Purification and properties of an extracellular lipase fromPythium ultimum

An extracellular triacylglycerol lipase (EC 3.1.1.3) fromPythium ultimum strain No. 144 was purified by ammonium sulfate precipitation, and by diethylaminoethyl Sepharose CL-6B and Sephacryl S−200 chromatography. The purified enzyme preparation showed a prominent polypeptide band in polyacrylamide gel electrophoresis, associated with esterase activity according to activity staining. Molecular weight of the protein was estimated at 270 kD using gel filtration on Sephacryl S−200, and 68 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicating that the enzyme may be a tetramer. The optimum pH and temperature for activity of the enzyme were 8.0 and 30°C, respectively. Activity was reduced by Co²⁺, Fe²⁺, Sn²⁺ and Mn²⁺ and stimulated by Ca²⁺, Mg²⁺, Na⁺, K⁺ and surfactants such as taurocholic acid, Triton X−100,n-octyl glucoside,n-dodecyl-β-D-maltoside, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate(CHAPS), and 3-[-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate. The apparent maximum specific activity was 42 μmole/min/mg in the absence of CHAPS and 77 μmole/min/mg in its presence. The reaction rate was progressively higher with increasing number of double bonds in the substrate, and the enzyme showed a preference for triacylglycerols containing fatty acids having thecis double bond configuration. AAES publication No. 6-933419.     

Partial purification and characterization of sphingosine N-acyltransferase (ceramide synthase) from bovine liver mitochondrion-rich fraction

Sphingosine N-acyltransferase (ceramide synthase, E.C. 2.3.1.24) was solubilized from bovine liver mitochondrion-rich fraction with n-ocytl β-d-thioglucoside as the detergent and partially purified by sequential chromatography on columns of DE-32, shingosine affinity, and Sepharose CL-6B. The partially purified preparation migrated on SDS-polyacrylamide gel electrophoresis as two major protein bands of 62 and 72 kDa. The molecular mass of the enzyme estimated by gel filtration was 240–260 kDa, suggesting that the partially purified enzyme is present in a subunit form or simply has an aggregative nature. The specific activity of the final preparation for the condensation of sphingosine with stearoyl-CoA increased by 98.7-fold compared with the starting material. The optimal pH value for the ceramide synthesis was 7.5. The partially purified enzyme had an apparent K _m of 146 μM and a V _max of 11.1 nmol/min/mg protein for stearoyl-CoA. The K _m and V _max values toward sphingosine were 171 μM and 11.3 nmol/min/mg protein, respectively. Interestingly, sphinganine was also a good substrate for this enzyme, and the K _m and V _max values were 144 μM and 8.5 nmol/min/mg protein, respectively.     

Purification and Biochemical Characterization of Lipase from <Emphasis Type="Italic">Ficus carica</Emphasis> Latex of Tunisian East Coast Zidi Variety

Lipase from Ficus carica L. (Moraceae) latex of the Zidi variety was purified 80.5-fold with 68.5 % recovery using silica gel chromatography. The molecular weight of the enzyme was 29 kDa as determined by SDS-PAGE. High lipolytic activity was found in the crude extract during the fruit ripening process. The activity of purified lipase (ZL) seemed to depend strongly on chain length and showed a preference to long chain triacylglycerols. Indeed, ZL specific activity was 370.3 UI/mg using olive oil as a substrate at 45 °C and pH 5.5. In contrast, activity towards short chain triacylglycerols (tributyrin) was 12-fold lower (32 UI/mg). The enzyme was quite stable in the pH range 4–8, and thermally stable at 60 °C displaying t _1/2 about 90 min using olive oil as a substrate. The values of K _m app and V _m were found to be 14.3 mM and 294.1 μmol/min/mg, respectively. ZL activity was strongly reduced by Fe²⁺, Mg²⁺ and Zn²⁺, while significantly increased by Ca²⁺ and Cu²⁺. The enzyme was stimulated by sodium dodecyl sulfate, and Tween-80, while Triton X-100 and EDTA had a slight inhibitory effect. No Effect was observed in addition of PMSF and iodoacetic acid.     

Immobilization and stabilization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SRT9 lipase on tri(4-formyl phenoxy) cyanurate

Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 °C to 70 °C. The immobilized lipase showed 20–25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ∼60% when incubated with 30% butanol. The kinetic studies revealed increase in K _M value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the V _max of immobilized enzyme (142.8 μmol min⁻¹ mg⁻¹), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 °C in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.     

