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Elastase-1 vs trypsin, lipase and amylase serum levels in pancreatic diseases.

Serum levels of elastase-1 were measured in 174 patients with pancreatic diseases and in 131 controls and were compared with the circulating levels of trypsin, lipase and amylase and with clinical data. In 48 patients with chronic pancreatitis serum enzyme levels were also compared with the pancreatic exocrine capacity. About 50% of the patients with chronic pancreatic disease showed increased levels of serum elastase, sometimes even in the face of long lasting pain-free periods, and/or of severe pancreatic impairment. On the contrary, serum trypsin and lipase were almost always either normal or below the normal range in the absence of painful relapses and/or in the presence of an impairment of the exocrine pancreatic function. A strict correlation was found between elastase-1 and trypsin (r = 0.778) and lipase (r = 0.834). However, controls and patients with chronic pancreatic diseases behaved differently, an increase in trypsin and in lipase levels being associated in the patients with chronic pancreatitis with an increase in elastase-1 values significantly larger than that observed in controls. These findings raise the hypothesis, at present unproven, that trypsin and lipase serum assays are more reliable indices of pancreatic exocrine function, whereas serum elastase-1 levels may indicate the presence of acute pancreatic episodes, even if subclinical, the importance of which, in the natural history of the disease, remains unknown.

Acute Disease↗

Monoacylglycerol lipase. Regulation and increased activity during hypoxia and ischemia.

The presence of monoacylglycerol lipase was established in extracts of acetone-dried powders from rat and bovine brains using thioester substrate analogs. At pH 7.4, the apparent Km and Vmax values for 1-S-decanoyl-1-mercapto-2,3-propanediol were 56 microM and 227 nmol/h/mg protein in bovine gray matter. The divalent metal ions Ca2+ and Mg2+ had no effect on enzymic activity, but Zn2+ at 500 microM produced a 50% inhibition of this enzyme. Free fatty acids also caused a marked inhibition of monoacylglycerol lipase activity. Norepinephrine and 5-hydroxytryptamine slightly stimulated the enzymic activity. Hypoxic-hypoxia and 30-s postdecapitation ischemia resulted in a considerable increase in monoacylglycerol lipase activity of rat brain. However, the increased activity of monoacylglycerol lipase returned to normal after 5 min of ischemia. The increased activity of monoacylglycerol lipase during hypoxic-hypoxia and short-time ischemia may be partially responsible for increased levels of free fatty acids during these processes.

Acetylcholine↗

Lipase from a Brazilian strain of Penicillium citrinum.

A lipases (glycerol ester hydrolases E. C. 3.1.1.3) from a brazilian strain of Penicillium citrinum has been investigated. When the microorganism was cultured in the simple medium (1.0% olive oil and 0.5% yeast extract), using olive oil in as carbon source in the inocula, the enzyme extracted showed maximum activity (409 IU/mL). In addition, decrease of yeast extract concentration also reduces the lipase activity. Nevertheless, when yeast extract was replaced by ammonium sulfate, no activity was detected. Purification by precipitation with ammonium sulfate showed best activity in the 40-60% fraction. The optimum temperature for enzyme activity was found in the range of 34-37 degrees C. However, after 30 min at 60 degrees C, the enzyme was completely inactivated. The enzyme showed optimum at pH 8.0. The dried concentrated fraction (after dialysis and lyophilization) maintained its lipase activity at room temperature (28 degrees C) for 8 mo. This result in lipase stability suggests an application of lipases from P. citrinum in detergents and other products that require a high stability at room temperature.

Brazil↗

Isolated rat pancreatic acini as a model to study the potential role of lipase in the pathogenesis of acinar cell destruction.

