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PubMed2005

Cell surface display of lipase in Pseudomonas putida KT2442 using OprF as an anchoring motif and its biocatalytic applications.

We developed a new cell surface display system in Pseudomonas putida KT2442 using OprF, an outer membrane protein of Pseudomonas aeruginosa, as an anchoring motif in a C-terminal deletion-fusion strategy. The Pseudomonas fluorescens SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of P. putida KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47 degrees C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47 degrees C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (R)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in P. putida KT2442 and consequently for various biocatalytic applications.

Catalysis↗
PubMed1996

Physiological factors affecting production of extracellular lipase (LipA) in Acinetobacter calcoaceticus BD413: fatty acid repression of lipA expression and degradation of LipA.

The extracellular lipase (LipA) produced by Acinetobacter calcoaceticus BD413 is required for growth of the organism on triolein, since mutant strains that lack an active lipase fail to grow with triolein as the sole carbon source. Surprisingly, extracellular lipase activity and expression of the structural lipase gene (lipA), the latter measured through lacZ as a transcriptional reporter, are extremely low in triolein cultures of LipA+ strains. The explanation for this interesting paradox lies in the effect of fatty acids on the expression of lipA. We found that long-chain fatty acids, especially, strongly repress the expression of lipA, thereby negatively influencing the production of lipase. We propose the involvement of a fatty acyl-responsive DNA-binding protein in regulation of expression of the A. calcoaceticus lipBA operon. The potential biological significance of the observed physiological competition between expression and repression of lipA in the triolein medium is discussed. Activity of the extracellular lipase is also negatively affected by proteolytic degradation, as shown in in vitro stability experiments and by Western blotting (immunoblotting) of concentrated supernatants of stationary-phase cultures. In fact, the relatively high levels of extracellular lipase produced in the early stationary phase in media which contain hexadecane are due only to enhanced stability of the extracellular enzyme under those conditions. The rapid extracellular degradation of LipA of A. calcoaceticus BD413 by an endogenous protease is remarkable and suggests that proteolytic degradation of the enzyme is another important factor in regulating the level of active extracellular lipase.

Acinetobacter calcoaceticus↗
PubMed1997

Characterization of the Vibrio cholerae El Tor lipase operon lipAB and a protease gene downstream of the hly region.

We have cloned and sequenced a region encoding a lipase operon and a putative, previously uncharacterized metalloprotease of Vibrio cholerae O1. These lie downstream of hlyA and hlyB, which encode the El Tor hemolysin and methyl-accepting chemotactic factor, respectively. Previous reports identified the hlyC gene downstream of hlyAB, encoding an 18.3-kDa protein. However, we now show that this open reading frame (ORF) encodes a 33-kDa protein, and since the amino acid sequence is highly homologous to the triacylglyceride-specific lipase of Pseudomonas spp., hlyC has been renamed lipA. LipA contains the highly conserved pentapeptide and catalytic triad amino acid regions of the catalytic sites of other lipases. The region downstream of lipA has been sequenced and has revealed ORFs lipB and prtV. The amino acid sequence of lipB is homologous to those of the accessory lipase proteins (lipase-specific foldase) required by Pseudomonas and various other bacterial species for the production of mature active lipase, and in agreement with this, we show that both lipA and lipB are required to restore a lipase-deficient lipA null mutant of V. cholerae. The intergenic stop codon for lipA overlaps the ribosome-binding site for lipB, and a stem-loop resembling a rho-independent terminator is present immediately downstream from lipB, suggesting that lipA and lipB form a lipase operon in V. cholerae. prtV lies downstream of lipAB but is transcribed in the opposite direction and is predicted to share the same putative transcriptional terminator with lipAB. The zinc-binding and catalytic domains conserved among many metalloproteases are present in PrtV, which is highly homologous to the immune inhibitor A (InA) metalloprotease of Bacillus thuringiensis. PrtV was visualized as approximately 102 kDa, which is consistent with the coding capacity of the gene. The genetic organization of this region suggests that it is possibly part of a pathogenicity island, encoding products capable of damaging host cells and/or involved in nutrient acquisition by V. cholerae. However, neither lipA nor prtV null mutants were attenuated in the infant mouse model, nor did they exhibit reduced colonization potential compared with wild type in competition experiments.

