Phospholipid Hydrolysis and Transphosphatidylation by Phospholipase D from Indian Mustard Seeds in Two-Phase Systems

Phospholipase D (PLD), isolated from Indian mustard seeds and purified to homogeneity, has recently been identified as typical α‐type PLD with a high activity toward phosphatidylcholine (PC) in an aqueous mixed micellar system (Khatoon et al. 2014, Phytochemistry 117, 65–75). In light of biocatalytical application, we have now studied the enzyme in aqueous‐organic two‐phase systems and compared the results with the properties of the enzyme in aqueous mixed micellar systems. n‐Hexane containing 5–10 mol% of 1‐ or 2‐octanol proved to be optimal as an organic solvent in the two‐phase system, whereas anionic detergents such as sodium dodecyl sulfate or sodium deoxycholate were favorable components in the mixed micellar system. The optimum pH value (5.5–5.6) and the optimum Ca²⁺ ion concentration (70 mM) were independent of the reaction system. The head group selectivity in terms of activity toward different phospholipids (PC > phosphatidylethanolamine > phosphatidylglycerol) was similar in both types of reaction systems. Also, the K_M values toward PC were on the same order of magnitude. In contrast, the V_max value in the two‐phase system was 20‐fold lower than in the mixed micellar system. The enzyme was able to catalyze the substitution of choline in PC by ethanolamine, glycerol, and ethylene glycol with high efficiency. l ‐Serine was exchanged for choline with low activity. Myo‐inositol was not an alcohol acceptor, but promoted the hydrolysis of PC.     

Structural Importance of the Acyl Group in Substrate Specificity of Purified Bovine Lysophospholipase D

The structural importance of the acyl group in lysophosphatidylcholine (LPC) as substrate of purified bovine lysophospholipase D (lysoPLD) was investigated. Among LPCs with saturated acyl chains, the K _m value decreased according to the length of the acyl chain (C12–C16) up to the palmitoyl group, while the V _m value showed no remarkable change. But, the extension of the acyl size to C18, as observed with 1-stearoyl LPC (K _m, 8.5 mM), rather resulted in a remarkable increase in the K _m value. Meanwhile, the introduction of one double bond in the C18 saturated acyl chain led to a remarkable reduction in the K _m value, as observed with 1-oleoyl LPC (K _m, 0.48 mM). Furthermore, 1-linoleoyl LPC (K _m, 56 μM) with two double bonds exhibited a smaller K _m value than 1-oleoyl LPC, suggesting that the unsaturation degree might be important in augmenting the binding affinity of LPCs. A similar phenomenon was also observed with 1-arachidonoly LPC (K _m, 79 μM) or 1-docosahexaenoyl LPC (K _m, 36 μM). Overall, the order of catalytic efficiency (V _m/K _m value) of those LPCs seemed to be affected by the K _m value rather than the V _m value, which differed by at most threefold among LPC derivatives. Next, the introduction of a hydroperoxide group into 1-linoleoyl-LPC or 1-arachidonoyl LPC led to a further reduction in K _m values (1-hydroperoxylinoleoyl LPC, 26 μM; 1-hydroperoxyarachidonoyl LPC, 33 μM), accompanied by a further increase in the V _m/K _m values. Additionally, phosphatidylcholines (PCs) with an oxidized acyl chain at sn-2 position were found to be efficient as 1-palmitoyl LPC as substrates of lysoPLD. Taken together, the catalytic efficiency of LPCs or oxidized PCs as substrates of lysoPLD seems to be determined by the property of the acyl chain, length of the acyl chain, unsaturation degree and oxidation status.     

Over-expression of the Anti-apoptotic Protein Bcl-2 Affects Membrane Lipid Composition in HL-60 Cells

We studied modifications induced at the membrane lipid level by over-expression of the anti-apoptotic protein Bcl-2. When total cell phospholipids were analyzed, the transformation led to a moderate decrease in poly-unsaturated fatty acids, compensated by an increase in mono-unsaturated species. At the mitochondrial membrane level, the changes were more important and occurred in saturated and dimethyl acetal fatty acids, which became more abundant, while unsaturated fatty acid content diminished. This indicates a decline in oxidation-sensitive fatty acids (unsaturated species) together with a gain in oxidation-insensitive saturated fatty acids and in plasmalogen (as detected by dimethyl acetal fatty acids) considered as oxygen species scavengers. Theses changes, combined with the protective role of Bcl-2 against oxidation due to its effect on the redox potential, should protect cells from apoptosis starting in mitochondria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acyl Chain Specificity of Marine Streptomyces klenkii PhosPholipase D and Its Application in Enzymatic Preparation of Phosphatidylserine