We have recently reported that lipase may play a role in the pathogenesis of acute pancreatitis by its ability to release fatty acids from triglycerides. The aim of this study was to further investigate the effect of lipase and its various digestive products on the integrity of isolated pancreatic rat acini. Pancreatic acini were prepared by collagenase digestion and their newly synthesized proteins labeled with 35S-methionine. Acini were later incubated in buffer to which various factors were added: Products of lipolytic digestion, such as various fatty acids and monoglycerides, fat tissue, nonactivated or trypsin activated homogenized pancreatic tissue, and a specific lipase inhibitor (THL, tetrahydrolipstatin). Cellular destruction was quantified by the degree of radiolabeled proteins released. Short chain fatty acids and monoglycerides (up to C-12) caused cellular destruction, whereas long chain fatty acids and their respective monoglycerides were not harmful. With regard to unsaturated fatty acids, long chain fatty acids (C-18 to C-22) were also able to destroy cells. The degree of cellular necrosis correlated with incubation time and fatty acid concentration. The cellular damage caused by incubation of acini with either inactive or trypsin activated pancreatic homogenates together with triglycerides could be completely inhibited by the specific lipase inhibitor THL. Bile alone caused no damage. When bile was combined with activated-pancreatic homogenates, about 25% of newly synthesized proteins were released by acini within 30 min. Incubation with a combination out of bile activated pancreatic homogenates and triglycerides resulted in the most pronounced damage. This acinar destruction could only be partly inhibited by THL. These studies suggest that both lipase and phospholipase-A2 may play an important role in the pathogenesis of acinar cell destruction.

Animals↗

Determination of the kinetic properties of platycodin D for the inhibition of pancreatic lipase using a 1,2-diglyceride-based colorimetric assay.

A 1,2-diglyceride-based multi-step colorimetric assay to measure the pancreatic lipase activity was applied for the determination of the kinetic profiles of the lipase inhibition with a slight modification and the validity verification. With this assay method, our study revealed that platycodin D, one of major constituents of Platycodi Radix, inhibits the pancreatic lipase activity in a competitive type, with the value of KI being 0.18+/-0.02 mM. In addition, PD has affected the values of K(m,app) and Kcat/Km in a dose-dependent manner. The results shed a meaningful light on how PD mediates lipid metabolism in the intestinal tracts. On the other hand, since the revised assay is sensitive, rapid, and does not affect the accuracy to the kinetic properties, it is applicable not only to evaluation of the kinetic properties of the pancreatic lipase, but also to high-throughput screening of pancreatic lipase activity.

Colorimetry↗

Lipase mediated chiral resolution of 4-arylthio-2-butanol as an intermediate for beta-lactam antibiotics.

This paper deals with chiral enzymatic resolution of 4-arylthio-2-butanols by lipase to prepare potential intermediates of beta-lactam antibiotics. Among several lipases employed, lipase P type enzyme gave the highest ee value to prepare (R)-4-arylthio-2-butyl acetate. The enzymatic resolution of phenyl substituted alcohol (6a) using lipase P showed the highest ee value (99.7%) among those of 4-arylthio-2-butanol derivatives. Lipase P mediated hydrolysis of acylester 7a gave also (R)-alcohol 6a selectively. For determination of enantiomeric purity of these enzymatic resolved analytes, liquid chromatographic analysis was performed using two coupled Chiralcel OD and (R,R)-WhelkO chiral column.

Butanols↗

Insulin sensitivity is impaired in heterozygous carriers of lipoprotein lipase deficiency.

AIMS/HYPOTHESIS: Several studies have investigated the lipoprotein phenotype in heterozygous carriers of a defective lipoprotein lipase allele. We studied whether heterozygosity for lipoprotein lipase deficiency also affects glucose metabolism beyond its effect on plasma lipids. METHODS: To address this question 85 heterozygous carriers of either a missense mutation (Gly188Glu) or a splice site mutation (C-->A in position -3 at the acceptor splice site of intron 6) in the LPL gene which both result in a catalytically inactive product were compared with 108 unaffected subjects from the same families. RESULTS: Carriers for one of these mutations had higher fasting insulin levels but only a trend towards increased fasting blood glucose concentrations could be detected. HOMA index values were significantly higher in carriers than in non-carriers. Furthermore, in carriers, a significantly higher BMI and a trend towards higher systolic and diastolic blood pressure were observed. Carriers also had significantly higher fasting triglycerides, lower HDL cholesterol, and lipoprotein lipase particles of smaller size, confirming previous reports. Among carriers, subjects with one rare allele of the SstI polymorphism in the apo CIII gene had significantly higher plasma triglyceride levels than those with two common SstI alleles. This difference could not be observed in non-carriers of a mutant lipoprotein-lipase allele. The mean intima media thickness of the carotid arteries was slightly, but not significantly higher in carriers when compared with non-carriers. CONCLUSION/INTERPRETATION: This study shows that carrier status of one defective lipoprotein-lipase allele is associated with impaired insulin sensitivity, an atherogenic lipoprotein profile and other characteristics of the metabolic syndrome, which are risk factors for atherosclerotic vascular disease. A higher incidence of atherosclerotic vascular disease, however, could not be firmly established in carriers of this study population.