Amino Acid Sequence↗
PubMed1989

Acid resistant lipase as replacement therapy in chronic pancreatic exocrine insufficiency: a study in dogs.

Conventional treatment of pancreatic steatorrhoea in man has been unsatisfactory because 90% of the lipase content of therapy is inactivated by acid in the stomach and large doses of replacement treatment are needed to provide adequate supplementation. An acid stable agent (fungal lipase) was investigated in the treatment of pancreatic deficiency steatorrhoea in 11 pancreatectomised dogs maintained on a fixed dietary intake of fat and treated with pancreatin or fungal lipase. Ten grams (60,000 U lipase) of pancreatin was compared with 400mg (4800 U lipase) of fungal lipase administered with each meal against a no treatment group. There was no significant difference in stool bulk and faecal fat excretion between pancreatin and lipase treated animals. Both groups showed a significant reduction in stool bulk and fat excretion when compared with the no treatment group (p less than 0.01). A markedly diminished treatment volume, in the form of fungal lipase, is as effective in controlling steatorrhoea as pancreatin and may prove to be a potentially valuable therapy for patients with pancreatic insufficiency.

Animals↗
PubMed1992

Separation and characterization of 61- and 57-kDa lipases from Geotrichum candidum ATCC 66592.

Two lipolytic proteins (61 and 57 kDa) present in a Sephadex G-100 fraction of extracellular lipase from Geotrichum candidum ATCC 66592 were separated using high-performance liquid chromatography. Crossed electrofocusing immunoelectrophoresis was used to demonstrate that the 61-kDa lipase fraction contained two forms of lipase with pI 4.5 and 4.7. However, when deglycosylated with endoglycosidase H, the two forms gained an identical pI, 4.6. The 57-kDa lipase fraction contained one form of lipase with pI close to 4.5. Although the 61- and 57-kDa lipases were immunologically identical, the substrate specificity differed. Thus, the 61-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was 60% of the initial velocity of hydrolysis of oleic acid methyl ester, whereas the 57-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was only 7% of the initial velocity of hydrolysis of oleic acid methyl ester.

Chromatography, High Pressure Liquid↗
PubMed1986

Diacylglycerol lipase and kinase activities in rabbit aorta and coronary microvessels.

Diacylglycerol lipase and kinase activities were measured in particulate and soluble fractions from rabbit aorta (intima-media) and coronary microvessels. With rabbit aorta, the hydrolysis at the sn-1 position of 1-palmitoyl-2-oleoyl-sn-glycerol had a pH optimum of 5-6 and was greater than hydrolysis at the sn-2 position (pH optimum of 6.5). Only the 2-monoacylglycerol accumulated during incubations at pH 5 and 6.5. These results are consistent with an ordered two-step reaction sequence where the fatty acid at the sn-1 position is released first, followed by the hydrolysis of the fatty acid from the 2-monoacylglycerol by a monoacylglycerol lipase with a neutral pH optimum. Lipase activity (sn-2 hydrolysis) at pH 6.5 was greater than kinase activity at all substrate concentrations. The presence of arachidonate at the sn-2 position of the diacylglycerol increased kinase activity but had little effect on lipase activity. Kinase activity was mainly particulate, whereas 50-60% of diacylglycerol lipase and 50% of monoacylglycerol lipase activity were soluble. Diacylglycerol lipase and kinase were also present in coronary microvessel preparations. Diacylglycerol lipase (sn-2 hydrolysis) activity in coronary microvessels was not enhanced by preincubation of the enzyme preparation with cAMP-dependent protein kinase.

Animals↗
PubMed1985

Rat lingual lipase: effect of proteases, bile, and pH on enzyme stability.

