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PubMed1995

A lipase of Aeromonas hydrophila showing nonhemolytic phospholipase C activity.

Extracellular lipase activity detected on tributyrin agar has been identified in a cosmid clone, JM3084, constructed from the chromosome of Aeromonas hydrophila and vector pHC79. This lipase, named apl-1, also exhibits nonhemolytic phospholipase C activity on lecithin and p-nitrophenylphosphorylcholine. Subcloning of the cosmid JMP3084 with partial Sau3a1 digestion localized the lipase gene to a 3.4-kb DNA fragment. Southern blot analysis shows the gene apl-1 to exist in single copy on the A. hydrophila chromosome. Expression of apl-1 in the pT7 system identified a single protein of molecular weight 70 kDa. Nucleotide sequencing of apl-1 has identified an open reading frame of 2055 bases predicting a protein of 73 kDa. The presence of an amino terminal signal sequence of 18 amino acids accounts for this molecular weight disparity. Further analysis of the lipase amino acid sequence revealed the presence of a classical serine active lipase site (Gly-X-Ser-X-Gly) located between residues 561 and 570. The A. hydrophila chromosomal copy of apl-1 has been inactivated by use of the mutagenesis vector pJP5603, resulting in the complete removal of phospholipase C activity and lowered levels of lipase activity detected on tributyrin agar.

Aeromonas hydrophila↗
PubMed1987

Radioimmunoassay for human pancreatic lipase in acute pancreatitis.

We examined the utility of serum pancreatic lipase by radioimmunoassay as a diagnostic test for acute pancreatitis and its correlation with serum total amylase, pancreatic isoamylase, and lipase activity. Data were analyzed on 11 patients with documented acute pancreatitis, three groups of patients (N = 104) with nongastrointestinal, gastrointestinal, and renal diseases, and 30 healthy controls. Patients with acute pancreatitis had significantly (P less than 0.01) higher mean serum lipase by radioimmunoassay than all other groups. Using a serum lipase of 112 ng/ml as a cutoff point in all patients, the test was 91% sensitive and 96% specific for the diagnosis of acute pancreatitis. The correlation coefficients of serum lipase by radioimmunoassay with respect to total amylase, pancreatic isoamylase, and lipase activity were 0.86, 0.98, and 0.79, respectively.

Acute Disease↗
PubMed1984

Serum lipase response to cerulein secretin stimulation test in patients with pancreas-associated diseases as measured by sensitive colorimetric assay (a BALB-DTNB method).

We have compared responsiveness of serum lipase and amylase activity to the pancreatic exocrine stimulation with cerulein and secretin (CS test) in normal subjects and patients with pancreas-related and other diseases. The lipase and amylase activities were measured by a sensitive colorimetric method, the BALB-DTNB method and the Caraway method, respectively. The percentage of positive lipase and amylase response cases was as follows: confirmed chronic pancreatitis (N = 22), 27 and 14%; suspected chronic pancreatitis (N = 37), 46 and 32%; pancreatic cancer (N = 16), 44 and 25%; biliary tract diseases (N = 11), 14 and 14%; miscellaneous (N = 11), 0 and 18%; normal subjects (N = 13), and partial pancreatectomy (N = 5), 0 and 0%, respectively. The serum lipase response cannot be regarded as specific for pancreatic diseases because the lipase response cases were found in biliary tract diseases as well. However, in view of frequent, fast, and intense responsiveness to the CS test, the serum lipase activity measured by the BALB-DTNB method may be more useful than the serum amylase as an auxiliary diagnostic aid for suspected pancreatitis which might develop into confirmed chronic pancreatitis or cancer of the head or body of the pancreas.

Adolescent↗
PubMed1983

Determination of lipase specificity.