Adipose Tissue↗

Enhanced secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae by translational fusion to cellulose-binding domain.

The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the fusion protein in S. cerevisiae was constructed using the alpha-amylase signal peptide and the galactose-inducible GAL10 promoter. Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion. The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures. Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus. Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S. cerevisiae, since in vivo breakage at the linker was almost negligible.

Amino Acid Sequence↗

Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil.

A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp. strain IHI-91 growing optimally at 65 degrees C. Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l-1 h-1 at a condition rate of 0.4 h-1. Lipase productivity was increased by 50% compared to data from batch fermentations. Up to 70% of the total lipase activity measured was associated to cells and by-products or residual substrate. Kinetic and stoichiometric parameters for the utilisation of olive oil were determined. The maximal biomass output method led to a saturation constant Ks of 0.88 g/l. Both batch growth data and a washout experiment yielded a maximal specific growth rate, mu max, of 1.0 h-1. Oxygen uptake rates of up to 2.9 g l-1 h-1 were calculated and the yield coefficient, Y X/O, was determined to be 0.29 g dry cell weight/g O2. From an overall material balance the yield coefficient, Y X/S, was estimated to be 0.60 g dry cell weight/g olive oil.

Bacillus↗

Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology.

The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol-1. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol-1 at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h.

Analysis of Variance↗

Spacer-mediated display of active lipase on the yeast cell surface.

We have constructed a Saccharomyces cerevisiae strain displaying an active lipase on the cell surface by cell surface engineering. The gene encoding Rhizopus oryzae lipase (ROL) was fused with the genes encoding the pre-alpha-factor leader sequence and the C-terminal half of alpha-agglutinin including the glycosylphosphatidylinositol-anchor attachment signal. The constructed gene was overexpressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance lipase activity by preserving the conformation of the active site near the C-terminal portion. Localization of the expressed ROL on the cell surface was confirmed by immunofluorescence microscopy. The ROL displayed on the yeast cell wall exhibited activity toward soluble 2,3-dimercaptopropan-1-ol tributyl ester (BALB) and insoluble triolein. The insertion of linker peptides effected the activity towards BALB, thereby demonstrating that the optimal length of linker peptides was present. The activity towards triolein was higher in lipases with longer linker peptides. ROL displayed on the cell wall exhibited a comparable and/or higher activity towards triolein than the secreted form of the enzyme. This is the first report of an active lipase displayed on the cell surface. Furthermore, insertion of a linker peptide of the appropriate length as a spacer may be an improved method to effectively display enzymes, especially those having the active region at the C-terminal portion, on the cell surface.

Agglutinins↗

Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production.

Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomvces cerevisiae MT8-1. The gene encoding lipase from R. orvzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rProROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 degrees C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 degrres C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization.

Amino Acid Sequence↗

Optimal design for the maximization of Penicillium cyclopium lipase production.

Penicillium cyclopium triacylglycerol lipase production was maximized in stationary batch culture. We used a surface response methodology based on a Doehlert experimental design to study the effect on the lipase activity released in the culture medium of the three most important factors: substrate concentration, pH and inoculum. Besides reducing the number of experiments required for optimization, this technique allowed us to quantify the lipase activity in any part of the experimental domain.We determined an optimal set of conditions for high lipase production: 1% substrate (corn steep), pH 5.5 and an inoculum of 10(4) spores/ml. Between conditions giving the minimum and the maximum lipase production, we observed a nine-fold increase of both the predicted and measured values.

Lipase↗

Regioselectivity of new bacterial lipases determined by hydrolysis of triolein.