Hydrolysis of neutral lipid substrates by rat hepatic lipase

Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). V_max values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The V_max values obtained were 144 μmol/min/mg for 1-oleoyl-sn-glycerol, 163 μmol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 μmol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 μmol/min/mg for PA, 50 μmol/min/mg for PE and 4 μmol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at thesn-1 andsn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereo-selective with respect to 1,3-diacyl-sn-glycerol substrates. To determine if the presence of one lipid could affect the hydrolysis of the other, all possible dual combinations of 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol, in the same micelle were made and the hydrolysis rate of each substrate was determined. Interaction occurred only for the 1,2-dioleoyl-sn-glycerol/1,3-dioleoyl-sn-glycerol mixture where the hydrolysis of 1,2-dioleoyl-sn-glycerol was slightly inhibited and that of 1,3-dioleoyl-sn-glycerol slightly activated compared to the predicted theoretical rate. These findings demonstrate that when presented in similar physical states, the neutral lipid substrates tested were hydrolyzed at a higher rate by hepatic lipase than the phospholipid substrates.     

Continuous production of fatty acids from palm olein by immobilized lipase in a two-phase system

Commercial lipases were tested for the ability to hydrolyze palm olein in isooctane in a two-phase system. Lipase OF (from Candida rugosa) showed the highest specific activity of 209 U/mg protein where 1 U is the amount of lipase enzyme required to produce 1 μmol of fatty acid (as palmitic acid) per minute. The enzyme was adsorbed completely on Accurel EP 100 (particle size <200 μm) with 20.5% activity retained. The soluble and the immobilized lipase OF showed optimal activity at the same pH and temperature (pH 6.5–7.5 and 35°C). However, the immobilized lipase had a wider range of pH and higher temperature stability. Continuous hydrolysis of palm olein was performed in a packed-bed reactor with 656 U of immobilized enzyme. The substrate (20% palm olein in isooctane) and Tris/maleate buffer were fed concurrently at the flow rates of 0.08 and 0.04 mL/min, respectively. The system gave a degree of hydrolysis (DH) of 90–100% for up to 250 h. A more stable system allowing for more than 300 h operation at DH>95% was achieved by mixing the immobilized enzyme with 1000–1500 μm Accurel EP100 to increase the system porosity and continuous feeding of the aqueous phase recycling from the product mixture. A similar result was also obtained using 1007 U of the immobilized enzyme and 60% palm olein in isooctane fed at 0.06 mL/min.     

Characterization of partially purified extracellular lipase fractions from Pseudomonas fragi CRDA 037

The purification of extracellular lipases from the culture medium of Pseudomonas fragi CRDA 037 was obtained by ammonium sulfate precipitation, followed by ion-exchange chromatography and then by size exclusion chromatography, and re-size exclusion chromatography, which resulted in two enzymatic fractions, FIVa′ and FIVb′. The fractions FIVa′ and FIVb′ had specific activities of 105.5 and 121.6 U/mg, respectively, with purification folds of 169.3 and 195.2, respectively, using triacetin as a substrate. The two purified fractions showed optimal activities at pH 9.5 and 10.0, respectively, at 80°C. Three bands were found in fraction FIVa′ and two bands in fraction FIVb′ by native polyacrylamide gel electrophoresis; these results indicated that homogeneity of the purified fractions was not achieved. The enzyme efficiency values, calculated as the ratio of V _max to K _m value for fractions FIVa′ and FIVb′, were 72.16 × 10⁻² and 38.15 × 10⁻², respectively. The lipase activity of fraction FIVa′ was more specific for the hydrolysis of fatty acid esters with fatty acid chain lengths of C12 to C18, whereas that of fraction FIVb′ showed a relatively broader range of specificity. The lipase activity of fraction FIVa′ showed higher specificity toward triacetin, tristearin, and tripalmitin as the substrate, whereas that of fraction FIVb′ exhibited higher affinity toward triacetin, trimyristin, and triolein. The effect of selected salts and detergents on the lipase activity of the purified fractions was also investigated. The lipase activity of the purified lipase fractions was completely inhibited by 10 mM of FeCl₂, FeCl₃, and Ellman’s reagent. However, 10 mM of CaCl₂ and EDTA activated the two purified lipase fractions by 20 to 50%.     

Immobilization and enzymatic activity of β-glucosidase on mesoporous SBA-15 silica

The mesoporous silicate SBA-15 has shown to be a good support for the immobilization of β-glucosidase from almonds, an enzyme with high molecular weight (ca. 130 kDa for the dimer). An enzyme loading of 430 mg per gram of support (3.2 ± 0.2 μmol g⁻¹ of SBA-15) was achieved at 7 h. The optimum pH for the immobilization was 3.5. The electrostatic interactions between the surface of SBA-15 and the enzyme molecules were the driving force of the adsorption process. The immobilized β-glucosidase presented enzymatic activity on the hydrolysis of the 4-nitrophenyl-β-d-glucopyranoside at 3.5 pH. The catalytic activity was similar to the free enzyme for reaction time of 30 min. When the reaction pH was higher (5.5 pH) the enzyme was desorbed.     

