Crude Porcine Pancreatic Lipase Research Articles

Anti-pancreatic lipase and anti-adipogenic effects of 5,7,3′,4′,5′-pentamethoxy and 6,2′,4′-trimethoxy flavone - An in vitro study

In this study, the anti-obesity effects of 5,7,3′,4′,5′-pentamethoxyflavone (PMF) and 6,2′,4′-trimethoxyflavone (TMF) were evaluated through two distinct mechanisms of action: inhibition of crude porcine pancreatic lipase (PL), and inhibition of adipogenesis in 3T3-L1 pre-adipocytes. Both flavones show dose dependent, competitive inhibition of PL activity. Molecular docking studies revealed binding of the flavones to the active site of PL. In 3T3-L1 adipocytes, both flavones reduced the accumulation of lipids and triglycerides. PMF and TMF also lowered the expression of adipogenic and lipogenic genes. They both reduced the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer-binding protein α and β (C/EBP α and β), sterol regulatory element-binding protein 1 (SREBF 1), fatty acid synthase (FASN), adipocyte binding protein 2 (aP2), and leptin gene. In addition, these flavones enhanced adiponectin mRNA expression, increased lipolysis and enhanced the expression of lipolytic genes: adipose triglycerides lipase (ATGL), hormone sensitive lipase (HSL) and monoglycerides lipase (MAGL) in mature 3T3-L1 adipocytes. Overall, PMF was seen to be a more potent inhibitor of both PL activity and adipogenesis versus TMF. These results suggest that PMF and TMF possess anti-obesity activities and can be further evaluated for their anti-obesity effects.

Immobilization of porcine pancreatic lipase on poly-hydroxybutyrate particles for the production of ethyl esters from macaw palm oils and pineapple flavor

Poly-hydroxybutyrate particles (PHB) were used as support to immobilize porcine pancreatic lipase (PPL). The biocatalysts prepared were tested in the synthesis of pineapple flavor by esterification of butanol and butyric acid in heptane medium, and in the synthesis of ethyl esters by transesterification of macaw palm pulp (MPPO) and macaw palm kernel (MPKO) oils with ethanol in solvent-free systems. The effect of protein loading on the biocatalyst activity was assessed in olive oil hydrolysis. Maximum hydrolytic activity of 292.8±8.60IU/g was observed. Langmuir isotherm model was applicable to the adsorption of PPL on PHB particles. Maximum immobilized protein amount was 24.3±1.70mg/g. The optimal pH and temperature in hydrolysis reaction for the immobilized PPL were at pH 8.5 and 50°C, while for the crude PPL extract were at pH 8.0 and 45°C. Immobilized PPL exhibited full hydrolytic activity after 2h of incubation in non-polar solvents. In esterification reaction, optimal conversion was around 93% after 2h of reaction. After six esterification cycles, the biocatalyst retained 63% of its initial activity. The biocatalyst prepared attained transesterification yield of 50% after 48h of reaction for MPKO and 35% after 96h of reaction for MPPO.

Purification of porcine pancreatic lipase by aqueous two-phase systems of polyethylene glycol and potassium phosphate

An aqueous two-phase system (ATPS) was applied for the purification of porcine pancreatic lipase (PPL) from crude PPL using polyethylene glycol (PEG) and potassium phosphate. Phase diagrams for polyethylene glycol (PEG) and potassium phosphate dibasic were determined at room temperature to find an operating region to first form the ATPS. The PPL was preferentially partitioned into the PEG-rich phase in systems with molecular weights of 1000 and 1500 and concentrated in the phosphate-rich phase in systems with PEG of 4000. Moreover, instead of tie line length (TLL), we used a stability ratio without NaCl in the system, and we first applied fluorescence spectroscopy for the protein conformational analysis of the ATPS. The molecular weight of the purified lipase was determined to be approximately 52 kDa by SDS-PAGE. The enzyme was efficiently purified in PEG 1500/potassium phosphate (17/13, %) at a pH of 7.0 at 4 °C. This system obtained an enzyme partition coefficient of 12.7, an extraction efficiency of 94.7% and a purification factor of approximately 4. These results demonstrate that the aqueous two-phase system is a highly efficient method for PPL purification.

Enzymatic kinetic resolution of primary allenic alcohols. Application to the total synthesis and stereochemical assignment of striatisporolide A

Crude Porcine pancreatic lipase was successfully used for the kinetic resolution of axially chiral primary allenic alcohols providing very high enantioselectivities with E values above 200. This simple access to optically active allenes was applied to the total synthesis of the fungal metabolite (-)-striatisporolide A, allowing its unambiguous stereochemical assignment.