In addition to initiating fat digestion in the stomach, lingual lipase may play a significant digestive role in the upper small intestine. By in vitro incubation techniques, the stability of rat lingual lipase at various physiological pH values, as well as the effects of pure proteases, rat gastric juice, bile, pancreatic juice, and mixed duodenal contents, on enzyme activity was explored. There were no changes in base-line activity of porcine pepsin, bovine carboxypeptidase-treated lipase, or heat-denatured proteases compared with controls after incubation at pH 2-6 at 37 degrees C for up to 1 h. In contrast, porcine trypsin-treated lipase demonstrated a significant loss from base-line activity to 59 +/- 12% (mean +/- SE) at pH 4, 34 +/- 11% at pH 6, and 41 +/- 4% at pH 8, and bovine chymotrypsin caused a loss in lipase activity to 11 +/- 7% at pH 8. Rat gastric juice containing 5,000 U pepsin reduced lipase activity to 17 +/- 5% of initial activity at pH 2 and to 45 +/- 3% at pH 4. Rat bile alone diminished activity only 35%, but rat pancreatic juice or mixed duodenal juice reduced lingual lipase activity to 1-12% of initial activity after 60 min at pH 6. Lingual lipase is particularly important in fat digestion in the stomach; however, its role in quantitative fat digestion under small intestinal conditions may be limited.

Animals↗
PubMed1994

Regulation of pancreatic amylase and lipase gene expression by diet and insulin in diabetic rats.

Although insulin has been proposed to mediate the dietary regulation of pancreatic amylase, its interaction with diet in the regulation of amylase and lipase is not well understood and was examined in diabetic rats fed diets high in carbohydrate (HC), protein (HP), or fat (HF) and treated with insulin. Diabetes, independent of diet, decreased amylase content (97%; P < 0.0001) and mRNA (90%; P < 0.0001), but insulin only restored amylase content and mRNA to respective dietary control values. Diabetes, independent of diet, also increased lipase mRNA 1.6-fold (P < 0.004) but interacted (P < 0.0003) with diet on lipase content, resulting in opposite effects in HC- (increased 202%) and HF-diabetic rats (decreased 40%). Insulin partially restored lipase content and mRNA to respective dietary control values. Diet, independent of diabetes, regulated amylase content (P < 0.0001) and mRNA (P < 0.0003), which were three- to fourfold greater in HC- than in HF-fed rats, and lipase content (P < 0.001) and mRNA [rat pancreatic lipase 1 (rPL-1), P < 0.04; rPL-3, P < 0.0001], which were 1.8-fold greater in HF- than in HC- or HP-fed rats. Insulin failed to stimulate maximal amylase gene expression in HP- or HF-fed diabetic rats, suggesting that it is necessary, but not sufficient, for this dietary regulation. Differential regulation of lipase activity and mRNA by diet and insulin raises the possibility that lipase gene expression is regulated by a complex interaction of diet and insulin.

Amylases↗
PubMed1993

Thyroidal regulation of lingual lipase development in suckling rats.

Rat lingual lipase undergoes maturational increases during postnatal development. The role of thyroxine (T4) in the control of lingual lipase during development was evaluated. T4 given at an early suckling stage (starting day 4 or 5) moderately increased lingual lipase (20-30%) compared to age-matched controls. A similar dose of T4 given later (age > 2 weeks) was ineffective. The T4-sensitive period coincides with a time of low circulating T4, suggesting a role of T4 in modulating the development of lingual lipase in rat pups. Since simultaneous treatment with U486, a type II glucocorticoid receptor antagonist only partially blocked the T4 induction of lingual lipase, T4 appeared to have a direct action on the lingual gland. Pups of propylthiouracil (PTU)-treated dams (previously found to be hypothyroid) showed a delay in the maturation of lingual lipase compared to age-matched pups whose dam was not given PTU. Pups were most sensitive to PTU in the early suckling stage. PTU-induced delayed maturation of lingual lipase was a result of hypothyroidism, since T4 replacement when given early (at the age of 5 days) abolished most of the effect of PTU. When T4 was given later (at the age of 10 days), recovery was much less. This suggests the presence of an early period that is critically dependent on T4 for the full expression of lingual lipase in the rat tongue serous glands.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Hepatic lipase, lipoprotein metabolism, and atherogenesis.