The newly identified lipases of 67 bacterial strains, primarily Bacillus and Pseudomonas, from the ARS Culture Collection have been characterized on the basis of their positional specificity for triglycerides (triolein). Lipase was produced by growing the cultures in tryptone-glucose-yeast extract medium for 24 h at 30 degrees C before addition of triglyceride. The lipase was allowed to act on the triglyceride for 3 days before analysis by thin-layer chromatography. Of the bacterial lipases tested, 55 displayed random specificity, 9 were 1,3-specific, and 3 showed no apparent lipase activity under these conditions.

Bacillus↗

Cloning and expression of a novel lipase gene from Bacillus sphaericus 205y.

A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene.

Amino Acid Sequence↗

Serum lipase levels pre and post Lundh meal: evaluation of exocrine pancreatic status in cystic fibrosis.

Determination of pancreatic function is essential in cystic fibrosis. The most-reliable method is by measuring pancreatic enzymes in the duodenum following intravenous or oral stimulation. However, this is invasive, time consuming, and expensive. Indirect tests are non-invasive but lack accuracy. This study examines a simple test which combines pancreatic stimulation by Lundh meal and sequential serum lipase measurements. The test was performed on three groups: group A, 36 cystic fibrosis patients carrying two mutations associated with severe disease and pancreatic insufficiency (delta F508, W1282X, G542X, N1303K, S549R); group B, 8 compound heterozygote cystic fibrosis patients carrying one mutation causing mild disease with pancreatic sufficiency (3849 + 10 kb C-->T); group C, 17 healthy individuals. Basal lipase levels were 2-16.5, 16.4-73, and 8.5-27.8 U/l in groups A, B, and C, respectively, with some overlapping between groups. There were three patterns of lipase activity (1) consistently low levels (group A) suggested a severely affected insufficient pancreas; (2) normal basal levels followed by a linear rise peaking 30 min after the meal (found in 16 of 17 healthy individuals and 3 patients of group B) reflecting an unaffected sufficient pancreas; (3) elevated lipase levels not influenced by the meal (5 patients of group B). This reflects an ongoing destructive process in the pancreas which will eventually result in conversion from pancreatic sufficiency to pancreatic insufficiency. Hence serum lipase activity prior to and 30 min after Lundh meal is a good indicator of pancreatic status allowing categorization of cystic fibrosis patients as pancreatic insufficient, pancreatic sufficient, or pancreatic sufficient with late conversion to insufficiency.

Adolescent↗

DNA polymorphisms of lipase related genes.

We previously determined sequenced the PNLIP gene encoding pancreatic lipase in cell lines of subjects with clinical deficiency of pancreatic lipase (MIM 246600) and found no putative disease-causing mutations. As part of the ongoing analysis of the genomic DNA of these subjects, we now report the development of genomic amplification primers to sequence the coding regions of CLPS, CEL, PLRP1, and PLRP2, encoding pancreatic co-lipase, carboxyl-ester lipase, and pancreatic-lipase-related proteins-1 and -2, respectively. Whereas we found no putative disease-causing missense or nonsense mutations in these samples, we discovered a total of 13 common polymorphisms (12 single nucleotide polymorphisms) in these four genes. Genotypes of these polymorphisms may be useful in future association analyses.

Carboxylesterase↗

Cultivation conditions and properties of extracellular crude lipase from the psychrotrophic fungus Penicillium chrysogenum 9'.

Among 97 fungal strains isolated from soil collected in the arctic tundra (Spitsbergen), Penicillium chrysogenum 9' was found to be the best lipase producer. The maximum lipase activity was 68 units mL(-1) culture medium on the fifth day of incubation at pH 6.0 and 20 degrees C. Therefore, P. chrysogenum 9' was classified as a psychrotrophic microorganism. The non-specific extracellular lipase showed a maximum activity at 30 degrees C and pH 5.0 for natural oils or at pH 7.0 for synthetic substrates. Tributyrin was found to be the best substrate for lipase, among those tested. The Km and Vmax were calculated to be 2.33 mM and 22.1 units mL(-1), respectively, with tributyrin as substrate. The enzyme was inhibited more by EDTA than by phenylmethylsulfonyl fluoride and was reactivated by Ca2+. The P. chrysogenum 9' lipase was very stable in the presence of hexane and 1,4-dioxane at a concentration of 50%, whereas it was unstable in presence of xylene.

Arctic Regions↗