Mining of phospholipase D (PLD) with altered acyl group recognition except its head group specificity is also useful in terms of specific acyl size phospholipid production and as diagnostic reagents for quantifying specific phospholipid species. Microbial PLDs from Actinomycetes, especially Streptomyces, best fit this process requirements. In the present studies, a new PLD from marine Streptomyces klenkii (SkPLD) was purified and biochemically characterized. The optimal reaction temperature and pH of SkPLD were determined to be 60 °C and 8.0, respectively. Kinetic analysis showed that SkPLD had the relatively high catalytic efficiency toward phosphatidylcholines (PCs) with medium acyl chain length, especially 12:0/12:0-PC (67.13 S⁻¹ mM⁻¹), but lower catalytic efficiency toward PCs with long acyl chain (>16 fatty acids). Molecular docking results indicated that the different catalytic efficiency was related to the increased steric hindrance of long acyl-chains in the substrate-binding pockets and differences in hydrogen-bond interactions between the acyl chains and substrate-binding pockets. The enzyme displayed suitable transphosphatidylation activity and the reaction process showed 26.18% yield with L-serine and soybean PC as substrates. Present study not only enriched the PLD enzyme library but also provide guidance for the further mining of PLDs with special phospholipids recognition properties.     

Phospholipase D Activity in Relation to the Size of Substrate Micelles

The activity of phospholipase D (PLD) is strongly dependent on the aggregation state of its substrate. Artificial substrates, such as phosphatidyl‐p‐nitrophenol (PpNP), are preferably used in a mixture with Triton^® X‐100 (Serva) and sodium dodecyl sulfate (SDS) in the form of mixed micelles. In this paper the activities of PLD from cabbage (PLD_cab), which needs Ca²⁺ ions for its activity, and PLD from Streptomyces sp. (PLD_Str), which is independent of Ca²⁺ ions, are compared as a function of the detergent and substrate concentrations. While the variation of the Triton^® X‐100 and PpNP contents changed the activities of both enzymes in similar way, SDS showed activation effects on plant PLD but inactivation effects on microbial PLD. The activity decreased to 50% at a 7‐tenfold molar excess of Triton^® X‐100 related to PpNP. SDS induced a strong activation (up to fourfold) of PLD_cab but decreased the activity of PLD_Str with increasing concentration. Activity‐substrate profiles of both PLDs passed optima at 3–4 mM PpNP. In all experiments, the activity changes correlated with changes of the micelle sizes determined by dynamic light scattering. These results highlight the immense importance of the micelle structure, which is not considered in most studies.     

Investigation of Structural Features of Two Related Lipases and the Impact on Fatty Acid Specificity in Vegetable Fats

One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL), show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand.     

Dietary Crude Lecithin Increases Systemic Availability of Dietary Docosahexaenoic Acid with Combined Intake in Rats

Crude lecithin, a mixture of mainly phospholipids, potentially helps to increase the systemic availability of dietary omega‐3 polyunsaturated fatty acids (n‐3 PUFA), such as docosahexaenoic acid (DHA). Nevertheless, no clear data exist on the effects of prolonged combined dietary supplementation of DHA and lecithin on RBC and plasma PUFA levels. In the current experiments, levels of DHA and choline, two dietary ingredients that enhance neuronal membrane formation and function, were determined in plasma and red blood cells (RBC) from rats after dietary supplementation of DHA‐containing oils with and without concomitant dietary supplementation of crude lecithin for 2–3 weeks. The aim was to provide experimental evidence for the hypothesized additive effects of dietary lecithin (not containing any DHA) on top of dietary DHA on PUFA levels in plasma and RBC. Dietary supplementation of DHA‐containing oils, either as vegetable algae oil or as fish oil, increased DHA, eicosapentaenoic acid (EPA), and total n‐3 PUFA, and decreased total omega‐6 PUFA levels in plasma and RBC, while dietary lecithin supplementation alone did not affect these levels. However, combined dietary supplementation of DHA and lecithin increased the changes induced by DHA supplementation alone. Animals receiving a lecithin‐containing diet also had a higher plasma free choline concentration as compared to controls. In conclusion, dietary DHA‐containing oils and crude lecithin have synergistic effects on increasing plasma and RBC n‐3 PUFA levels, including DHA and EPA. By increasing the systemic availability of dietary DHA, dietary lecithin may increase the efficacy of DHA supplementation when their intake is combined.     