Specificity of lipases is controlled by the molecular properties of the enzyme, structure of the substrate and factors affecting binding of the enzyme to the substrate. Types of specificity are as follows. I. Substrate: (a) different rates of lipolysis of TG, DG, and MG by the same enzyme; (b) separate enzymes from the same source for TG, DG and MG. II. Positional: (a) primary esters; (b) secondary esters; and (c) all three esters or nonspecific hydrolysis. III. Fatty acid, preference for similar fatty acids. IV. Stereospecificity: faster hydrolysis of one primary sn ester as compared to the other. V. Combinations of I-IV. Lipases with these specificities are: Ia, pancreatic; Ib, postheparin plasma. IIa, pancreatic; IIb, Geotrichum candidum, for fatty acids with cis-9-unsaturation, and IIc, Candida cylindracea. III, G. candidum for unsaturates. IV. sn-1, postheparin plasma and sn-3 human and rat lingual lipases. V. Rat lingual lipase. Methods for determination involve digestion of natural fats of known structure and synthetic acylglycerols followed by analysis of the lipolysis products. All of the types of specificity have been detected with use of synthetic acylglycerols. Detection of stereospecificity requires enantiomeric acylglycerols which are difficult to synthesize, so other methods have been developed. These involve the generation of 1,2-(2,3)DG and resolution of the enantiomers. Trioleoylglycerol or racemic TG can be used as substrates. If the lipase is stereospecific, then either the sn-1,2- or 2,3-enantiomer will predominate. The relative amounts of the enantiomers can be determined by measurement of specific rotation, and nuclear magnetic resonance spectra. The DG can also be separated by conversion to phospholipids and hydrolysis with phospholipases A-2 or C. Applications of these procedures are discussed and data on the specificity of various lipases presented.

Animals↗
PubMed1989

A novel spectrophotometric assay for lipase activity utilizing cis-parinaric acid.

A new spectrophotometric assay for determining the activity of acylglycerol hydrolases (lipases, E.C. 3.1.1.3) was developed and optimized for yeast lipase (Candida cylindracea). Studies with porcine pancreatic lipase were also conducted and the influence of various detergents and divalent cations on the assay was evaluated. The assay uses cis-parinaric acid (PnA), a naturally occurring fatty acid that has unique spectroscopic properties, and takes advantage of the reversible binding of fatty acids to bovine serum albumin (BSA). Free PnA has an ultraviolet absorption peak at 321.2 nm. When PnA is bound to BSA, however, the peak shifts to 324.2 nm. The assay mixture contains 6 microM PnA, 1 microM BSA, 75 microM triolein, and 0.3 mM taurocholate in a 50 mM tris-HCl buffer with 1 microM EDTA. The release of oleic acid from triolein is monitored over time by measuring the ratio of optical densities (OD) at 319.0 and 329.0 nm. Initially, there is maximum binding of PnA to BSA, and the OD ratio is approximately 1.0. Upon addition of lipase, PnA is displaced from the BSA by oleic acid released from triolein, and the OD ratio increases to a maximum of about 1.8. However, when calcium is present in the reaction mixture an insoluble calcium-PnA complex forms, resulting in a progressive decrease in OD at both 319.0 and 329.0 nm. The kinetic assay described here is simple, rapid, sensitive, reproducible, inexpensive, and it can be adapted to measure the activity of a variety of calcium-independent lipases. Under similar assay conditions, activities for Candida cylindracea lipase obtained with this assay are similar to those obtained with 14C-labelled triolein.

Animals↗
PubMed1978

Acid triacylglycerol lipase from bovine thyroid gland.

An acid lipase has been detected in bovine thyroid tissue using triolein as a substrate. The activity, probably associated with the lysosomes, displays a rather broad pH-optimum in the pH 4 to pH 6.5 range. The lipase activity can be partially purified by cosedimentation with lysosomes followed by solubilization through detergent and chromatography on Sephadex G-200 and carboxymethyl cellulose. The elution profile on Sephadex G-200 shows one peak (moleculare weight 67,000 +/- 2,000). In the final CM-cellulose step, two lipase peaks (lipase LA and lipase LB) are found. Sulhydryl reagents (iodoacetate, iodoacetamide, and N-ethylmaleimide) as well as mercuric ions markedly reduce both enzyme activities. Calcium ions, EDTA, and heparin have no effect. Sodium fluoride and diisopropylfluorophosphate are only slightly inhibitory. Sodium chloride causes a slight increase in both lipase activities. Anionic phospholipids such as cardiolipin and phosphatidylserine are not essential for enzyme activity.

Animals↗
PubMed1991

Geotrichum candidum NRRL Y-553 lipase: purification, characterization and fatty acid specificity.

