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PubMed2005

A study of lipoprotein lipase gene intron 8 polymorphisms in Chinese Han race essential hypertension patients.

OBJECTIVE: To investigate the association of lipoprotein lipase gene intron 8 polymorphisms and Essential Hypertension in Han race Chinese. METHODS AND RESULTS: 116 patients with Essential Hypertension were enrolled and another 116 normal people were served as controls. All cases were examined for the genotypes of intron 8 in lipoprotein lipase gene by the methods of polymerase chain reaction restriction fragment length polymorphism, and the serum lipoprotein levels were also observed. Results showed that body mass index blood pressure and the serum triglyceride level were obviously increased in the Essential Hypertension group. The genotype and allele frequency of intron 8 in lipoprotein lipase in the Essential Hypertension group showed obvious differences compared with the control group. Serum triglyceride levels were higher in the patients with H+H+ genotype than in those in non H+H+ genotype of intron 8 in lipoprotein lipase by HindIII digestion. The systolic blood pressure showed a decreasing tendency among the H+H+ genotype, the H+H- genotype and the H-H- genotype individuals. CONCLUSION: The results suggest that lipoprotein lipase may be an important genetic factor associated with the Chinese Han race Essential Hypertension patients. The polymorphisms of intron 8 in lipoprotein lipase influence the blood-lipid metabolism, induce blood vessel rebuilding and play an important role in the invasion and development of Essential Hypertension.

Asian People↗
PubMed2004

Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica.

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering. In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene. We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures. Batch culture led to a lipase production of 26450 U ml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources. Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246 U ml(-1) after 80 h of cultivation. In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.

Bioreactors↗
PubMed2005

Surfactant tail length-dependent lipase activity profile in cationic water-in-oil microemulsions.

The catalytic activity of Chromobacterium viscosum lipase (CV-lipase) was estimated across varying surfactant tail lengths (C-10-C-18) in water-in-oil (w/o) microemulsions of cationic surfactants containing four different hydroxyethyl-substituted head groups. An attempt to find a correlation, if any, between the activity of interfacially solubilized lipase and the varying surfactant tails was made for the first time in micellar enzymology. The second-order rate constant, k2, in lipase-catalyzed hydrolysis of p-nitrophenyl-n-hexanoate at pH 6.0 and 25 degrees C shows an improvement in enzyme activity (approximately 30-140%) across different head groups of amphiphiles with increasing tail lengths in varying solution compositions. Improvement of enzyme activity is prominent in ascending from C-10 to C-14/C-16, depending on the nature of polar head group. The hydrolytic activity of lipase in different surfactant (50 mM)/water/isooctane/n-hexanol with varying z= [alcohol]/[surfactant] (6.4 or 4.8) was amplified by 25-250% with increment in surfactant tail length in comparison with widely used cationic w/o microemulsions having solution compositions (z=16). As a notable outcome of this research, we found w/o microemulsions of 25 mM tetradecyltrimethylammonium bromide/water/isooctane/n-hexanol (z=8) producing the highest ever activity of lipase in any w/o microemulsions.

Catalysis↗
PubMed2006

Identification and over-expression of a thermostable lipase from Geobacillus thermoleovorans Toshki in Escherichia coli.

A newly isolated thermophilic strain producing thermostable lipase was identified based on 16S rRNA sequencing, where phylogenetic analysis revealed its closeness to Geobacillus thermoleovorans. Thermostable lipase from this bacterium was cloned using consensus degenerate PCR primers. For over-expression in Escherichia coli, the lipase gene was sub-cloned in pET 15b vector with a strong T7 promotor. Lipase activity was approximately 4.5-fold higher than in the wild-type strain. The lipase enzyme was thermostable at 60 degrees C and pH 8, whereas a 30% residual activity was retained when incubated for 1h at 100 degrees C. Optimum lipase expression was obtained in 2 x YT medium after 70min of induction by IPTG.

Bacillaceae↗
PubMed2005

Expression, purification, and characterization of His-tagged Staphylococcus xylosus lipase wild-type and its mutant Asp 290 Ala.

The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR technique. The sequence corresponding to the mature lipase was subcloned in the pET-14b expression vector, with a strong T7 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. High level expression of the lipase by Escherichia coli BL21 (DE3) cells harbouring the lipase gene containing expression vector was observed upon induction with 0.4 mM IPTG at 37 degrees C. One-step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified His-tagged SXL was 1500 or 850 U/mg using tributyrin or olive oil emulsion as substrate, respectively. It has been proposed that the region near the residue Asp290 could be involved in the selection of the substrate. Therefore, we also mutated the residue Asp 290 by Ala using site-directed mutagenesis. The mutant SXL-D290A was overexpressed in E. coli BL21 (DE3) and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged SXL-D290A mutant was 1000 U/mg using either tributyrin or olive oil emulsion as substrate. A comparative study of the wild type (His(6)-SXL) and the mutant (His(6)-SXL-D290A) proteins was carried out. Our results confirmed that Asp290 is important for the chain length specificity and catalytic efficiency of the enzyme.