Phospholipase A2-catalyzed hydrolysis of lecithin in a continuous reversed-micellar membrane bioreactor

Lysophospholipids and free fatty acids produced by lecithin hydrolysis are important natural compounds with high potential for application in the food, chemical, and pharmaceutical industries. In this work, the enzymatic hydrolysis of lecithin (essentially phosphatidylcholine) catalyzed by porcine pancreatic phospholipase A₂ (phosphatide 2-acyl-hydrolase, EC 3.1.1.4), encapsulated in mixed reversed micelles of lecithin andbis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, was carried out in a continuous reversed-micellar membrane bioreactor. A tubular ceramic membrane with a 10,000 molecular weight (MW) cutoff was installed in an ultrafiltration module to retain the phospholipase A₂ (MW 14,000) and to continuously separate the products from the reaction media. Water and co-factor (Ca⁺⁺)-containing reversed micelles of lecithin/AOT in isooctane were supplemented to the reactor to compensate for the permeation of reversed micelles and to continuously supply the substrate. The influence of relevant parameters, such as substrate, AOT and enzyme concentrations, water content and fluid hydrodynamics, on the performance of the ultrafiltration membrane bioreactor was investigated. Fluid axial velocity and substrate concentration were the major factors that affected the transport processes through the membrane. Permeate flow rate increased significantly with fluid axial velocity and decreased with substrate concentration; on the other hand, water and enzyme concentrations were identified as critical parameters for the final conversion of lecithin. The relationship between productivity and normalized residence time was analyzed for each set of experimental parameters tested. Operational stability of the bioreactor was tested in a long-term operation to confirm the high stability of this catalytic system.     

Crystal Structure of a Phospholipase D from the Plant-Associated Bacteria Serratia plymuthica Strain AS9 Reveals a Unique Arrangement of Catalytic Pocket

Phospholipases D (PLDs) play important roles in different organisms and in vitro phospholipid modifications, which attract strong interests for investigation. However, the lack of PLD structural information has seriously hampered both the understanding of their structure–function relationships and the structure-based bioengineering of this enzyme. Herein, we presented the crystal structure of a PLD from the plant-associated bacteria Serratia plymuthica strain AS9 (SpPLD) at a resolution of 1.79 Å. Two classical HxKxxxxD (HKD) motifs were found in SpPLD and have shown high structural consistence with several PLDs in the same family. While comparing the structure of SpPLD with the previous resolved PLDs from the same family, several unique conformations on the C-terminus of the HKD motif were demonstrated to participate in the arrangement of the catalytic pocket of SpPLD. In SpPLD, an extented loop conformation between β9 and α9 (aa228–246) was found. Moreover, electrostatic surface potential showed that this loop region in SpPLD was positively charged while the corresponding loops in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) were neutral. The shortened loop between α10 and α11 (aa272–275) made the SpPLD unable to form the gate-like structure which existed specically in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) and functioned to stabilize the substrates. In contrast, the shortened loop conformation at this corresponding segment was more alike to several nucleases (Nuc, Zuc, mZuc, NucT) within the same family. Moreover, the loop composition between β11 and β12 was also different from the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9), which formed the entrance of the catalytic pocket and were closely related to substrate recognition. So far, SpPLD was the only structurally characterized PLD enzyme from Serratia. The structural information derived here not only helps for the understanding of the biological function of this enzyme in plant protection, but also helps for the understanding of the rational design of the mutant, with potential application in phospholipid modification.     

Characteristic distributions of fatty acids in different lipids from Jack beans (Canavalia gladiata DC.)

Extracted lipids obtained from Jack beans (white and red) were fractionated by TLC into nine subfractions. The major components were TAGs (TAG: 43.8–45.7 wt%) and phospholipids (PL: 46.7–47.0 wt%), while other components were also present in minor proportions (0.3–2.7 wt%). The principal fatty acids (FA) are generally palmitic (18.8–28.8%), stearic (0.7–6.8%), oleic (42.0–51.8%), linoleic (16.2–22.8%), and α‐linolenic (3.0–8.2%) acids, the distribution of which differs according to these lipid classes. There were no significant differences (p>0.05) in the positional distribution of FA in the TAG; unsaturated FA (97.5%) were predominantly concentrated in the sn‐2 position while saturated FA (33.3%) primarily occupied the sn‐1 position or sn‐3 position. However, significant differences (p<0.05) in FA distribution existed when the individual PL were compared between the white and red beans. Based on the FA composition of these lipids, it seems that the two cultivars of Jack beans are very similar to each other with a few exceptions. The results could be useful to both producers and consumers for our daily diet to improve value of the Japanese diet. Practical applications : The lipid composition suggests that these beans could be a good source of nutraceuticals with providing heath benefits. The white and red beans may be well incorporated into our daily Japanese diets to improve nutritional value. The data obtained in this study provide valuable information for manufacturing functional drinks such as Jack bean tea in Japan.     

