum-free substrate. The heparin dose-response curve indicated maximum relase of both activities at a heparin dose of 500 IU/kg. The lipase activities in rat postheparin plasma were 3 to 4-fold higher than those in human postheparin plasma. The LPL activity in female rats was significantly higher than in males whereas there was no sex difference for hapatic lipase.

Animals↗
PubMed1980

Decline of postheparin plasma lipoprotein lipase in acromegalic patients.

Lipoprotein lipase and hepatic triglyceride lipase in postheparin plasma were measured in seven patients with active acromegaly by an immunochemical method utilizing antiserum prepared against hepatic triglyceride lipase. A mild or moderate hypertriglyceridemia was shown, with plasma triglyceride concentrations between 156 and 544 mg/dl. Lipoprotein lipase was found to be decreased in all patients (p less than 0.001). Hepatic triglyceride lipase was also low in these patients (p less than 0.001). We speculate that acromegalic hypertriglyceridemia is mediated, at least in part, by the decline in lipoprotein lipase and possibly by the decline in hepatic triglyceride lipase activities.

Acromegaly↗
PubMed1992

Type I hyperlipoproteinemia caused by lipoprotein lipase defect in lipid-interface recognition was relieved by administration of medium-chain triglyceride.

We have previously reported lipoprotein lipase with a defect of lipid-interface recognition in a patient with type I hyperlipoproteinemia. In this patient, lipoprotein lipase from post-heparin plasma (PHP) hydrolyzed monomeric substrate tributyrin, but scarcely hydrolyzed triolein emulsified with Triton X-100 and that in very-low-density lipoproteins ([VLDL] d < 1.006 g/mL), and did not bind to VLDL. The triglyceride (TG) level of this patient did not decrease to less than 1,000 mg/dL with a low-fat diet (1,400 kcal containing 10 g fat/d). When the patient took 30 g medium-chain TG (MCT) in addition to the 1,400-kcal diet, her serum TG level decreased to 250 mg/dL and her clinical signs improved. The low clearance rate of serum TG with heparin injection improved after intake of MCT. Caproic acid levels were maintained at 1.4% and 2.6% in chylomicrons and VLDL after MCT intake, respectively. The patient's lipoprotein lipase hydrolyzed triolein emulsified with 2% tricaprin at the same rate as that of control lipoprotein lipase. The patient's lipoprotein lipase-catalyzed hydrolyzing rate of triolein in chylomicrons obtained after MCT administration was also enhanced up to 70% of that of control lipoprotein lipase. These findings suggest that hypertriglyceridemia caused by lipoprotein lipase with a defect in lipid-interface recognition could be relieved with the administration of medium-chain TG, and that one of the mechanisms of this effect might be a modification of TG-rich lipoproteins by MCT.

Adolescent↗
PubMed1977

Selective deficiency of hepatic triglyceride lipase in uremic patients.

To investigate the pathogenesis of hypertriglyceridemia in patients with renal disease we measured plasma lipoprotein composition as well as hepatic triglyceride lipase and lipoprotein lipase in post-heparin plasma. Three groups with renal disease were studied: conservatively treated chronic uremia; patients undergoing maintenance hemodialysis; and renal-allograft recipients. A selective decrease of hepatic triglyceride lipase with normal lipoprotein lipase was found in conservatively treated uremia and in patients undergoing hemodialysis. Elevated levels of very-low-density lipoproteins and increased triglycerides in low-density lipoproteins occurred in these patients. In contrast, hepatic triglyceride lipase and lipoprotein lipase were both normal in patients after renal transplantation who had Type II hyperlipoproteinemia as a common lipoprotein pattern with increased low-density-lipoprotein cholesterol and decreased high-density-lipoprotein cholesterol concentrations. The accumulation of a triglyceride-rich low-density lipoprotein in the majority of patients with renal disease may be the consequence of low hepatic triglyceride lipase.

Adult↗