Amino Acid Substitution↗
PubMed2005

Monoacylglycerol lipase inhibition by organophosphorus compounds leads to elevation of brain 2-arachidonoylglycerol and the associated hypomotility in mice.

Three components of the cannabinoid system are sensitive to selected organophosphorus (OP) compounds: monoacylglycerol (MAG) lipase that hydrolyzes the major endogenous agonist 2-arachidonoylglycerol (2-AG); fatty acid amide hydrolase (FAAH) that cleaves the agonist anandamide present in smaller amounts; the CB1 receptor itself. This investigation considers which component of the cannabinoid system is the most likely contributor to OP-induced hypomotility in mice. Structure-activity studies by our laboratory and others rule against major involvement of a direct toxicant-CB1 receptor interaction for selected OPs. Attention was therefore focused on the OP sensitivities of MAG lipase and FAAH, assaying 19 structurally diverse OP chemicals (pesticides, their metabolites and designer compounds) for in vitro inhibition of both enzymes. Remarkably high potency and low selectivity is observed with three O-alkyl (C1, C2, C3) alkylphosphonofluoridates (C8, C12) (IC50 0.60-3.0 nM), five S-alkyl (C5, C7, C9) and alkyl (C10, C12) benzodioxaphosphorin oxides (IC50 0.15-5.7 nM) and one OP insecticide metabolite (chlorpyrifos oxon, IC50 34-40 nM). In ip-treated mice, the OPs at 1-30 mg/kg more potently inhibit brain FAAH than MAG lipase, but FAAH inhibition is not correlated with hypomotility. However, the alkylphosphonofluoridate-treated mice show dose-dependent increases in severity of hypomotility, inhibition of MAG lipase activity and elevation of 2-AG. Moderate to severe hypomotility is accompanied by 64 to 86% MAG lipase inhibition and about 6-fold elevation of brain 2-AG level. It therefore appears that OP-induced MAG lipase inhibition leads to elevated 2-AG and the associated hypomotility.

Amidohydrolases↗
PubMed1998

Inhibition of lipases from Chromobacterium viscosum and Rhizopus oryzae by tetrahydrolipstatin.

Tetrahydrolipstatin is known as an inhibitor for pancreatic lipase but not for microbial lipases. In this paper we demonstrate that in the presence of water-insoluble substrates like tributyrin or olive oil, tetrahydrolipstatin inhibits the lipases of Chromobacterium viscosum and Rhizopus oryzae, although with different potency. In contrast to porcine pancreatic lipase, which forms an irreversible and covalent enzyme-inhibitor complex with tetrahydrolipstatin, the inhibition of the microbial lipases is reversible as the inhibitor can be removed from the enzyme-inhibitor complex by solvent extraction. Moreover, after inhibition of Chromobacterium viscosum lipase tetrahydrolipstatin remains chemically unchanged.

2-Propanol↗
PubMed2003

Fluorescent organophosphonates as inhibitors of microbial lipases.

Short- and long-chain 1-O-alkyl-2-acylaminodeoxyglycero- and alkoxy-alkylphosphonic acid p-nitrophenyl esters were synthesized as inhibitors for analytical and mechanistic studies on lipolytic enzymes. The respective compounds contain perylene or nitrobenzoxadiazole as reporter fluorophores covalently bound to the omega-ends of the respective 2-acylamino- and alkoxy- residues. Their inhibitory effects on the activities of three selected lipases showing different substrate preferences were determined, including the lipases from Rhizopus oryzae, Pseudomonas species, and Pseudomonas cepacia. R. oryzae lipase reacted much better with the single-chain inhibitors than the two-chain deoxyglycerolipids. In contrast, P. cepacia lipase was inactivated by perylene-containing two-chain phosphonate (XXII) to a larger extent as compared to the other inhibitors whereas Pseudomonas species lipase interacted efficiently and without any preferences with all inhibitors used in this study. In summary, the different lipases show a very characteristic reactivity pattern not only with respect to triacylglycerol substrates but also to their structurally related inhibitors. Thus, the novel phosphonates might be useful tools not only for analysis and discrimination of known lipolytic enzymes but also for discovery of yet unknown lipases/esterases in biological samples.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

A new fluorometric method for measuring the action of C apolipoproteins on milk lipoprotein lipase.