Lipases from Geotrichum candidum NRRL Y-553 are of interest because of their unique specificity for cis-9-unsaturated fatty acids relative to both stearic and palmitic acids. The lipases were partially purified by chromatography on Octyl Sepharose, AG MP-1 macroporous anion exchanger, and chromatofocusing resin. The preparation was found to contain multiple, glycosylated lipases varying slightly in pI (pI 4.88, 4.78, 4.65, 4.57 and 4.52) as judged by both activity and silver staining. The molecular mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 64 kilodaltons for the main species, with minor species of 60 and 57 kilodaltons present as well. The specificity of the crude lipases for hydrolysis of 4-methylumbelliferyl esters of oleic vs. palmitic acid was 20-to-1. The specificity of the purified, partially separated lipases was similar to that of the crude preparation. Thus the lipases could be used even in crude form for the hydrolysis and restructuring of triacylglycerols on a large scale.

Geotrichum↗
PubMed1990

The anticoordinate changes of pancreatic lipase and colipase activity to amylase activity induced by adrenalectomy in normal and diabetic rats.

The effect of adrenalectomy (Adx) on pancreatic lipase and colipase activities in normal, streptozotocin-induced diabetic, and obese Zucker rats was studied. It was found that Adx induced a rapid decrease in amylase activity, a rapid increase in lipase activity, and a slow increase in colipase activity in normal rats. Twenty days after Adx, the levels of amylase activity were 70% lower than control, whereas the activities of lipase and colipase were 55 and 25% higher than control, respectively. The replacement of hydrocortisone normalized the changes in the three enzyme activities. Injection of insulin partially corrected the amylase activity, but had no significant effect on the activities of lipase and colipase. In streptozotocin-induced diabetic rats and obese Zucker rats, amylase activity was decreased, and lipase and colipase activities increased, compared to normal rats. These changes of enzyme activity were further enhanced by Adx. It is concluded that Adx results in an anticoordinate change in pancreatic lipase, colipase, and amylase activities, suggesting that glucocorticoid also influences the synthesis of the pancreatic lipolytic enzymes.

Adrenal Glands↗
PubMed2006

Human adipose triglyceride lipase (PNPLA2) is not regulated by obesity and exhibits low in vitro triglyceride hydrolase activity.

AIMS/HYPOTHESIS: The recent identification of murine adipose triglyceride lipase (ATGL, now known as patatin-like phospholipase domain containing 2 [PNPLA2]), gene product of Pnpla2, has questioned the unique role of hormone sensitive lipase (HSL, now known as LIPE), gene product of Lipe, in fat cell lipolysis. Here, we investigated human ATGL and HSL adipose tissue gene expression and in vitro lipase activity. SUBJECTS, MATERIALS AND METHODS: Levels of mRNA in adipose tissue from healthy obese and non-obese subjects were measured and lipase activity and adipocyte lipolytic capacity determined. HSL and ATGL cDNAs were transfected into Cos-7 cells and the relative tri- and diglyceride hydrolase activities were measured. RESULTS: Obesity was associated with a decreased subcutaneous and increased omental adipose tissue level of HSL mRNA. Subcutaneous HSL mRNA content was normalised upon weight reduction. In contrast, ATGL mRNA levels were unaffected by obesity and weight reduction. A high adipose tissue lipase activity was associated with increased maximal lipolysis and increased HSL, but not with ATGL mRNA levels. The in vitro triglyceride hydrolase activity of HSL was markedly higher than that of ATGL and contrary to HSL, ATGL was devoid of diglyceride hydrolase activity. The use of a selective HSL-inhibitor resulted in complete inhibition of HSL-mediated tri- and diglyceride hydrolase activity. The pH profile of human white adipose tissue triolein hydrolase activity was identical to that of HSL but differed from the ATGL profile. CONCLUSIONS/INTERPRETATION: HSL, but not ATGL gene expression shows a regulation according to obesity status and is associated with increased adipose tissue lipase activity. Moreover, HSL has a higher capacity than ATGL to hydrolyse triglycerides in vitro.

Adult↗
PubMed2000

The lipoprotein lipase activator, NO-1886, suppresses fat accumulation and insulin resistance in rats fed a high-fat diet.