Identification of Cyclopropaneoctanoic Acid 2-Hexyl in Human Adipose Tissue and Serum

Fatty acids containing a cyclopropane ring in their structure (cyclopropane FA) have been found in a wide variety of bacteria, a number of protozoa, and Myriapoda. Little is known about cyclopropane FA in mammal, especially in human tissues. The present study deals with the identification of cyclopropane FA in adipose tissue and serum of humans and rats. Fatty acids extracted from the adipose tissue and serum obtained from obese women during bariatric surgery were methylated and analyzed on GC–MS. We have identified: cyclopropaneoctanoic acid 2-hexyl, cyclopropaneoctanoic acid 2-octyl, cyclopropanenonanoic acid, and 2-[[2-[(2-ethylcyclopropyl)methyl]cyclopropyl]methyl] acid in human adipose tissue. We confirmed the presence of cyclopropaneoctanoic acid 2-hexyl by derivatization of FA extracted from human adipose tissue to picolinyl esters. Cyclopropaneoctanoic acid 2-hexyl was the main cyclopropane FA (approximately 0.4 % of total fatty acids in human adipose tissue, and about 0.2 % of total fatty acids in the serum). In adipose tissue cyclopropaneoctanoic acid 2-hexyl was found mainly in triacylglycerols, whereas in serum in phospholipids and triacylglycerols. The cyclopropaneoctanoic acid 2-hexyl has also been found in serum, and adipose tissue of rats in amounts comparable to humans. The content of cyclopropaneoctanoic acid 2-hexyl decreased in adipose tissue of rats maintained on a restricted diet for 1 month. In conclusion, we demonstrated that cyclopropaneoctanoic acid 2-hexyl is present in human adipose tissue and serum. Adipose tissue cyclopropaneoctanoic acid 2-hexyl is stored mainly in triacylglycerols and the storage of this cyclopropane FA is affected by food restriction.     

Process for the Recovery of Cobalt and Nickel from Sulphate Leach Liquors with Saponified Cyanex 272 and D2EHPA

Abstract

In this paper, a process is reported for the recovery of cobalt and nickel from copper raffinate solutions using partially saponified Cyanex 272 and D2EHPA as the extractants. The aqueous feed contains 1.65 g/L cobalt and 16.42 g/L nickel. More than 99.9% cobalt separation was achieved with 0.13 M Cyanex 272 (60% neutralized with alkali) in two counter‐current stages at an aqueous to organic phase ratio of 1.1:1. Co‐extraction of nickel was 0.18% only. Stripping of cobalt from a loaded organic phase was carried out with synthetic spent electrolyte solution at an organic to aqueous phase ratio of 2.5 in two counter‐current stages to generate a pregnant electrolyte solution to produce cobalt metal by electrowinning. Similarly, optimum conditions for nickel extraction with 60% neutralized 1 M D2EHPA at O/A ratio of 1.4 in 2 two stages and stripping of metal with synthetic spent electrolyte at O/A ratio of 1.6 in two stages were standardized. Extraction and stripping efficiencies were >99% and the flowsheet of the process is demonstrated.     

Detection of 430 Fatty Acid Methyl Esters from a Transesterified Butter Sample

Milk fat is known to contain one of the highest number of fatty acids of all edible oils. Some of these fatty acids are known to be valuable (e.g. conjugated linoleic acids, furan fatty acid) and other as undesirable (e.g. saturated and some trans-fatty acids) food ingredients. However, a comprehensive picture on the presence of many trace fatty acids has not been achieved. For this reason we have developed an analysis scheme based on the conversion of the fatty acids into methyl esters. The fatty acid methyl esters were then fractionated by urea complexation. Both the filtrate of the urea complexation (~4 % of the sample weight) and the original sample were fractionated by high-speed counter-current chromatography (HSCCC). The resulting fractions were analyzed by GC/MS analysis. With this method 430 fatty acids were detected in one single butter sample. More than 230 fatty acids had two or more double bonds. In addition to the widely known spectrum of fatty acids we also detected a range of cyclohexyl fatty acids (five homologues) and methyl-branched fatty acids (including short chain and even-numbered anteiso-fatty acids), conjugated tetradecadienoic acids along with the novel ω-oxo-fatty acids (seven homologues). The reported relative retention time on the polar column may serve as a data base for the screening of other samples for this profusion of fatty acids.     