Monolayer vesicles containing pyrene-labelled nonanoyltriglyceride (1-2 ditetradecyl 3-pyrene nonanoyl glyceride) were used as a substrate to measure bovine milk lipoprotein lipase activity. The activation of lipoprotein lipase by synthetic fragments of apolipoprotein C II and apo C III was measured. Fragments 30-78 and 43-78 had actions similar to that of the entire apo C II. Fragments 50-78 and 55-78 were 50% active, fragment 60-78 was 10% active and fragment 66-78 was inactive. Thus the activating capacity depended on the length of the carboxyterminal fragment. Replacing tyrosine 62 in apo C II by glycine removed all lipoprotein lipase activating capacity, while making Tyr 62 less accessible for binding to lipids and enzyme decreased apo C II activating capacity. Apo C III1 inhibited both basal lipoprotein lipase activity (no apo C II) and lipoprotein lipase activated by apo C II. Apo C III, fragment A (1-40) which did not bind lipids, had no inhibitory effect, while fragment B(41-79) had the same effect as whole apo C III,. Apo AI, AII and C I also inhibited lipoprotein lipase. The fluorometric assay is easy to perform, and suitable for metabolic studies such as fatty-acid exchanges between lipoproteins, as it produces no alteration in the reaction products. It also avoids the use of a radio-labelled substrate.

Animals↗
PubMed1991

Hepatic lipase activity during oral and parenteral 17 beta-estradiol replacement therapy: high-density lipoprotein increase may not be antiatherogenic.

OBJECTIVE: Hepatic lipase activity is involved in the removal of cholesterol and phospholipid from plasma high-density lipoprotein (HDL) to the liver. Changes in hepatic lipase are responsible for some lipoprotein modifications observed during estrogen administration (i.e., increased HDL levels). The aim of this study was to compare the effects of alternative routes of administration of estrogen on hepatic lipase activity and lipoprotein metabolism. DESIGN, PATIENTS: The effects of oral and parenteral estradiol (E2) replacement therapy on post-heparin hepatic lipase were studied in the short-term (6 weeks) in postmenopausal women. INTERVENTIONS: Ten patients received 2 mg/d or oral micronized E2 and 10 patients 3 mg/d of percutaneous E2. RESULTS: Both treatments significantly increased plasma E2 levels. Hepatic lipase decreased (-33%) significantly (P less than 0.05), and the phospholipids and free cholesterol content of HDL and HDL3 increased significantly (P less than 0.05) during oral but not during parenteral treatment. CONCLUSIONS: The short-term pharmacological effect of E2 on hepatic lipase depends on the route of administration. The inhibition of this enzyme may reduce the removal of HDL-cholesterol by the liver. The expected vascular benefits of such a pharmacological increase in HDL are questionable.

Administration, Cutaneous↗
PubMed1999

Hydrophobic interaction chromatography of Chromobacterium viscosum lipase on polypropylene glycol immobilised on Sepharose.

The fractionation of Chromobacterium viscosum lipase was performed using a polypropylene glycol-Sepharose gel. The influence of mobile phase composition on the adsorption of lipase on the gel was studied and it was found that the retention of lipase depends on the salt used and increased with increasing the ionic strength. The retention was not strongly affected by changing the pH value of the mobile phase. By using 20% (w/v) ammonium sulphate in phosphate buffer a total retention of lipase on the column was obtained and by simply decreasing the ionic strength of the buffer, desorption of lipase could be achieved. The chromatographic purification of Chromobacterium viscosum lipase by hydrophobic interaction chromatography on Sepharose CL-6B modified by covalent immobilisation of 1,4-butanediol diglycidyl ether, polyethylene glycol and polypropylene glycol was also compared.

Butylene Glycols↗
PubMed1987

Protein kinase inhibitor blocks the activation of a myocardial triacylglycerol lipase.