Screening of selected food and medicinal plant extracts for pancreatic lipase inhibition.

Lipids are important components in human nutrition; however, their increased intake contributes to the development of obesity and can lead to multiple long-term complications. Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a key enzyme for the absorption of dietary triglycerides. Interference with fat hydrolysis results in the reduced utilization of ingested lipids, therefore inhibition of lipases decreases fat absorption. Extracts from 106 species of medicinal plants, vegetables and fruits were screened for potential lipase inhibitory activity. p-Nitrophenylpalmitate and 5-bromo-4-chloro-3-indoxylpalmitate were used as substrates in an in vitro test with crude porcine pancreatic lipase. Bearberry (Arctostaphylos uva-ursi), garden pea (Pisum sativum), Norway spruce (Picea abies) and large-leaved lime (Tilia platyphyllos) extracts were the most active. Additionally, the activity of selected extracts with removed polyphenols was measured. Extracts of bearberry, garden pea and large-leaved lime are a promising source for developing functional foods or isolating active compounds.

Gelatin Blends with Alginate: Gels for Lipase Immobilization and Purification

Blends of natural polysaccharide sodium alginate (5%) with gelatin (3%) cross-linked with glutaraldehyde provide beads with excellent compressive strength (8 x 10(4) Pa) and regular structure on treatment with calcium chloride. Lipases from porcine pancreas, Pseudomonas cepacia, and Candida rugosa were immobilized in such a blend with excellent efficiency. The immobilized enzymes were stable and were reused several times without significant loss of enzyme activity both in aqueous and reverse micellar media. The beads were functionalized with succinic anhydride to obtain beads with extra carboxylic acid groups. These functionalized beads were then successfully used for 7.4-fold purification of crude porcine pancreatic lipase in a simple operation of protein binding at pH 5 and release at pH 8.5.

Quality improvement of crude porcine pancreatic lipase preparations by treatment with humid supercritical carbon dioxide

Crude preparations of porcine pancreatic lipase (PPL) contain apart from the PPL a significant number of other hydrolases as impurities. The treatment of crude PPL preparations with humid supercritical carbon dioxide leads to a quality improvement of the preparations. Five hundred milligrams of PPL preparation were incubated in a high pressure reactor (140 ml) at 150 bar and 75°C for 24 h. Different amounts of water were put into the reactor prior incubation. A set of different assays based on hydrolytic activities was used after the treatment. It was found out that the treated preparations are significantly more active with the long chain triglyceride triolein (675% residual activity with 100 μl water added) and an artificial substrate (1,2- o-dilauryl-rac-glycero-3-glutaric acid-resorufin ester (DGGR)), while there is a loss of activity towards the short chain triglycerides, tributyrin and triacetin. Treatment with humid supercritical carbon dioxide activates the PPL that is active towards long chain triglycerides and denatures the other hydrolases that are active only towards short chain triglycerides. Repeated incubation in dry supercritical carbon dioxide causes no loss of activity towards short chain triglycerides indicating the important role of the water in the supercritical phase for the denaturing effect on the hydrolase impurities. The treated preparations have a high temperature stability (no activity loss after 72 h at 60°C).

A simple process for increasing the specific activity of porcine pancreatic lipase by supercritical carbon dioxide treatment

Treatment of crude porcine pancreatic lipase preparations (powders) with supercritical carbon dioxide (SC-CO 2) at 75°C and 150 bar leads to a time-dependent increase of enzyme activity (activities were measured prior and after SC-CO 2 treatment). After 24 h of incubation in SC-CO 2, a maximum activity of 860% was measured compared to the untreated enzyme (using 1,2- O-dilauryl- rac-glycero-3-glutaric acid resorufin ester as substrate). SC-CO 2 treatment caused a weight loss of about 4%. 0.47% of the weight loss (mean of three independent experiments) were separated as an oily extract in a tube that was connected with the extractor and through which depressurization was performed. In the extract, free fatty acids were found (mainly palmitic acid, stearic acid and oleic acid). No chemical modification on the protein reactive groups were found as studied by determination of reactive free amino groups, determination of free –SH groups and by determination of the carbonyl content (as an oxidation marker). No significant difference in the protein content between the untreated and SC-CO 2 processed lipase powders were found. Tryptophan fluorescence emission spectra showed no change of the emission maximum indicating no larger protein conformational change. SC-CO 2-treated lipase showed no loss of its increased activity after storage in a freezer for 6 months.

