Aqueous dissolution of crystalline and amorphous amylose-alcohol complexes

Complexes of amylose, the linear starch polysaccharide, with linear alcohols having chain lengths varying from 4 to 8 carbon atoms, were prepared. Either crystalline or amorphous complexes could be formed depending on preparation conditions. Crystalline complexes gave sharp X-ray diffraction patterns, characteristic of the V_H form of amylose, whereas no observable pattern was obtained from the amorphous form. Thermal dissociation of the complexes occurred at increasing temperatures with increasing alcohol chain length. Crystalline complexes dissociated at temperatures approximately 23°C higher than their amorphous counterparts and the enthalpy of dissociation was also greater for the crystalline samples. Enthalpy values were independent of alcohol chain length. Differences in thermal behaviour of the two types of complex may be described in terms of the polymer crystal lattice energy and may explain the variability of reported results for complex dissociation in the literature.     

Temperature effect on retrogradation rate and crystalline structure of amylose

Commercial potato amylose was used to study temperature effects on the retrogradation of amylose solutions (3.5mg/ml). The retrogradation rate decreased as incubation temperature increased (5 to 45 °C). The degree of retrogradation within 24 h decreased from 58.8 to 7.1% as incubation temperature increased from 5 to 45 °C. In the amylose solution, different-sized molecular subfractions retrograded at different rates. After incubating at 5 °C for 100 days, the majority of the amylose molecules retrograded and precipitated from the solution; at 45 °C, only amylose of the small-molecular subfraction (number average, DP_n = 110; weight average, DP_w = 150) retrograded and precipitated. Entanglement of molecules was observed in size exclusion chromatograms. The morphology of retrograded amylose observed by using a scanning electron microscope differed with the retrogradation temperature. The chain length of amylose crystalline segments, prepared by hydrolysis of retrograded amylose, showed a narrow distribution (polydispersity from 1.21 to 1.67). The chain lengths of resistant segments increased DP_n from 39 to 52 and DP_w from 47 to 72 for α-amylolysis and DP_n from 34 to 40 and DP_w from 48 to 67 for 16% sulfuric acid hydrolysis, when incubation temperature increased from 5 to 45 °C.     

Amylose–lipid complexation: a new fractionation method

Amylose fractions of different peak Degree of Polymerisation (DP) (DP20, DP60, DP400, DP950) were complexed with docosanoic acid (C22) and glyceryl monostearate (GMS) at 60 and 90 °C. Complexation yields, relative crystallinities, dissociation temperatures and enthalpies increased with amylose chain lengths (DP20–DP60–DP400). Relative crystallinities and thermal stabilities of the DP950-complexes were slightly lower than those of the other amylose fractions, probably due to increased conformational disorders, resulting in crystal defaults. Molecular weight distributions of the complexes revealed that, irrespective to the complexation temperature, the critical DP for complex formation and precipitation was 35 and 40 for complexes with GMS and C22, respectively, corresponding to the length needed to accommodate two GMS- or C22-molecules within an amylose helix. Complexation of dextrins with a well-chosen lipid, allows to separate starch derived dextrins with a predictable critical chain length as border. Dextrins, of sufficient DP will complex and precipitate, while the shorter dextrins will remain in solution.     

Solubility of Saturated Fatty Acids in Water at Elevated Temperatures

The solubility in water of saturated fatty acids with even carbon numbers from 8 to 18 was measured in the temperature range of 60 to 230°C and at a pressure of 5 or 15 MPa. The pressure had no significant effect on the solubility. The solubility of the fatty acids increased with increasing temperature. At temperatures higher than about 160°C, the logarithm of the solubility in mole fraction was linearly related to the reciprocal of the absolute temperature for each fatty acid, indicating that the water containing solubilized fatty acid molecules formed a regular solution at the higher temperatures. The enthalpy of a solution of the fatty acids in water, which was evaluated from the linear relationship at the given temperatures, increased linearly with the carbon number of the fatty acid.     