Purified inhibitor of the cyclic AMP-dependent protein kinase (PKI) has been used as a probe to determine if hormone and cyclic AMP-induced activation of the cardiac alkaline triacylglycerol (TG) lipase is mediated through the cAMP-dependent protein kinase. Addition of CAM (cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine) to any of the four fractions (homogenate, 10,000 g supernatant, 105,000 g supernatant, or heparin-Sepharose eluate) from heparin perfused heart activated the TG lipase 60% to 110%. Preincubation of these fractions with 33 ng of PKI had no effect on control enzyme activity. Addition of PKI (33 ng) to extracts following CAM activation had little effect on homogenate TG lipase activity, but reduced activities in 10,000 g and 105,000 g supernatant fractions to their respective control levels, and inhibited TG hydrolase activity of activated heparin-Sepharose eluate to 50% below the control activity. If extracts were preincubated with PKI prior to CAM addition, TG lipase activity was reduced to approximately 50% below control levels in all fractions. PKI addition (33 ng) to 105,000 g supernatant obtained from hearts stimulated 60% by epinephrine perfusion reduced activity to 50% below the control level. PKI inhibition of TG lipase activity of 105,000 g supernatant could be reversed by adding 0.5 microgram of catalytic subunit of protein kinase (PKC) to the extract. The inhibition below control levels caused by CAM and PKI indicate that the PKI-PKC complex by itself or in combination with other extract molecules, has an inhibitory effect on the TG lipase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Is decreased activity of C-II activated lipoprotein lipase in type III hyperlipoproteinemia (broad-beta-disease) a cause or an effect of increased apolipoprotein E levels?

Apolipoprotein E (ApoE; "arginine-rich" polypeptide) strongly inhibited both C-I and C-II activated lipoprotein lipases but not the protamine insensitive triglyceride lipase. Inhibition of lipoprotein lipases by ApoE in contrast to inhibition by C-III was not reversed to any significant extent by either increased concentration of activator or triglyceride in the substrate. Our previous studies have shown that in a type III hyperlipoproteinemia (broad-beta-disease) a post-heparin plasma lipoprotein lipase activated by C-II polypeptide of lipoprotein C is decreased in enzyme activity and exhibits an impaired ability to hydrolyze triglycerides in very low density lipoproteins. Type III patients are characterized by elevated concentrations of ApoE in the serum. The data presented in this report suggest that the decreased C-II activated lipoprotein lipase may be further aggravated by increased ApoE levels. Since this enzyme is involved in the catabolism and removal of lipoproteins, decreased activity of C-II activativated lipoprotein lipase may presumably be responsible for increased ApoE.

Apolipoproteins↗
PubMed1983

Lung lipoprotein lipase: inhibition by barbiturates.

The action of barbiturates on the enzymatic activity of lung lipoprotein lipase has been studied in vitro and in vivo. Mouse lung preparations can be separated into two fractions containing lipoprotein lipase activity and designated as soluble and membrane-bound. In vitro, both enzyme fractions were inhibited in a graded manner by the barbiturates when present in concentrations of 1-4 mg/ml. Experiments to determine the mechanism for the inhibition of lipoprotein lipase suggest that it may be related to non-specific binding of the drug to the enzyme since other drugs e.g., phenylbutazone and a coumarin derivative which bind non-specifically to serum albumin showed similar inhibitory activity. When rat lungs were perfused in situ with heparin, 49% of the total lipoprotein lipase activity was released into the perfusate. However, when pentobarbital was perfused prior to heparin, no lipoprotein lipase activity was observed in the perfusate. The possible clinical implications resulting from inhibition of pulmonary lipoprotein lipase by barbiturates are discussed in this paper.

Animals↗
PubMed1997

The synthetic pentasaccharide SR 90107A/Org 31540 does not release lipase activity into the plasma.

The present study was designed to find out whether the synthetic pentasaccharide SR 90107A/Org 31540, which is presently being evaluated in clinical trials as an antithrombotic agent, influences lipoprotein metabolism in rats as determined by plasma triglyceride (TG) lipase activity. A comparison with three clinically used sulphated polysaccharides-unfractionated heparin (UFH), low molecular weight heparin (LMWH) and pentosan polysulphate (PPS)- was performed. UFH evoked a dose-dependent increase in plasma TG lipase activity which plateaued at doses > or = 1 mg/kg i.v.. PPS and LMWH demonstrated a lower efficacy than heparin at 0.3 and 1 mg/kg i.v., but the maximum lipase releasing effect at 3 mg/kg i.v. was identical for UFH, PPS and LMWH. SR 90107A/Org 31540 did not release TG lipase activity at single i.v. doses up to 3 mg/kg. Repeated-dose experiments with SR 90107A/Org 31540 (1 mg/kg s.c. for 9 days) revealed no influence on the lipase releasing effect of UFH (1 mg/kg i.v. on day 10). These results demonstrate that SR 90107A/Org 31540 does not influence lipid metabolism in rats through lipase release, suggesting that SR 90107A/Org 31540 may offer an advantage over UFH and LMWH in clinical situations where an anticoagulant/antithrombotic effect is desired, but both an increase in plasma free fatty acids and atherogenic alterations of lipoprotein metabolism are considered harmful.