Lipase purification by various techniques and its thermostabilization in presence of surface active additives

Crude porcine pancreatic lipase was purified by removing water-insoluble impurities (residual lipid material) associated with it by a conventional method, using hydrotropic additives and by liquid coacervate extraction. Maximum yield with good recovery of activity was obtained when hydrotropes were used to separate the associated lipids from lipase. The thermostability of the enzyme was also checked in the solutions of additives such as sodium butyl monoglycol sulfate (Na-BMGS), proline and Triton X-114. In Na-BMGS solutions above a concentration of 0·2 mol dm−3 the lipase activity decreased beyond 50°C whereas in 1 mol dm−3 proline solution it was retained even at 80°C, showing a good thermostabilizing effect. However, in the presence of Triton X-114 the enzyme was completely inactivated with increase in temperature. © 1998 SCI.

Lipase‐Catalyzed Regio‐ and Enantioselective Esterification of rac‐1,2‐O‐Cyclohexylidene‐myo‐inositol

The hydroxyl group at C‐5 in racemic 1,2‐O‐Cyclohexylidene‐myo‐inositol (rac‐1) was regio‐ and enantioselectively acylated to give 5‐O‐acetyl‐ (2a)‐ or 5‐O‐butyryl‐2,3‐O‐cyclohexylidene‐myo‐inositol (3a), respectively, by treatment of 1 with vinyl acetate or 2,2,2‐trifluoroethyl butyrate in the presence of crude porcine pancreatic lipase in various organic solvents. Compound 2a was phosphorylated and deproted to give myo‐inositol 1,4,6‐trisphosphate (8). Compound 2a was deacetylated and the resulting 2,3‐O‐cyclohexylidene‐myo‐inositol (1) was converted to myo‐inositol 1,4,5,6‐tetrakisphosphate (6).

Enzymatic polyesterification in organic media. II. Enzyme‐catalyzed synthesis of lateral‐substituted aliphatic polyesters and copolyesters

AbstractEnzymatic polymerization of hydroxyesters in organic media has been the subject of a previous and ongoing research in our laboratory. As part of that study, four ϵ‐substituted‐ϵ‐hydroxyesters were synthesized, and then polymerized using crude porcine pancreatic lipase (PPL) in n‐hexane. These lateral‐substituted hydroxyesters polymerized enantioselectively to produce optically active oligomers and a resolved optically active unreacted monomer. It was found that with increasing bulkiness of the lateral substituent, in the order Me >Et >Ph, the enzymatic reaction becomes slower, yet the enantioselectivity is higher. The lateral‐substituted hydroxyesters were also copolymerized enzymatically with the more reactive linear methyl ϵ‐hydroxyhexanoate. Optically active copolymers were obtained, higher in molecular weight than the analogous homopolymers. © 1993 John Wiley & Sons, Inc.

Enzymatic polyesterification in organic media. Enzyme‐catalyzed synthesis of linear polyesters. I. Condensation polymerization of linear hydroxyesters. II. Ring‐opening polymerization of ϵ‐caprolactone

AbstractWith the object to synthesize polyesters by enzymatic catalysis in organic media, two directions have been investigated: (1) the condensation polymerization of linear ω‐hydroxyesters and (2) the ring‐opening polymerization of lactones. The commercially‐available crude porcine pancreatic lipase (PPL), suspended in organic solvents, was the preferred enzyme for the reactions. In order to determine the optimal conditions for the condensation polymerization, the bifunctional methyl 6‐hydroxyhexanoate was used as a model compound to study the influence of the following parameters: type of the enzymecatalyst, kind of solvent, concentration, temperature, duration, size of the reaction mixture, and stirring. Film‐forming polyesters with a degree of polymerization (DP) up to about 100 were obtained from linear aliphatic hydroxyesters in n‐hexane at reflux temperature (69°C). Yet concurrently with the intermolecular condensation polymerization, macrolactones were also formed by intramolecular reaction. Two aromatic hydroxyesters did not react under these conditions. For the ring‐opening polymerization of lactones the reaction of ϵ‐caprolactone with methanol as the preferred nucleophile, was studied. Polyesters with a DP of up to 35 were obtained in n‐hexane at temperatures between 25 and 40°C. The degrees of polymerization of the polyesters were determined by comparative analyses of the end groups in the 1H‐NMR spectra and by determination of molecular weights either by vapor phase osmometry, gel permeation chromatography, or intrinsic viscosity. © 1993 John Wiley & Sons, Inc.

Activities in Crude Porcine Pancreatic Lipase: Enantioselectivity in Hydrolysis of the Diacetate of 2-Phenylpropane-1,3-Diol

The hydrolysis of a prochiral diacetate by porcine pancreatic lipase is catalysed by the purified enzyme, not by an enzyme present in the crude enzyme but absent from the purified enzyme, as previously reported.