Nature of the Elements Transporting Long-Chain Fatty Acids Through the Red Cell Membrane

Docosahexaenoic acid is found to be bound to three equivalent sites on albumin with the same affinities as palmitic acid at 0–38°C, which demonstrates that ethene-1,2-diyl- and methylene-groups contribute equally to the affinity. The equilibrium dissociation constants (K _dm s) for red cell membrane binding sites of linoleic- and docosahexaenoic acid at pH 7.3 are determined at temperatures between 0 and 37°C. The temperature-independent capacities for binding are 12 ± 1 and 25.4 ± 3.0 nmoles g⁻¹ ghosts respectively. Double isotope binding experiments reveal that the unsaturated fatty acids: arachidonic-, linoleic-, docosahexaenoic-, and oleic acid have partially shared capacities in ratios approximately 1:2:4:5, in contrast to the noncompetitive binding of palmitic acid. The observations suggest a two-tier binding limitation. One is the number of protein sites binding fatty acid anions electrostatically and the other is the number of suitable annular lipids adaptively selected among membrane lipids by the hydrocarbon chain. These competition conditions are confirmed by measurements of the tracer exchange efflux at near 0°C from albumin-free and albumin-filled ghosts of linoleic- and docosahexaenoic acid, either alone or in the presence of arachidonic- and palmitic acid. Under equilibrium conditions, the calculated ratios of inside to outside membrane binding is below 0.5 for four unsaturated fatty acids. The unidirectional rate constants of translocation between the inside and the outside correlate with the number of double bonds in these fatty acids, which are also correlated with the dissociation rate constants of the complexes with albumin. The membrane permeation occurs presumably by binding of the anionic unsaturated fatty acids to an integral protein followed by channeling of the neutral form between opposite binding sites of the protein through annular lipids encircling the protein. Received: 30 June 1997/Revised: 23 February 1998     

Potato phosphorylase catalyzed synthesis of amylose-lipid complexes

On-line size-exclusion chromatography monitoring of potato phosphorylase catalyzed amylose synthesis--starting from alpha-D-glucose-1-P and maltohexaose--revealed rather monodisperse amylose populations. In the presence of lipids, amylose-lipid complexes spontaneously formed and precipitated. They were recovered by centrifugation, freeze-dried, and characterized by wide-angle X-ray diffraction and differential scanning calorimetry. The presence of lipids during amylose synthesis led to lower amylose degrees of polymerization (DP). Lipid chain length defined amylose DP, which increased in the order myristic acid (C14), glyceryl monostearate (GMS), stearic acid (C18), and docosanoic acid (C22). The thermal stability of the complexes increased in the same manner, with the C22 complexes having the highest dissociation temperature. In addition, we hypothesized that these results provide additional evidence for the fringed micellar organization of (semi-enzymically synthesized) amylose-lipid complexes.     

Substrate specificity of fatty-acyl-CoA ligase in liver microsomes

The substrate specificity of fatty-acyl-CoA ligase in liver microsomes has been studied in a system in which fatty acids are present initially as complexes with unilamellar vesicles of phosphatidylcholine. The latter were prepared by cosonication of phospholipids and different fatty acids. As compared with previous studies of the enzyme the activity of acyl-CoA ligase is several-fold higher for assays carried out with fatty acid substrates added as components of a bilayer. This was true for all fatty acids studied. Also as compared with data reported previously in the literature there was a systematic relationship between the structure of fatty acids, activity at V_max for synthesis of acyl-CoA and avidity of binding to the ligase. Activity at V_max was greatest for lauric acid and decreased with increasing chain length. The apparent avidity of enzyme for fatty acids was greatest for octanoic acid and decreased as chain length increased.     

