Synthesis of structured phospholipids by immobilized phospholipase A2 catalyzed acidolysis

Acyl modification of the sn-2 position in phospholipids (PLs) was conducted by acidolysis reaction using immobilized phospholipase A(2) (PLA(2)) as the catalyst. In the first stage we screened different carriers for their ability to immobilize PLA(2). Several carriers were able to fix the enzyme and maintain catalytic activity; however, the final choice of carrier for the continued work was a non-ionic weakly polar macroreticular resin. Response surface methodology was applied to evaluate the influence of substrate ratio, reaction temperature, and water addition during acidolysis reaction between caprylic acid and soybean phosphatidylcholine (PC). Reaction temperature and water addition had significant effect on acidolysis reaction, however no effect was observed for substrate ratio (mol caprylic acid/mol PC) in range tested. In general an inverse relationship between incorporation of caprylic acid and PC recovery was observed. Highest incorporation obtained during acidolysis reactions was 36%. Such incorporation could be obtained under reaction temperature, 45 degrees C; substrate ratio, 9mol/mol caprylic acid/PC; water addition of 2%; 30wt.% immobilized enzyme; and reaction time, 48h. The yield under these conditions was however only 29%. Lysophosphatidylcholine (LPC) was the major by-product formed during the reaction. Incorporation of acyl donor into LPC was very low (<4%), which indicates that acyl migration is only a minor problem for PLA(2) catalyzed synthesis reaction. Conjugated linoleic acid and docosahexaenoic acid were also tested as acyl donors, and were able to be incorporated into PC with 30 and 20%, respectively.     

Lipase-catalyzed acyl exchange of soybean phosphatidylcholine in n-Hexane: a critical evaluation of both acyl incorporation and product recovery

Lipase-catalyzed acidolysis was examined for the production of structured phospholipids in a hexane system. In a practical operation of the reaction system, the formation of lyso-phospholipids from hydrolysis is often a serious problem, as demonstrated from previous studies. A clear elucidation of the issue and optimization of the system are essential for the practical applications in reality. The effects of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio were optimized in terms of the acyl incorporation, which led to the products, and lyso-phospholipids formed by hydrolysis, which led to the low yields. The biocatalyst used was the commercial immobilized lipase Lipozyme TL IM and substrates used were phosphatidylcholine (PC) from soybean and caprylic acid. A response surface design was used to evaluate the influence of selected parameters and their relationships on the incorporation of caprylic acid and the corresponding recovery of PC. Incorporation of fatty acids increased with increasing enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio. Enzyme dosage had the most significant effect on the incorporation, followed by reaction time, reaction temperature, solvent amount, and substrate ratio. However the parameters had also a negative influence on the PC recovery. Solvent amount had the most negative effect on recovery, followed by enzyme dosage, temperature, and reaction time. Individually substrate ratio had no significant effect on the PC recovery. Interactions were observed between different parameters. On the basis of the models, the reaction was optimized for the maximum incorporation and maximum PC recovery. With all of the considerations, the optimal conditions are recommended as enzyme dosage 29%, reaction time 50 h, temperature 54 degrees C, substrate ratio 15 mol/mol caprylic acid/PC, and 5 mL of hexane per 3 g substrate. No additional water is necessary. Under these conditions, an incorporation of caprylic acid up to 46% and recovery of PC up to 60% can be obtained from the prediction. The prediction was confirmed from the verification experiments.     