Porcine pancreatic lipase-catalized enantioselective hydrolysis of N-protected amino acid methyl-esters

A preparative-scale enantioselective hydrolysis of racemic methyl esters of several N-protected amino acid has been carried out by using crude porcine pancreatic lipase (Triacylglycerol lipase, EC 3.1.1.3) PPL as a hydrolytic enzyme. In all cases 50% of the racemic methyl ester was hydrolysed to the N-protected L-amino acid with high yield and high optical purity.Hydrolysis rates were very close related not only to the amino acid structure but also to the steric and/or electronic nature of the ester and N-protecting groups. Thus, the very convenient ester methyl group can be enantioselectively hydrolysed with PPL when N-protecting group is a carbonyl derivative, as it is the usual benzoyl group.

Enzymatic resolution of sterically demanding bicyclo[3.2.0]heptanes: evidence for a novel hydrolase in crude porcine pancreatic lipase and the advantages of using organic media for some of the biotransformations

Sterically demanding 7,7-dimethylbicyclo[3.2.0]hept-2-en-6-one 1 was enzymatically resolved via the exo-acetate 11a using crude porcine pancreatic lipase. By employing different fractions of hydrolases from the crude enzyme, evidence was obtained that an enzymatic ‘impurity’ was responsible for the highly selective reaction (E > 300). Alternatively, 7,7-dimethyl and 7,7-diphenyl derivatives 1 and 2 were equally well resolved, via bromohydrins 12b and 13b, by lipases from Pseudomonas cepacia and Candida cylindracea but only when acylations were conducted in organic media.

Stereoselective enzymatic hydrolysis of 2-cyclohexyl-and 2-phenyl-1,3-propanediol diacetate in biphasic systems

A key intermediate (S(−) 2-cyclohexyl-1,3-propanediol monoacetate) was made with high optical purity for the total synthesis of a new angiotensin converting enzyme inhibitor, Fosinopril. The stereoselective hydrolysis of 2-cyclohexyl-1,3-propanediol diacetate (I) and 2-phenyl-1,3-propanediol diacetate (II) was carried out with lipases. Among various lipases evaluated, only porcine pancreatic lipase (PPL) and Chromobacterium viscosum lipase demonstrated efficient conversion and gave the desired enantiomer of monoacetate. In aqueous solution, the desired S(−) monoacetate exhibited an optical purity of 65%–80% (30%–60% enantiomeric excess [e.e.]). However, when the same reactions were conducted in a biphasic system, the product S(−) monoacetate exhibited an optical purity of 99%–100% (98%–100% e.e.). The high purity product was achieved with 65 mol% yield at 1% substrate concentration. Among various solvents evaluated in biphasic systems, efficient hydrolysis was achieved in toluene, cyclohexane, and trichloro-trifluoroethane. The crude PPL was partially purified and two lipase fractions (A and B) were identified. Lipases A and B had a molecular mass of 38 000 and 40 000 daltons, respectively, and both were found to catalyze the hydrolysis of I and II to the appropriate monoacetate in a biphasic system.

Hydrolysis of N-protected amino acid allyl esters by enzymatic catalysis

Enzyme catalysis as an alternative to Pd(O) cleavage of N-α-protected amino acid allyl esters is presented; α-chymotrypsin, papain and crude porcine pancreatic lipase were succesfully used, but pure lipases were not able to perform the reaction.

Morphology and ultrastructure of Mycoplasma pneumoniae spherules.

During growth in fluid medium, most strains of Mycoplasma pneumoniae produce free-floating granules which become larger with time. We have called these granules "spherules." This study describes the morphological and ultrastructural features of M. pneumoniae strain AP-164 spherules, both free and in association with HeLa cells in cell culture. In thin section, spherules were composed of lobulated cells, connected together by membranes, and ring-shaped cells. The two-dimensional morphology observed varied according to the plane of section and to the age of the culture. In HeLa cell cultures, mycoplasmata were found attached to plasma membranes of cells; in sections, individual mycoplasmata were often aligned in radial apposition to the membranes. Mycoplasmata were not found intracellularly. The three-dimensional morphology of spherules was examined by the critical point method and by scanning electron microscopy. Both methods revealed lobulated forms, ring-shaped forms, and star-shaped forms. Treatment of the spherules with crude porcine pancreatic lipase effectively released large numbers of free organisms. Phosphotungstic acid preparations of these uncentrifuged forms revealed a morphology in agreement with the other methods used. Lobulated ring forms with "beaded" filaments were prominent. In respect to morphology, M. pneumoniae under our conditions resembles that of the type species M. mycoides.