The role of hepatic lipase as a multifunctional protein that modulates lipoprotein metabolism and atherosclerosis has been extensively documented over the last decade. Hepatic lipase functions as a lipolytic enzyme that hydrolyzes triglycerides and phospholipids present in circulating plasma lipoproteins. Hepatic lipase also serves as a ligand that facilitates lipoprotein uptake by cell surface receptors and proteoglycans, thereby directly affecting cellular lipid delivery. Recently, another process by which hepatic lipase modulates atherogenic risk has been identified. Bone marrow transplantation studies demonstrate that hepatic lipase present in aortic lesions markedly alters aortic lesion formation even in the absence of changes in plasma lipids. These multiple functions of hepatic lipase, which facilitate not only plasma lipid metabolism but also cellular lipid uptake, can be anticipated to have a major and complex impact on atherogenesis. Consistently, human and animal studies support proatherogenic and antiatherogenic roles for hepatic lipase. The concept of hepatic lipase as mainly a lipolytic enzyme that reduces atherogenic risk has evolved into that of a complex protein with multiple functions that, depending on genetic background and sites of expression, can have a variable effect on atherosclerosis.

Animals↗
PubMed1975

Determinants of human adipose tissue lipoprotein lipase. Effect of diabetes and obesity on basal- and diet-induced activity.

The role of insulin in the regulation of human adipose tissue lipoprotein lipase was evaluated. Adipose tissue heparin-releasable lipoprotein lipase (thought to be related to peripheral clearance of plasma triglycerides) was low in insulin-deficient, untreated hyperglycemic diabetic subjects (P less than 0.001) and treatment of hyperglycemia returned the activity to normal. In chronic hyperinsulinism, represented by obesity, heparin-releasable activity among control subjects was correlated to percent of ideal body weight (r=0.53, P less than 0.05) and to fat cell size (r=0.61, P less than 0.02). Acetone-ether powder lipoprotein lipase activity (presumed to reflect total tissue enzyme) was also related to percent of ideal body weight (r=0.76, P less than 0.001 for controls; r=0.67, P less than 0.05 for diabetics) and to fat cell size (r=0.71, P less than 0.01 for controls; r=0.85, P less than 0.01 for diabetics. Postprandial-stimulated insulin secretion was related to diet-induced changes in lipoprotein lipase in control subjects; both were dependent upon the amount of dietary carbohydrate. In contrast, the diabetic patients with low insulin responses, failed to increase lipoprotein lipase activity with feeding. The changes in heparin-releasable (r=0.66, P less than 0.01) and acetone-ether powder (r=0.69, P less than 0.01) activity during feeding were related to the percent increase in plasma insulin. Thus, insulin appears to be important in the regulation of human adipose tissue lipoprotein lipase activity. Elevated insulin levels in obesity and increased insulin secretion after eating were associated with increased lipoprotein lipase activity. Defects in insulin secretion, both in postabsorptive and postprandial states, are associated with low adipose tissue lipoprotein lipase and may lead to hypertriglyceridemia in diabetic man.

Adipose Tissue↗
PubMed1982

Colipase and maximally activated pancreatic lipase in normal subjects and patients with steatorrhea.

Human pancreatic lipase in duodenal secretions was studied under conditions of maximal activation by porcine colipase and maximal inhibition by sodium taurodeoxycholate. In almost all samples, total lipase activity in 4 mM sodium taurodeoxycholate was activated by the addition of porcine colipase. Activation was linear until saturation by cofactor was reached, and maximum activity was greater than that obtained in the absence of bile salts. At pH 8.0 in 4 mM sodium taurodeoxycholate, lipase activity was due to pancreatic lipase in samples from normal and steatorrheic individuals and was proportional to the concentration of endogenous colipase in samples that could be activated by exogenous colipase. In these samples, therefore, colipase activity could be conveniently assayed as the lipase activity at pH 0.8 in 4 mM sodium taurodeoxycholate. Colipase to total pancreatic lipase ratios varied widely from individual to individual and on average were significantly lower in steatorrheic patients. In individual samples, colipase secretion was stimulated by pancreozymin and secretin roughly in parallel with total pancreatic lipase, but some variation in the ratio of the two was often seen in successive collection periods. Because pancreatic lipase is usually unsaturated with respect to cofactor, lipolytic activity in duodenal secretions may be finely controlled by modulation of colipase secretion.