无溶剂体系中脂肪酶改造猪油制备功能性脂的研究

我国有丰富廉价的动植物油资源,但这一资源并未得到有效的利用,为了充分利用这些资源,开展了无溶剂系统脂肪酶催化猪油与辛酸酸解制备功能性脂的研究工作。脂肪酶筛选实验表明,在所选用的五种脂肪酶中,来自T .languginosa的固定化脂肪酶LiopzymeTLIM的催化效果最好。以LipozymeTLIM为催化剂,进一步研究了酶量、底物比率、反应时间、反应温度和水分添加量对猪油中辛酸插入率的影响。反应产物通过高效液相色谱法(HPLC)进行分析。研究结果表明,当脂肪酶量为20% (底物重量百分比)时,猪油中辛酸插入率最高。反应时间研究表明,当反应时间达到24h时,辛酸的插入率最高,达到38.77mol%。当猪油与辛酸的比率为1∶2 (摩尔比)时,辛酸的插入率最高,达到30 95mol%。在4 5～6 0℃范围之内,反应温度对辛酸插入率没有明显的影响,温度高于6 0℃时,辛酸插入率降低。水分添加量为2 5 %时,辛酸插入率最高,高达35 76mol%。     

Synthesis of DHA/EPA-rich phosphatidylcholine by immobilized phospholipase A<Subscript>1:</Subscript> effect of water addition and vacuum condition

DHA/EPA-rich phosphatidylcholine (PC) was successfully synthesized by immobilized phospholipase A₁ (PLA₁)-catalyzed transesterification of PC and DHA/EPA-rich ethyl esters in a solvent-free system. Effects of reaction temperature, water addition and substrate mass ratio on the incorporation of DHA/EPA were evaluated using response surface methods (RSM). Water addition had most significant effect on the incorporation. Reaction temperature and substrate mass ratio, however, had no significant effect on the incorporation. The maximal incorporation was 19.09 % (24 h) under the following conditions: temperature 55.7 °C, water addition 1.1 wt % and substrate mass ratio (ethyl esters/PC) 6.8:1. Furthermore, effects of water addition (from 0 to 1.25 wt %) on DHA/EPA incorporation and the composition of products were further investigated. The immobilized PLA₁ was more active when water addition was above 0.5 wt %. By monitoring the reaction processes with different water addition, a possible reaction scheme was proposed for transesterification of PC with DHA/EPA-rich ethyl esters. In summary, PC and sn2-lysophosphatidylocholine (LPC) were predominant in the mixtures at early stages of reaction, whereas sn1-LPC and glycerophosphocholine (GPC) predominant at later stages. The vacuum employed after 24 h significantly increased the incorporation of DHA/EPA and the composition of PC, and the highest incorporation (30.31 %) of DHA/EPA was obtained at 72 h and the yield of PC was 47.2 %.     

Head group precursors modify phospholipid synthesis in Schistosoma mansoni

The two predominant phospholipids in schistosomula of Schistosoma mansoni are phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are found in a molar ratio of 0.52 (PE/PC). The incorporation of four fatty acids (arachidonic, myristic, oleic, and palmitic) and glycerol into phospholipids of schistosomula was measured. In two different media (one containing ethanolamine, the other without), all four fatty acids were predominantly incorporated into PC with a PE/PC ratio of approximately 0.1 in a 90-min label. After a 24-h chase, PC remained the predominant labeled phospholipid but the fatty acid-labeled PE/PC ratio increased slightly, the specific activity of labeled neutral lipids decreased, and the specific activity of labeled PE increased. Glycerol was incorporated with a ratio of 0.55 in the presence of ethanolamine but only 0.19 in its absence. Schistosomula also incorporate fatty acids into phosphatidylmonomethylethanolamine (PMME) and phosphatidyldimethylethanolamine (PDME) at rates intermediate to that into PE and PC in the presence of the respective head group precursor; this incorporation was inhibited by choline. Relative to PC, oleic acid is incorporated into PE, PMME, and PDME at rates higher than for palmitic acid. These results suggest that schistosomula possess acyltransferase(s) with head group specificity and that acyl chains are transferred from neutral lipids to phospholipids over time.     