Purification of the acyl-CoA elongase complex from developing rapeseed and characterization of the 3-ketoacyl-CoA synthase and the 3-hydroxyacyl-CoA dehydratase

Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration, and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported. A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20∶0-CoA and (E) 2,3–20∶1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities showed that maximal activity could be measured by using 15 to 30 μM 18∶1-CoA and 30 μM malonyl-CoA, and that 18∶0-CoA and 18∶1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence of 75 μM Triton X-100 and 25 μg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex.     

Partial purification and properties of lipase from germinating seeds of Jatropha curcas L.

Lipase present in the seeds of Jatropha curcas L. was isolated and some of its properties studied. Lipase activity was detected in both dormant and germinating seeds. The lipase was partially purified using a combination of ammonium sulfate precipitation and ultrafiltration, which increased the relative activity of the lipase by 28- and 80-fold, respectively. The lipase hydrolyzed palm kernel, coconut, and olive oils at comparable rates (approximately 5 μg FFA/μg protein/min); palm—Raphia hookeri and Jatropha curcas L.—oils at about twice the rate of the first group of oils; and palm and fish oils at a higher rate than all other oils. The lipase, however, had the highest activity with monoolein. Optimal pH and temperature for maximal lipase activity were 7.5 and 37°C, respectively. The addition of ferric ion (15 mM) to the lipase assay medium caused 90% inhibition of lipase activity, whereas calcium and magnesium ions enhanced lipase activity by 130 and 30%, respectively.     

Biochemical and structural characterization of triacylglycerol lipase from Penicillium cyclopium

An extracellular lipase, active on water-insoluble triacylglycerols, has been isolated from Penicillium cyclopium. The purified enzyme has a molecular mass of 29 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis. It hydrolyzes emulsions of tributyrin, trioctanoin, and olive oil at the same rate as pancreatic lipase and shows very low activity against partial acylglycerols (monooctanoin and dioctanoin) and methyl esters. It is stable at 35°C for 60 min and has maximal activity in a pH range of 8–10. Hydrolysis of triacylglycerols by P. cyclopium lipase is inhibited by detergents such as Triton X-100. Comparison of the sequence of the 20 first amino acid residues of P. cyclopium triacylglycerol lipase with other Penicillium lipases indicates a high homology with previously characterized lipases produced by P. expansum and P. solitum which are enzymes of comparable size and substrate specificity. Conversely, homology between P. cyclopium lipase and P. simplicissimum lipase, a nonspecific lipolytic enzyme, is low. Penicillium cyclopium triacylglycerol lipase shows no homology with P. camembertii lipase which is specific to monoacylglycerol and diacylglycerol.     

Isolation and identification of acetyl-CoA carboxylase from rainbow trout (Salmo gairdneri) liver

Acetyl-CoA carboxylase is the pivotal enzyme in the de novo synthesis of fatty acids and is the only carboxylase with a biotin-containing subunit greater than 200,000 daltons. The biotin moiety is covalently linked to the active site and has a high affinity (K_d=10⁻¹⁵ M) for the protein avidin. This relationship has been used in previous studies to identify acetyl-CoA carboxylase isolated from mammalian species. However, acetyl-CoA carboxylase has not been isolated and characterized in a poikilothermic species such as the rainbow trout. The present study describes the isolation and identification of acetyl-CoA carboxylase in the cytosol of rainbow trout (Salmo gairdneri) liver. The enzyme was isolated using two distinct procedures—polyethylene glycol precipitation and avidin-Sepharose affinity chromatography. Identification of the isolated protein as acetyl-CoA carboxylase was made by the following: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (2) avidin binding; (3) in vivo labeling with [¹⁴C]biotin; and (4) acetyl-CoA carboxylase-specific activity. The subunit molecular weight of the major protein was 230,000 daltons ±3.3%. This protein was shown to bind avidin (M_r=16,600) prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of biotin. In addition, protein isolated from fish that had previously received intraperitoneal injections of [¹⁴C]biotin, showed the majority of radioactivity associated with the 230,000 dalton protein. The polyethylene glycol precipitation yielded 200 μg protein (4.4 μg/g liver), with a specific activity of 5 nmol malonyl-CoA/min/mg protein, whereas avidin affinity chromatography yielded 1.75±1.1 mg protein (9.0 μg/g liver), with a specific activity of 1.37±0.18 μmol malonyl-CoA/min/mg protein. The enzyme was citrate dependent showing maximum activity between 10 and 20 mM. Acetyl-CoA carboxylase-specific activity decreased by 50% in the presence of 0.2 M NaCl. These findings suggest that the major protein (M_r=230,000) purified from rainbow trout liver is acetyl-CoA carboxylase with enzyme characteristics comparable to mammalian acetyl-CoA carboxylase.     