Long-chain acyl thioesters prepared by solvent-free thioesterification and transthioesterification catalysed by microbial lipases

Long-chain acyl thioesters (thio wax esters) have been prepared in high (80% to more than 90%) yields by solvent-free esterification of fatty acids (lauric, myristic, palmitic and stearic acids) with long-chain thiols, such as decane thiol, dodecane thiol, tetradecane thiol and hexadecane thiol, catalysed by lipases from Candida antarctica (Novozym) and Rhizomucor miehei (Lipozyme) in the presence of a 0.4-nm molecular sieve. In the thioesterification reaction Novozym was a more effective biocatalyst than Lipozyme. The extent of thioesterification increased with increasing molar ratio of fatty acid to alkane thiol (1:1 to 3:1) and with temperature (40 °C compared to 60 °C), as well as with the amount of the enzyme preparation and the amount of 0.4-nm molecular sieve. Decreasing the chain length of the alkane thiol from C₁₆ to C₁₀ also increased the extent of thioesterification. Lipase-catalysed solvent-free transthioesterification of fatty acid methyl esters with alkane thiols was less effective for the preparation of acyl thioesters than was thioesterification of fatty acids with alkane thiols. In transthioesterification, Lipozyme was slightly more effective as a biocatalyst than Novozym. Received: 3 September 1998 / Received revision: 18 November 1998 / Accepted: 21 November 1998     

Mobility of lipid in complexes of amylose–fatty acids by deuterium and C solid state NMR

Palmitic and lauric acid complexes with amylose were studied by solid state methods: ¹³C CP/MAS NMR, deuterium NMR, X-ray powder diffraction and differential scanning calorimetry (DSC). The crystalline amylose complexes were found to be in a V-type sixfold single chain helix. The melting points of the complexes were over 100°C, at least 40–50°C higher than the melting points of the free fatty acids. CP/MAS ¹³C NMR spectra revealed two resonance peaks at 33.6 and 32.4 ppm for the palmitic acid, which were assigned as free and complexed fatty acid, respectively. A single resonance peak at 32.4 ppm was found for the lauric acid and assigned to the complex. The chemical shift of 32.4 ppm for the complexed fatty acids suggests a combined trans and gauche conformation for the fatty acid chain in the complex. T₁ relaxation measurements on the two palmitic acid resonances show different behavior: a very slow relaxation for the 33.6 ppm and a much faster relaxation (1.2 s) for the 32.4 ppm resonances. The latter was similar to the relaxation of the single resonance of the lauric acid (1.1 s). Temperature dependent deuterium spectra of the amylose–lauric acid and amylose–palmitic acid complexes suggest a complete complexation for the amylose–lauric acid, whereas the amylose–palmitic acid complex is partially disassociated by the thermal treatment. Based on the overall data, a partially disordered model is proposed: an imperfect helix with the fatty acid partly inside and partly out, depending on crystallization conditions and the necessity of placing the carboxyl head outside the V-helix.     

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

When amylose was synthesized using potato phosphorylase in the presence of amylose complexing lipids, monodisperse populations of amylose–lipid complexes were formed. Enzyme dosage and glucose-1-phosphate (glc-1-P)/primer ratio influenced the reaction rate of the enzymic synthesis, presumably by changing the balance between amylose synthesis and amylose–lipid complexation and precipitation, and impacted the molecular weight of the complexes. Lipid characteristics affected the dissociation properties and amylose chain lengths of the amylose–lipid complexes presumably by determining the minimal amylose chain length necessary for complexation and precipitation. Tailor made short chain amylose–lipid complexes can hence be produced by choosing the appropriate reaction conditions. We propose a synthesis mechanism in which the primer is elongated until an amylose chain is obtained which is of sufficient length to complex a first lipid. Further chain extension then occurs, together with subsequent complexation until the complex becomes insoluble and precipitates.     