Modeling and optimization of phospholipase A1‐catalyzed hydrolysis of phosphatidylcholine using response surface methodology for lysophosphatidylcholine production

Modeling the phospholipase A₁ (PLA₁)‐catalyzed partial hydrolysis of soy phosphatidylcholine (PC) in hexane for the production of lysophosphatidylcholine (LPC) and optimizing the reaction conditions using response surface methodology were described. The reaction was performed with 4 g of PC in a stirred batch reactor using a commercial PLA₁ (Lecitase Ultra) as the biocatalyst. The effects of temperature, reaction time, water content, and enzyme loading on LPC and glycerylphosphorylcholine (GPC) content in the reaction products were elucidated using the models established. Optimal reaction conditions for maximizing the LPC content while suppressing acyl migration, which causes GPC formation, were as follows: temperature, 60°C; reaction time, 3 h; water content, 10% of PC; and enzyme loading, 1% of PC. When the reaction was conducted with 40 g of PC under these conditions, the reaction products contained 83.7 mol % LPC and were free of GPC. LPC had a higher total unsaturated fatty acid content than original PC had and was mainly composed of linoleic acid (78.0 mol % of the total fatty acids). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:35–41, 2015     

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.     

Phospholipid effects on SGLT1-mediated glucose transport in rabbit ileum brush border membrane vesicles

In small intestine, sodium-glucose cotransporter SGLT1 provides the main mechanism for sugar uptake. We investigated the effect of membrane phospholipids (PL) on this transport in rabbit ileal brush border membrane vesicles (BBMV). For this, PL of different charge, length, and saturation were incorporated into BBMV. Transport was measured related to (i) membrane surface charge (membrane-bound MC540 fluorescence), (ii) membrane thickness (PL incorporation of different acyl chain length), and (iii) membrane fluidity (r_12AS, fluorescence anisotropy of 12-AS).Compared to phosphatidylcholine (PC) carrying a neutral head group, inhibition of SGLT1 increased considerably with the acidic phosphatidic acid (PA) and phosphatidylinositol (PI) that increase membrane negative surface charge. The order of PL potency was PI>PA > PE = PS > PC. Inhibition by acidic PA-oleate was 5-times more effective than with neutral PE (phosphatidylethanolamine)-oleate. Lineweaver-Burk plot indicated uncompetitive inhibition of SGLT1 by PA.When membrane thickness was increased by neutral PC of varying acyl chain length, transport was increasingly inhibited by 16:1 PC to 22:1 PC. Even more pronounced inhibition was observed with mono-unsaturated instead of saturated acyl chains which increased membrane fluidity (indicated by decreased r_12AS).In conclusion, sodium-dependent glucose transport of rabbit ileal BBMV is modulated by (i) altered membrane surface charge, (ii) length of acyl chains via membrane thickness, and (iii) saturation of PL acyl chains altering membrane fluidity. Transport was attenuated by charged PL with longer and unsaturated acyl residues. Alterations of PL may provide a principle for attenuating dietary glucose uptake.     

Transesterification of phosphatidylcholine in <Emphasis Type="Italic">sn</Emphasis>-1 position through direct use of lipase-producing <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> cells as whole-cell biocatalyst

The enzymatic process presents an advantage of producing specified phospholipids that rarely exist in nature. In this study, we investigated the regiospecific modification of phosphatidylcholine (PC) in the sn-1 position using immobilized Rhizopus oryzae. In a reaction mixture containing egg yolk PC and exogenous lauric acid (LA) in n-hexane, lipase-producing R. oryzae cells immobilized within biomass support particles (BSPs) showed a much higher transesterification activity than lipase powders. To improve the product yield, several parameters including substrate ratio and reaction time were investigated, resulting in the incorporation of 44.2% LA into the product PC after a 48-h reaction. The analysis of the molecular structure showed that a large proportion of exogenous LA (>90%) was incorporated in the sn-1 position of the enzymatically modified PC. Moreover, the BSP-immobilized R. oryzae maintained its activity for more than 12 batch cycles. The presented results, therefore, suggest the applicability of BSP-immobilized R. oryzae as a whole-cell biocatalyst for the regiospecific modification of phospholipids.     

Asymmetric distribution of phosphatidylcholine and sphingomyelin between micellar and vesicular phases. Potential implications for canalicular bile formation.