Purification of pregastric lipases of caprine origin

Pregastric lipases from kid (KPGL) and goat (GPGL) were purified from the commercial extracts by different chromatographic procedures. The total recovery of activity for both purification methods was ca. 10%, and the specific activities of KPGL and GPGL were 533 and 546 U/mg, respectively, at pH 6.5, 35°C for tributyrylglycerol (TBG) as substrate in a casein/lecithin emulsion. The purification factors were 130- and 76-fold for the goat and kid lipases, respectively. The purified lipases from kid and goat showed the same 50 kDa protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an identical sequence for the first 11 amino acids. The optimal pH for the lipases was within the pH range 6–7, with maximal activity at pH 6.5. The stability of the purified lipases was decreased dramatically at pH>6.5, but was enhanced by the addition of albumin. Presented at the AOCS 88th Annual Meeting & Expo, May 11–14, 1997, Seattle, Washington.     

Biocatalytic Properties of Lipase from Walnut Seed (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Lipase (E.C. 3.1.1.3) from walnut seed was purified 28.6-fold with 31% yield using Sephadex G-100 gel chromatography. Olive oil served as good substrate for the enzyme. The optimum pH and temperature were 9.0 and 70 °C, respectively. The lipase was stable between 30 and 80 °C for 5 min. K _m and V _max values were determined as 48 mM and 23.06 × 10⁻³ U/min mg for triolein as substrate. Lipase activity was slightly reduced by Cu²⁺, Ca²⁺, Hg²⁺, Mn²⁺, and Ni²⁺ ions, while Mg²⁺ and Zn²⁺ had no effects. Anionic surfactant sodium dodecyl sulfate stimulated lipase activity while non-ionic surfactants Tween-80 and Triton X-100 had negligible effects on enzymatic activity. The enzyme activity was not affected by 50 mM urea and thioacetamide. Potassium ferricyanide, n-bromosuccinamide and potassium cyanide reduced the enzyme activity. The enzyme showed a good stability in organic solvents, the best result being in n-hexane (113% residual activity). The activity of dialysate was maintained approximately 80% for 1 year at −20 °C.     

Bovine Brain Diacylglycerol Lipase: Substrate Specificity and Activation by Cyclic AMP-dependent Protein Kinase

Diacylglycerol lipase (EC 3.1.1.3) was purified from bovine brain microsomes using multiple column chromatographic techniques. The purified enzyme migrates as a single band on SDS-PAGE and has an apparent molecular weight of 27 kDa. Substrate specificity experiments using mixed molecular species of 1,2-diacyl-sn-glycerols indicate that low concentrations of Ca²⁺ and Mg²⁺ have no direct effect on enzymic activity and 1,2-diacyl-sn-glycerols are the preferred substrate over 1,3-diacyl-sn-glycerols. The enzyme hydrolyzes stearate in preference to palmitate from the sn-1 position of 1,2-diacyl-sn-glycerols. 1-O-Alkyl-2-acyl-sn-glycerols are not a substrate for the purified enzyme. The native enzyme had a V _max value of 616 nmol/min mg protein. Phosphorylation by cAMP-dependent protein kinase resulted in a threefold increase in catalytic throughput (V _max = 1,900 nmol/min mg protein). The substrate specificity and catalytic properties of the bovine brain diacylglycerol lipase suggest that diacylglycerol lipase may regulate protein kinase C activity and 2-arachidonoyl-sn-glycerol levels by rapidly altering the intracellular concentration of diacylglycerols.     

Purification and characterization of an extracellular lipase from <Emphasis Type="Italic">Geotrichum marinum</Emphasis>

An extracellular lipase (EC 3.1.1.3) from Geotrichum marimum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDSPAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V _max and K _m were 1000 μmol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40°C. At 6 mM, Na⁺, K⁺, Ca²⁺, and Mg²⁺ stimulated activity, but Na⁺, and K⁺ at 500 mM and Fe²⁺ and Mn²⁺ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.     