Animals↗
PubMed2003

Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy.

Lipoprotein lipase is the principal enzyme that hydrolyzes circulating triglycerides and liberates free fatty acids that can be used as energy by cardiac muscle. Although lipoprotein lipase is expressed by and is found on the surface of cardiomyocytes, its transfer to the luminal surface of endothelial cells is thought to be required for lipoprotein lipase actions. To study whether nontransferable lipoprotein lipase has physiological actions, we placed an alpha-myosin heavy-chain promoter upstream of a human lipoprotein lipase minigene construct with a glycosylphosphatidylinositol anchoring sequence on the carboxyl terminal region. Hearts of transgenic mice expressed the altered lipoprotein lipase, and the protein localized to the surface of cardiomyocytes. Hearts, but not postheparin plasma, of these mice contained human lipoprotein lipase activity. More lipid accumulated in hearts expressing the transgene; the myocytes were enlarged and exhibited abnormal architecture. Hearts of transgenic mice were dilated, and left ventricular systolic function was impaired. Thus, lipoprotein lipase expressed on the surface of cardiomyocytes can increase lipid uptake and produce cardiomyopathy.

Animals↗
PubMed1984

Fat digestion in the stomach: stability of lingual lipase in the gastric environment.

Digestion of dietary fat starts in the stomach, where lingual lipase hydrolyzes triglycerides to free fatty acids and partial glycerides at pH 3.0-6.0. Lingual lipase is secreted continuously from lingual serous glands and accumulates in the stomach between meals, when gastric pH is less than 3.0. We have, therefore, examined the resistance of lingual lipase to low pH and its possible protection by dietary components present in the stomach contents. Partially purified rat lingual lipase (7-15 micrograms enzyme protein) was preincubated at 37 degrees C for 10-60 min at pH 1.0-6.0 before incubation for assay of lipolytic activity, hydrolysis of tri-[3H]olein at pH 5.4. The data show that partially purified rat lingual lipase preparations are stable at 37 degrees C in the pH range of 2.5-6.0. Enzyme activity, however, is rapidly and irreversibly lost during preincubation at pH 1.0-2.4 for 10-30 min. Protein (gelatin 1% or albumin 1% or 2.5%) cannot prevent the inactivation of lingual lipase at low pH. The large molecular species (molecular weight greater than 500,000) of lingual lipase (thought to be an aggregate of enzyme with lipids) is slightly more resistant to inactivation than the 46,000 dalton preparation, suggesting that lipids might protect the enzyme from inactivation. Indeed, about 60% of the initial lipase activity is preserved during incubation at pH 2.0 in the presence of 50 mM lecithin or 10 mM triolein. The data indicate that triglycerides which are hydrolyzed by this enzyme as well as phospholipids that are not hydrolyzed can prevent the inactivation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Absence of triglyceride accumulation in lipoprotein lipase-deficient human monocyte-macrophages incubated with human very low density lipoprotein.