Both phosphatidylcholine (PC) and sphingomyelin (SM) are the major phospholipids in the outer leaflet of the hepatocyte canalicular membrane. Yet, the phospholipids secreted into bile consist principally (>95%) of PC. In order to understand the physical;-chemical basis for preferential biliary PC secretion, we compared interactions with bile salts (taurocholate) and cholesterol of egg yolk (EY)SM (mainly 16:0 acyl chains, similar to trace SM in bile), buttermilk (BM)SM (mainly saturated long (>20 C-atoms) acyl chains, similar to canalicular membrane SM) and egg yolk (EY)PC (mainly unsaturated acyl chains at sn-2 position, similar to bile PC). Main gel to liquid-crystalline transition temperatures were 33. 6 degrees C for BMSM and 36.6 degrees C for EYSM. There were no significant effects of varying phospholipid species on micellar sizes or intermixed-micellar/vesicular bile salt concentrations in taurocholate-phospholipid mixtures (3 g/dL, 37 degrees C, PL/BS + PL = 0.2 or 0.4). Various phases were separated from model systems containing both EYPC and (EY or BM)SM, taurocholate, and variable amounts of cholesterol, by ultracentrifugation with ultrafiltration and dialysis of the supernatant. At increasing cholesterol content, there was preferential distribution of lipids and enrichment with SM containing long saturated acyl chains in the detergent-insoluble pelletable fraction consisting of aggregated vesicles. In contrast, both micelles and small unilamellar vesicles in the supernatant were progressively enriched in PC. Although SM containing vesicles without cholesterol were very sensitive to micellar solubilization upon taurocholate addition, incorporation of the sterol rendered SM-containing vesicles highly resistant against the detergent effects of the bile salt. These findings may have important implications for canalicular bile formation.     

Solvent-free enzymatic synthesis of alitame precursor using eutectic substrate mixtures

N-benzyloxycarbonyl-L-aspartic acid ethyl ester-D-alanine amide, a derivative of alitame, was synthesized from a eutectic mixture of the substrates N-benzyloxycarbonyl-L-aspartic acid diethyl ester and D-alanine amide using alpha-chymotrypsin. The hydrophilic solvents DMSO and MEA were found to be the best adjuvants for formation of a eutectic substrate mixture. A low eutectic temperature of 27 degrees C was obtained for the substrate mixture containing 9% DMSO, 18% MEA, and 12% water. Under these conditions a conversion yield of 70.3% (mol/mol) was obtained at 37 degrees C. The optimum molar ratio of the acyl acceptor D-alanine amide and the acyl donor N-benzyloxycarbonyl-L-aspartic acid diethyl ester was 1:1.     

Kinetically controlled synthesis of ampicillin with immobilized penicillin acylase in the presence of organic cosolvents

Penicillin acylase (PA) is used in the industrial production of 6-amino penicillanic acid (6-APA). However, by proper control of reaction medium, the enzyme can be used in the reverse synthesis of β-lactam antibiotics from the corresponding β-lactam nuclei and suitable acyl donors. Under thermodynamically controlled strategy, the use of organic cosolvents can favor synthesis over hydrolysis by lowering water activity and favoring the non-ionic reactive species. Under kinetically controlled strategy using activated acyl donors, organic solvents can favor synthesis by depressing hydrolytic reactions. Results are presented on the synthesis of ampicillin from phenylglycine methyl ester and 6-APA with immobilized Escherichia coli PA in the presence of organic cosolvents. Several solvents were tested in terms of enzyme stability and solubility of substrates. Ethylene glycol, glycerol, 1–2 propanediol and 1–3 butanediol were selected accordingly and ampicillin synthesis was performed in all of them. Best results in terms of yield and productivity were obtained with ethylene glycol, with which further studies were conducted. Variables studied were enzyme to limiting substrate ratio, acyl acceptor to acyl donor ratio, organic solvent concentration, pH and temperature. Experimental design based on a two-level fractional factorial design was conducted. pH was determined as the most sensitive variable and was further optimized. The best conditions for ampicillin synthesis in terms of productivity, within the range of values studied for those variables, were pH 7.4, 28°C, 36 U_S PA/mmol 6-APA, 3 mol PGME/mol 6-APA and 45 % (v/v) ethylene glycol concentration. Productivity was 7.66 mM ampicillin/h, which corresponds to a specific productivity of 7.02 μmol ampicillin/h U_S at 55 % yield. Productivity was lower than in buffer but product yield was higher because of the much lower relative hydrolysis rates.     