AIMS/HYPOTHESIS: Fat balance is critical in the aetiology of obesity and related diseases. Lipoprotein lipase is of major importance in lipid metabolism. The aim of this study was to investigate the long-term effects of the lipoprotein lipase activator, NO-1886, on substrate utilisation, adiposity and insulin action in rats fed a high-fat diet. METHODS: Male, Sprague-Dawley rats were fed for 10 weeks on a chow diet or a high-fat diet with, or without, NO-1886 (50 mg x kg(-1) x day(-1)). Weight gain, fat accumulation and both hormone-sensitive and lipoprotein, lipase activities were measured. Insulin action was assessed by the euglycaemic hyperinsulinaemic clamp and metabolic rate/substrate utilisation by open-circuit respirometry. RESULTS: Compared with chow-fed controls, a high-fat diet increased weight gain, an effect lessened by NO-1886 [weight gain (g): chow, 37 +/- 3, high-fat, 222 +/- 9; high-fat + NO-1886, 109 +/- 6, all groups differed p < 0.001]. A similar pattern existed for fat accumulation [visceral fat (g): chow, 35.9 +/- 3.2; high-fat, 81.9 +/- 6.6; high-fat + NO-1886, 52.3 +/- 4.7, p < 0.01 high-fat vs the other groups]. A high-fat diet induced wholebody insulin resistance (clamp glucose infusion rate: 4.8 +/- 1.3 mg x kg(-1) x min(-1) vs 10.6 +/- 1.1 for the chow group, p < 0.01) with NO-1886 lessening this effect (8.3 +/- 0.5, p < 0.05 vs high-fat). The 24-h respiratory quotient was lower in the high-fat + NO-1886 group (0.825 +/- 0.010) compared with high-fat alone (0.849 +/- 0.004, p < 0.05). A high-fat diet increased lipoprotein and hormone-sensitive, lipase activities in epididymal fat, an effect not altered by NO-1886. In myocardium and skeletal muscle a high-fat diet lowered lipoprotein lipase activity, an effect lessened by NO-1886. CONCLUSION/INTERPRETATION: Lipoprotein lipase activators could have potential benefits for the treatment of obesity by increasing fat utilisation.

Adipose Tissue↗
PubMed2000

Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members.

Extracellular lipolytic activity enabled the human pathogen Candida albicans to grow on lipids as the sole source of carbon. Nine new members of a lipase gene family (LIP2-LIP10) with high similarities to the recently cloned lipase gene LIP1 were cloned and characterised. The ORFs of all ten lipase genes are between 1281 and 1416 bp long and encode highly similar proteins with up to 80% identical amino acid sequences. Each deduced lipase sequence has conserved lipase motifs, four conserved cysteine residues, conserved putative N-glycosylation sites and similar hydrophobicity profiles. All LIP genes, except LIP7, also encode an N-terminal signal sequence. LIP3-LIP6 were expressed in all media and at all time points of growth tested as shown by Northern blot and RT-PCR analyses. LIP1, LIP3, LIP4, LIP5, LIP6 and LIP8 were expressed in medium with Tween 40 as a sole source of carbon. However, the same genes were also expressed in media without lipids. Two other genes, LIP2 and LIP9, were only expressed in media lacking lipids. Transcripts of most lipase genes were detected during the yeast-to-hyphal transition. Furthermore, LIP5, LIP6, LIP8 and LIP9 were found to be expressed during experimental infection of mice. These data indicate lipid-independent, highly flexible in vitro and in vivo expression of a large number of LIP genes, possibly reflecting broad lipolytic activity, which may contribute to the persistence and virulence of C. albicans in human tissue.

Amino Acid Sequence↗
PubMed2004

Mono-thioesters and di-thioesters by lipase-catalyzed reactions of alpha,omega-alkanedithiols with palmitic acid or its methyl ester.

1- S-Mono-palmitoyl-hexanedithiol and 1- S-mono-palmitoyl-octanedithiol were prepared in high yield (80-90%) by solvent-free lipase-catalyzed thioesterification of palmitic acid with the corresponding alpha,omega-alkanedithiols in vacuo. Similarly, 1,6-di- S-palmitoyl-hexanedithiol and 1,8-di- S-palmitoyl-octanedithiol were prepared in moderate yield (50-60%) by solvent-free lipase-catalyzed thioesterification of palmitic acid with 1- S-Mono-palmitoyl-hexanedithiol and 1- S-mono-palmitoyl-octanedithiol, respectively. An immobilized lipase preparation from Rhizomucor miehei (Lipozyme RM IM) was more effective than a lipase B preparation from Candida antarctica (Novozym 435) or a lipase preparation from Thermomyces lanuginosus (Lipozyme TL IM). Lipase-catalyzed transthioesterifications of methyl palmitate with alpha,omega-alkanedithiols using the same enzymes were less effective than thioesterification for the preparation of the corresponding 1- S-mono-palmitoyl thioesters.