Lipoprotein lipase, a lipolytic enzyme essential for normal hydrolysis of triglycerides in very low density lipoprotein (VLDL) and chylomicrons, is found in several cell types, including macrophages. The role of lipoprotein lipase in mediating the uptake of normal VLDL triglycerides into human cultured monocyte-derived macrophages was studied using macrophage cells from a functionally lipoprotein lipase-deficient patient and macrophages of cells from a normal subject. After incubation with VLDL, massive accumulation of phase refractile (lipid) inclusions were noted by phase contrast microscopy within the normal, but not within the lipoprotein lipase-deficient, macrophages. Chemical determinations of intracellular lipid confirmed massive triglyceride accumulation within normal macrophages, but not in lipoprotein lipase-deficient macrophages. VLDL-derived cholesterol did not accumulate in either cell. These results confirm an additional role of lipoprotein lipase, that of mediating triglyceride accumulation into macrophages from normal human VLDL. Human monocyte-macrophages genetically deficient in a functional lipoprotein lipase will be useful to determine the role of lipoprotein lipase in macrophage accumulation of lipid from other forms of triglyceride-carrying lipoproteins, including hypertriglyceridemic VLDL, beta-VLDL, and chylomicrons.

Adult↗
PubMed2002

Cloning, characterization, and expression of cDNA encoding a lipase from Kurtzmanomyces sp. I-11.

A cDNA clone of the lipase secreted by Kurtzmanomyces sp. I-11 was isolated from a cDNA library of this yeast by PCR screening using oligonucleotide primers designed on the basis of the partial amino acid sequence of the lipase. The cloned cDNA (lip1) encoded a hydrophobic protein of 484 amino acids, where the first 20 amino acids and the following 6 amino acid sequences were predicted to be the signal sequence for secretion and a pro-sequence, respectively. The deduced amino acid sequence of the Kurtzmanomyces lipase was most similar to Candida antarctica DSM 3855 lipase A (74% identity) and weakly to other lipases. The consensus pentapeptide (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases was conserved. A high level of lipase was produced by Pichia pastoris transformed with the lip1 cDNA, indicating that the cloned cDNA indeed encodes a lipase.

Amino Acid Sequence↗
PubMed2003

Improvements in lipase production and recovery from Acinetobacter radioresistens in presence of polypropylene powders filled with carbon sources.

Polypropylene powders as the adsorbent for organic solution containing n-hexadecane and olive oil were employed as the carbon source for producing an alkaline lipase from Acinetobacter radioresistens. The best volumetric ratio of n-hexadecane to olive oil around 5 for lipase production was determined from shake-flask and fermentation cultivations. The existence of a maximum time course lipase activity of the aqueous phase was attributed to the compensation effects of olive oil on cell growth and lipase production, repression of lipase synthesis by oleic acid, and lipase adsorption on the supports. A linear relationship between the average cell growth rate in the exponential phase and the ratio of surface areas of the supports was found. The benefits of using the present fermentation process include less foaming and emulsion of the broth, less organic phase used, higher lipase production, and easy recovery of the lipase in the centrifugation step.

Acinetobacter↗
PubMed1993

Molecular characterization of an extracellular acid-resistant lipase produced by Rhizopus javanicus.

An extracellular lipase (triacylglycerol acylhydrolase EC 3.1.1.3), produced by the fungus Rhizopus javanicus was purified to homogeneity using an expeditious two-step isolation method. The enzyme, with a molecular mass of 36 kDa and a specific activity of 9260 microequivalent of fatty acid released per minute and mg under standard conditions, consists of three isoforms with isoelectric points of 7.8, 7.7, and 7.1, respectively. The purified lipase was digested using chemical and enzymatical procedures: CNBr cleavage, partial acid hydrolysis, and proteolytic cleavage by means of trypsin. Amino-acid sequencing of the resulting peptides indicates that the three lipases from Rhizopus javanicus, Rhizopus niveus and Rhizopus delemar are produced as identical proenzymes but processed differently. These Rhizopus lipases show 54% identity with the lipase from Rhizomucor miehei. Using the structure of the Rhizomucor miehei lipase, the molecular model of Rhizopus javanicus lipase was constructed. Both enzymes are alpha/beta type proteins with a central 8-stranded mixed beta-pleated sheet and have a remarkably similar distribution of hydrophobic amino acids at their surface. The tryptophan in the center of the helical lid covering the active site of Rhizomucor miehei lipase is mutated into an alanine, indicating that it is not essential for the proper movement of the helical lid.

Amino Acid Sequence↗