Phosphatidylcholine enrichment with medium chain fatty acids by immobilized phospholipase A1‐catalyzed acidolysis

Phospholipids are a biologically and industrially important class of compounds whose physical properties can be improved for diverse applications by substitution of medium‐chain fatty acids for their native fatty acid chains. In this study, phosphatidylcholine (PC) was enriched with medium‐chain fatty acids (MCFAs) by acidolysis with phospholipase A₁ (PLA₁) immobilized on Duolite A568. Response surface methodology was employed to evaluate the effects of the molar ratio of substrates (PC to free MCFAs), enzyme loading, and reaction temperature on the incorporation of free MCFAs into PC and on PC recovery. Enzyme loading and molar ratio of substrates contributed positively, but temperature negatively, to the incorporation of free MCFAs into PC. Increases in enzyme loading and the molar ratio of PC to free MCFAs led to increased incorporation of the latter into the former, but increased temperature had the opposite effect. By contrast, an increase in enzyme loading led to decreased PC recovery. Increased temperature had also a negative effect on PC recovery. Optimal conditions for maximum incorporation and PC recovery were molar ratio of PC to free MCFAs of 1:16, enzyme loading of 16%, and 50°C. Under these conditions, the incorporation of free MCFAs was 41% and the PC recovery was 53%. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Interesterification of phosphatidylcholine with lipases in organic media

Summary Lipases were investigated with respect to their ability to catalyse the incorporation of fatty acids into phosphatidylcholine (PC) by interesterification reactions. The enzymes were dried onto solid support materials and the conversions were carried out in water-saturated toluene. Three lipases (two fungal and one plant enzyme) had the desired activity; immobilized lipase from Mucor miehei (Lipozyme) was the most active enzyme. The Lipozyme-catalysed interesterification was selective for the sn-1 position of PC and during 48 h of reaction around 50% of the fatty acids in this position were replaced with heptadecanoic acid, a fatty acid which was practically absent in the original phospholipid. Due to adsorption on the support material and the competing hydrolysis reaction the total amount of PC in the reaction solution decreased to about 40% of the original amount. Higher interesterification rates were obtained with free fatty acids as acyl donors than with fatty acid esters. Offprint requests to: I. Svensson     

Lipase-catalyzed acylation of naringin with palmitic acid in highly concentrated homogeneous solutions

Acylation reactions of naringin with palmitic acid were performed by a lipase after formation of highly concentrated homogeneous solutions. Their initial naringin concentration was 840–950 mM, which is 20–60 times greater than that in organic solvent media. The overall productivity of highly concentrated solutions was more than 15 times greater than those of organic phase media. The addition of DMSO (20–40%, w/w) to substrate mixtures lowered the melting temperature of a naringin–palmitic acid mixture (1:1 molar ratio) to about 40 °C. Reactions at 80 °C apparently followed Michaelis–Menten kinetics despite extremely high substrate concentrations. As the temperature increased from 60 °C to 80 °C, the apparent viscosity of the highly concentrated solution decreased remarkably from 4.31 Pa s to 0.063 Pa s. An activation energy of 7.65 kcal/mol obtained in a range of 60–75 °C suggests a diffusion-control. On the other hand, an activation energy of 17.09 kcal/mol in a range of 75–90 °C indicates a reaction-control. The highest product conversion yield of 33% (mol/mol) was obtained in a 10 h reaction at 80 °C. Addition of activated molecular sieves to the highly concentrated solution increased the product conversion yield by 7% (mol/mol), suggesting that the original equilibrium was disrupted by removing water and then a new equilibrium was reached.     

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.     