Animals↗
PubMedDate not supplied

Ring-opening bulk polymerization of epsilon-caprolactone and trimethylene carbonate catalyzed by lipase Novozym 435.

The affects of lipase concentration on ring-opening bulk polymerizations of epsilon-caprolactone and trimethylene carbonate were studied by using Novozym 435 (immobilized form of lipase B from Candida antarctica) as biocatalyst. The polymerization of epsilon-caprolactone was carried out in bulk at 70 degrees C. Three lipase concentrations of 9.77, 1.80 and 0.50 mg/mmol epsilon-CL were used in the experiment. The results showed that increasing the lipase concentration used in the polymerization system resulted in an increased rate of monomer consumption. For an enzyme concentration of 9.8 mg lipase per mmol monomer, an 80% monomer conversion was achieved in a 4-h time period, while for the lower enzyme concentration of 1.8 mg lipase per mmol monomer, 48 h were needed to reach monomer conversion. Linear relationships between Mn and monomer conversions were observed in all three enzyme concentrations, suggesting that the product molecular weight may be controlled by the stoichiometry of the reactants for these systems. At the same monomer conversion level, however, Mn decreased with increasing enzyme concentration. After correcting for the amount of monomer consumed in initiation, the plot of ln[([M]o - [M]i)/([Mt] - [M]i)] versus reaction time was found to be linear, suggesting that the monomer consumption followed a first-order rate law and no chain termination occurred. For the TMC systems, the polymerization was carried out in bulk at 55 degrees C. Similar to the epsilon-CL systems, increasing the Novozym 435 concentration from 8.3 to 23.6 mg/mmol TMC increased the rate of monomer conversion. Unlike the epsilon-CL systems, however, nonlinear relationships were obtained between Mn and monomer conversion, indicating that possible chain transfer and/or slow initiation had taken place in these systems. Consistent with the above result, nonlinear behavior was observed for the plot of ln[[M]o/[M]t] versus reaction time.

Candida↗
PubMed2004

Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups.

A method for immobilization of Candida rugosa lipase to two types of chitosan beads by activating the hydroxyl groups of chitosan using carbodiimide coupling agent has been successfully developed. The ability of carbodiimide to activate the hydroxyl groups of chitosan was confirmed using the electron spectroscopy for chemical analysis (ESCA) technique. The properties of lipase immobilized using dry and wet chitosan beads were also investigated and compared. Immobilization enhanced the enzyme stability against changes of pH and temperature. High storage stability of 30 days and an increased enzyme activity of 2,110% were observed in wet immobilized lipase. Immobilized lipase using dry and wet chitosan beads retained 78% and 85% of its initial activity after 10 batch hydrolytic cycles. The kinetic parameters Km and Vmax were determined for the free and immobilized lipase. The activation energy (Ea) was found to decrease for immobilization of lipase on chitosan beads.

Candida↗
PubMed1997

Thermoalkalophilic lipase of Bacillus thermocatenulatus large-scale production, purification and properties: aggregation behaviour and its effect on activity.

Escherichia coli BL321 was transformed with the expression plasmid pCYTEXP1 carrying the BTL2 gene from Bacillus thermocatenulatus under the control of the strong temperature-inducible lambda pL promoter and was cultivated in a 100 1 bioreactor. The mature lipase was produced in large quantities (54,000 U g-1 wet cells) and further purified to homogeneity by a two-step purification protocol (hydrophobic chromatography and gel filtration chromatography). The pure enzyme was characterized and its physicochemical properties compared to those of the BTL2 lipase which had previously been weakly expressed in E. coli under the control of its native promoter on pUC18, yielding 600 U g-1 wet cells. The specific activity of the overexpressed enzyme was approx. 5-fold higher than that of the weakly expressed enzyme. The two proteins showed the same pI and N-terminal sequence and had very similar thermostability, pH stability, optimum pH and temperature activity, and substrate specificity. Both enzymes were extremely stable in the presence of several organic solvents and detergents. With trioleylglycerol as a substrate, the overexpressed lipase cleaves each of the three ester bonds. The purified BTL2 lipase shows a strong tendency to aggregate. Direct evidence for changes in the aggregation state was obtained by gel filtration chromatography. The effect of aggregation on lipase activity was strongly dependent on both substrate and temperature during the assay. Under certain conditions, a direct relationship was found between the molecular mass of the lipase aggregates and the increase in activity upon the addition of 1% (w/v) sodium cholate.

Bacillus↗