Ascomycota↗
PubMed1998

Stable production of human gastric lipase by chromosomal integration in the fission yeast Schizosaccharomyces pombe.

Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production of human gastric lipase. Integrant strains of S. pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media. Lipase activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated with copy number of the cassette in both complete and minimal media. Lipase activity is higher in complete medium than in minimal medium. Strains carrying three chromosomally integrated expression cassette copies can be grown without selection in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette on a multicopy plasmid.

Humans↗
PubMed1999

Substrate selectivity of various lipases in the esterification of cis- and trans-9-octadecenoic acid.

The substrate selectivity of numerous commercially available lipases from microorganisms, plants and animal tissue towards 9-octadecenoic acids with respect to the cis/trans configuration of the C=C double bond was examined by the esterification of cis- and trans-9-octadecanoic acid (oleic and elaidic acid respectively) with n-butanol in n-hexane. A great number of lipases studied, e.g. those from Pseudomonas sp., porcine pancreas or Carica papaya, were unable to discriminate between the isomeric 9-octadecenoic acids. However, lipases from Candida cylindracea and Mucor miehei catalysed the esterification of oleic acid 3-4 times faster than the corresponding reaction of elaidic acid and therefore have a high preference for the cis isomer. Of all biocatalysts examined, only recombinant lipases from Candida antarctica favoured elaidic acid as substrate. While the preference of Candida antarctica lipase B for the trans isomer was quite low, Candida antarctica lipase A had an extraordinary substrate selectivity and its immobilized enzyme preparation [Chirazyme L-5 (3) from Boehringer] esterified elaidic acid about 15 times faster than oleic acid.

Bacteria↗
PubMed1999

Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor.

Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.

Acinetobacter calcoaceticus↗
PubMed2004

A thermostable lipase produced by a newly isolated Geotrichum-like strain, R59.

Growth and production of lipase by a new Geotrichum-like strain, R59, were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, substances probably stimulating the lipase biosynthesis, the temperature, and time of growth were optimized. Sucrose and triolein were the most effective carbon sources for lipase production. Maximum lipase activity (146 U/ml(-1)) was obtained with urea as the nitrogen source. Growth at 30 degrees C, an initial pH of 6.0 and incubation time of 48 h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like strain R59. The enzyme was thermostable and exhibited very high activity after 1 h incubation at 60 degrees C.

Carbon↗
PubMed2004

Acinetobacter lipases: molecular biology, biochemical properties and biotechnological potential.

Lipases (EC 3.1.1.3) have received increased attention recently, evidenced by the increasing amount of information about lipases in the current literature. The renewed interest in this enzyme class is due primarily to investigations of their role in pathogenesis and their increasing use in biotechnological applications. Also, many microbial lipases are available as commercial products, the majority of which are used in detergents, cosmetic production, food flavoring, and organic synthesis. Lipases are valued biocatalysts because they act under mild conditions, are highly stable in organic solvents, show broad substrate specificity, and usually show high regio- and/or stereo-selectivity in catalysis. A number of lipolytic strains of Acinetobacter have been isolated from a variety of sources and their lipases possess many biochemical properties similar to those that have been developed for biotechnological applications. This review discusses the biology of lipase expression in Acinetobacter, with emphasis on those aspects relevant to potential biotechnology applications.

Acinetobacter↗
PubMed2006

Pretreatment of lipase with soybean oil before immobilization to prevent loss of activity.

Lipase was pretreated with soybean oil in order to allow fatty acids to bond to the active site before immobilization. This pretreated lipase exhibited steric hindrance around the active site such that during immobilization, covalent bonds were formed between the carrier and the lipase region far from the active site. The activity of the pretreated lipase immobilized covalently on a silica gel was 530 U/g-matrix, which is 16 times higher than that of the immobilized non-pretreated lipase. In addition, the immobilized lipase activity was maintained at levels exceeding 90% of its original activity after 10 reuses.

Binding Sites↗