Synthesis of geranyl acetate by esterification with lipase entrapped in hybrid sol-gel formed within nonwoven fabric

Candida cylindracea lipase was entrapped in organic-inorganic hybrid sol-gel polymers made from tetramethoxysilane (TMOS) and alkyltrimethoxysilanes. By forming the gels within the pores of a nonwoven polyester fabric, a novel immobilized biocatalyst in sheet configuration based on sol-gel en-trapment of the enzyme was obtained. Lipases immobilized in sol-gel matrices efficiently catalyzed the direct esterification reaction of geraniol and acetic acid in anhydrous hexane to produce geranyl acetate. The optimal formulation of the sol-gel solution for enzyme immobilization was at a 20∶1 molar ratio of water to total silane; a 4∶1 molar ratio of propyltrimethoxysilane to TMOS; hydrolysis time at 30 min; and enzyme loading of 200 mg lipase/g gel. Under these conditions, protein immobilization efficiency was 91%, and the specific activity of the immobilized enzyme was 2.6 times that of the free enzyme. Excellent thermal stability was found for the immobilized enzyme in dry form or in hexane solution in the presence of acetic acid, in which case severe inactivation of free enzyme was observed. The immobilized enzyme retained its activity after heating at 70°C for 2 h, whereas the free enzyme lost 80% of its activity.     

The Development of Flow-through Bio-Catalyst Microreactors from Silica Micro Structured Fibers for Lipid Transformations

This study demonstrates the utility of a flow-through enzyme immobilized silica microreactor for lipid transformations. A silica micro structured fiber (MSF) consisting of 168 channels of internal diameter 4–5 μm provided a large surface area for the covalent immobilization of Candida antartica lipase. The specific activity of the immobilized lipase was determined by hydrolysis of p-nitrophenyl butyrate and calculated to be 0.81 U/mg. The catalytic performance of the lipase microreactor was demonstrated by the efficient ethanolysis of canola oil. The parameters affecting the performance of the MSF microreactor, including temperature and reaction flow rate, were investigated. Characterization of the lipid products exiting the microreactor was performed by non-aqueous reversed-phased liquid chromatography (NARP-LC) with evaporative light scattering detector (ELSD) and by comprehensive two-dimensional gas chromatography (GC × GC). Under optimized conditions of 1 μL/min flow rate of 5 mg/mL trioleoylglycerol (TO) in ethanol and 50 °C reaction temperature, 2-monooleoylglycerol was the main product at >90% reaction yield. The regioselectivity of the Candida antartica lipase immobilized MSF microreactor in the presence of ethanol was found to be comparable to that obtained under conventional conditions. The ability of these reusable flow-through microreactors to regioselectively form monoacylglycerides in high yield from triacylglycerides demonstrate their potential use in small-scale lipid transformations or analytical lipids profiling.     

Release of lipoprotein lipase from cardiac myocytes by low-molecular weight heparin

Incubation of cardiac myocytes from rat heart with low-molecular weight heparin (LMWH; Mr approx. 3 kDa) for 30 min resulted in a concentration-dependent release of lipoprotein lipase (LPL) activity into the incubation medium. The release of lipoprotein lipase from cardiac myocytes isolated from both control and diabetic rat hearts induced by LMWH (10 μg/mL) following incubation times of 10 or 30 min was significantly greater than that produced by unfractionated heparin (10 and 30 μg/mL) or decavanadate (1 mM). Since LMWH released more LPL activity into the incubation medium than unfractionated heparin following a short (10 min) incubation time, LMWH is probably more effective in displacing LPL bound to heparan sulfate proteoglycan binding sites on the cell surface of cardiac myocytes.     

Purification of lysosomal cholesteryl ester hydrolase from rat liver by preparative isoelectric focusing

Ion-exchange chromatography and preparative isoelectric focusing (PIEF) were compared to produce a stable rat liver lysosomal cholesteryl ester hydrolase of high specific activity. The PIEF purification method proved to be more rapid and easier to perform. PIEF purification involved the following steps: i) osmotic shock of the lysosome fraction, ii) (NH₄)₂ SO₄ precipitation (10–70%, w/v), iii) Sepharose CL-6B gel filtration, and iv) PIEF. The enzyme was purified 60–120-fold with a yield of 2–4%. The activity of the purified enzyme was best restored by stabilizing with a 0.5% (w/v) albumin solution. The purified enzyme produced one major band on SDS-polyacrylamide gel electrophoresis having a MW of 58,500 daltons. Gel filtration showed a MW of 58,000 daltons. The optimum pH of the enzyme was 4.5, and the isoelectric point was 6.0–6.2. The specific activity of hydrolysis of cholesteryl oleate and triolein increased by similar rates during purification.