Phospholipase-mediated preparation of 1-ricinoleoyl-2-acyl-sn-glycero-3-phosphocholine from soya and egg phosphatidylcholine

1-Ricinoleoyl-2-acyl-sn-glycero-3-phosphocholine was prepared by incorporating ricinoleic acid completely in the sn-1 position of egg and soya phosphatidylcholine (PC) using immobilized phospholipase A(1) as the catalyst. The optimum reaction conditions for maximum incorporation of ricinoleic acid into PC through transesterification were 10% (w/w) immobilized enzyme (116 mg), a 1:5 mol ratio of PC (soya, 387 mg; egg, 384 mg) to methyl ricinoleate (780 mg) at 50 degrees C for 24 h in hexane.     

L-抗坏血酸洛芬酯非水相酶促合成的动力学与热力学

对酶法合成L-抗坏血酸洛芬酯(芬维C酯)的反应动力学与热力学进行研究,确定了最有效的酶促反应环境。合成布洛芬维C酯的最优条件:转速200r/min,温度65℃,加酶量5%(以底物的质量分数计),底物浓度1mol/L,平衡所需时间66h,平衡时产物质量分数为19.07%;合成酮洛芬维C酯的最优条件:200r/min,60℃,加酶量7.5%,底物浓度600mmol/L,平衡时间132h,产物质量分数为10.63%;合成氟比洛芬维C酯的最优条件:200r/min,65℃,加酶量5%,底物浓度400mmol/L,平衡时间144h,产物质量分数为6.76%。对底物进行了比较,得到了各自的动力学与热力学参数。布洛芬米氏常数为0.101μmol/L,vmax=32.68μmol/(min.g),热力学平衡常数为0.166;酮洛芬的分别为0.144μmol/L,12.97μmol/(min.g),0.091;氟比洛芬的分别为0.185μmol/L,9.35μmol/(min.g),0.055。     

Dynamics of uptake and channeling of arachidonic acid in hamster alveolar macrophages and the effect of bleomycin on arachidonyl distribution.

The uptake and distribution of [14C]-arachidonic acid (AA) by primary culture hamster alveolar macrophages (AM) were examined. The macrophages were incubated for 2 to 18 hrs in RPMI-1640 medium containing 0.1% BSA and [14C]-AA. The uptake of [14C]-AA by AM was rapid with 71% and 83% of exogenous [14C]-AA taken up after 2 and 4 hrs of incubation, respectively. Initially, the uptaken [14C]-AA was equally distributed between phospholipids (PL) and neutral lipids (NL). However, by 8 hrs, 86% and 14% of [14C]-AA was found in PL and NL, respectively. This distribution pattern remained constant through 18 hrs. Within the PL pool, most of the [14C]-AA was initially incorporated into phosphatidylcholine (PC). However, with time, as the percent of [14C]-AA incorporated in PC declined, the percent incorporated in phosphatidylethanolamine increased. The incorporation of [14C]-AA into sphingomyelin, phosphatidylinositol, and phosphatidylserine remained constant with time. Within the NL, most of the [14C)-AA was initially found incorporated into triacylglycerols (TG). After 4 hrs, the percent of [14C]-AA found in TG decreased markedly, while the percent found in cholesterol esters markedly increased. Incubation of AM prelabeled with [14C]-AA with bleomycin at 0.5, 5, 50 and 500 microM for 2 and 8 hrs failed to have any effect on the distribution of [14C]-AA in PL and NL pools.     

The enzymatic acyl exchange of phospholipids with lipases

The acyl exchange of phospholipids with lipases was investigated. The lipase from Rhizopus delemar catalyzed the acyl exchange reaction between various phospholipids and fatty acids. When we incubated 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) and oleic acid with lipase from R. delemar, the yield of diacyl phosphatidyl choline (PC) was 25% and the fatty acid composition of the converted PC was an oleic acid content of 25% and a palmitic acid content of 75%. This reaction exhibited 1-positional specificity. Three industrial lipases from Rhizopus sp., Mucor javanicus, and Candida cylindracea had the activity of the acyl exchange of phosphatidyl choline. The lipase from R. sp. gave the best result.