Solvent extraction reaction of hafnium(IV) from strong sulfuric acid solutions with D2EHPA and PC 88A

Hafnium can be selectively extracted over zirconium from strong sulfuric acid solutions by D2EHPA or PC 88A. Solvent extraction experiments have been performed to identify the reaction of Hf from strong sulfuric acid solutions (1 to 7 M) by these two extractants. Hafnium extraction was gradually decreased and then increased again with the increase of sulfuric acid concentration. By applying a slope method, the extracted Hf species was proposed to be HfA₄·(HA)₂ by D2EHPA and Hf(HSO₄)₂A₂·(HA)₂ by PC 88A, respectively. This difference in the nature of the extracted species was verified by FT-IR spectra.     

Formation of ordered microporous films with water as templates from poly(D,L‐lactic‐co‐glycolic acid) solution

Recently a method that uses water droplets at the air–solution interface as an ordered template was reported for the preparation of ordered micrometer‐size honeycomb structures. Here we show that the method can also be used for formation of honeycomb‐like porous films from random copolymers with certain hydrophilicity, besides those polymers with defined structures such as block copolymers, starlike homopolymers, amd amphiphilic polymers. This demonstrates that the stabilization of water droplets is the key factor for the regular structure. Also we indicate that size and structure of the films can be regulated by such variables as concentration and atmospheric humidity. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 1846–1850, 2003     

An efficient and expeditious synthesis of phytostanyl esters in a solvent‐free system

Esterification of natural phytostanols with various fatty acids by using Lewis acid‐surfactant combined catalyst was investigated. For synthesis of phytostanol esters of saturated fatty acids, cuprum dodecyl sulfate [Cu(DS)₂] was the most desirable catalyst due to its high selectivity, reusability, activity, and less corrosivity, whereas stanol selectivity with other catalysts, such as ZnCl₂ and tungstophosphoric acid. The substrate molar ratio of 1.2:1 (lauric acid/phytostanols) was the optimal. For synthesis of phytostanol esters of unsaturated fatty acids, cerium dodecyl sulfate [Ce(DS)₃] was better than [Cu(DS)₂] which was based on the oxidation of the unsaturated fatty acids during the reaction. The chemical structure of the sitostanyl stearate, sitostanyl oleate, and sitostanyl linoleate were confirmed by FTIR, MS, and NMR, respectively. As a result, the [Cu(DS)₂] and [Ce(DS)₃] were screened to synthesize phytostanyl esters of fatty acids for commercial production. Practical applications: Phytostanols are important for human health and nutrition. Unfortunately, due to the poor solubility of free stanols (unesterified) in fats and oils, there is a demand for a good way to improve the solubility or bioavailability of phytostanols, such as esterification of phytostanols with fatty acids. This study aims at finding an efficient and expeditious synthesis of phytostanyl esters. At the same time, environmental impact and the oxidation of the unsaturated fatty acids during the reaction should be considered.     

Extraction and Stripping Behavior of Iron (III) from Phosphoric Acid Medium by D2EHPA Alone and Its Mixtures with TBP/TOPO

In the present study an attempt has been made to understand the extraction and stripping behavior of iron (III) with D2EHPA alone and its mixture with TBP or TOPO in phosphoric acid medium. Effect of variables such as concentrations of iron (III), phosphoric acid, and phosphate in the aqueous phase, D2EHPA, TBP, and TOPO concentrations in the organic phase and temperature on the extraction process has been studied. The extraction of iron (III) decreased with increase in phosphoric acid concentration. The increase in D2EHPA concentration increased the extraction of iron (III). The presence of TOPO or TBP with D2EHPA showed antagonism. The increase in temperature decreased the extraction of iron (III) with D2EHPA alone and its mixture with either TOPO or TBP showing the exothermic nature of the extraction reaction. The stripping of iron (III) by various reagents followed the order: oxalic acid > phosphoric acid > hydrochloric acid > sulphuric acid > mixture of sulphuric and hydrofluoric acids > ascorbic acid > citric acid irrespective of extraction systems. Higher temperature favors the stripping. The effect of diluents on iron (III) extraction has also been studied. The mechanism of extraction has been explained in the light of the results obtained.