Thermostable alkaline lipase from a newly isolated thermophilic Bacillus,strain A30-1 (ATCC 53841)

A thermophilic bacterium was isolated from a hot spring area of Yellowstone National Park. The organism grew optimally at 60–65°C and in the pH range of 6–9. It was characterized as Bacillus sp. In the presence of corn or olive oil (1.0%) as the growth substrate, this Bacillus produced an extracellular lipolytic activity (EC 3.1.1.3). The enzyme activity could be efficiently recovered by ultrafiltration of cell-free culture supernatant. The partially purified lipase preparation had an optimum temperature of 60°C, at an optimum pH of 9.5. It retained 100% of the original activity after being heated at 75°C for half an hour. The half life of the enzyme was 8 h at 75°C. The enzyme retained at least 90% of the original activity after it was incubated at 60°C for 15 h at pH's in the range of 5 to 10.5. The enzyme was active on triglycerides containing fatty acids having a carbon chain length of C16 : 0 to C22 : 0 as well as on natural fats and oils. The enzyme activity was stable to both hydrogen peroxide and alkaline protease which are detergent ingredients. The purified enzyme had an isoelectric point of 5.15 and an approximate molecular weight of 65,000.     

Monte Carlo simulation of multiple attack mechanism of α-amylase

Porcine pancreatic α-amylase (EC 3.2.1.1) produces short maltooligosaccharides from a single enzyme-substrate complex without dissociation by multiple or repetitive attack. Multiple attack is caused by relative sliding of the enzyme along the product chain of the enzyme-product complex without dissociation to form another productive complex. The Monte Carlo method was applied to the multiple attack mechanism to predict product distribution from amylose and amylopectin molecules of arbitrary sizes. The position of the initial attack to make the enzyme-substrate complex and branched reaction paths from the enzyme-product complex were selected by random numbers and probabilities. A simulated product distribution from 100,000 samples of amylose of chain length greater than 80 agreed completely with experimental data at the early stage of hydrolysis of amylose of mean chain length 90. On the other hand, the simulated product distribution from amylopectin agreed with experimental data of potato amylopectin when the effective chain length of the A chain was 9. Since the mean chain length of the A chain of potato amylopectin is 15, it is possible that amylopectin is partially compact in solution, so that the enzyme can recognize and act only on the outer side of the A chain at the early stage of digestion. © 1996 John Wiley & Sons, Inc.     

Effect of Metal Cations and pH on the Antibacterial Activity and Uptake of Long Chain Fatty Acids

S ummary . The bactericidal activity of long chain saturated fatty acids was antagonized by alkaline earth metals. The activity of linoleic acid was less effectively antagonized but was more sensitive to reversal by ferric and stannous ions. With increasing pH value the bactericidal activity of lauric acid decreased but that of the longer chain saturated acids increased. Both Gram positive and Gram negative bacteria reversibly adsorbed fatty acids. Uptake increased with decreasing pH value and increasing chain length. Although adsorbed to a lesser extent, the intrinsic activity of linoleic acid was greater than lauric acid. The uptake appeared to be non-specific and governed by the physicochemical properties of both the acids and the bacterial cell surfaces. Sensitivity to the fatty acids increased with decreasing pH value. Protoplasts of Bacillus megaterium adsorbed fatty acids to a greater extent than whole cells. Resistance of the Gram negative Pseudomonas phaseolicola was not due to non-adsorption of the fatty acids.     

Clean synthesis of biolubricants for low temperature applications using heterogeneous catalysts

Biolubricants derived from vegetable oils are environmentally compatible products due to their low toxicity and good biodegradability. Synthetic esters based on polyols and fatty acids possess suitable properties for lubricant applications, even at extreme temperatures. In this work, synthesis of esters from trimethylolpropane (TMP) and carboxylic acids from C5 to C18 has been studied and compared using different heterogeneous catalysts (silica–sulphuric acid, Amberlyst-15, and immobilised lipase B from Candida antarctica). Silica–sulphuric acid was found to be the most efficient catalyst followed by Amberlyst-15, especially when using short chain carboxylic acids. The reaction efficiency decreased with increasing alkyl chain length. On the other hand, the immobilised lipase (Novozym^®435) did not exhibit any activity with C5 acid and the activity increased with increase in length of the fatty acid chain. For synthesis of C18-ester, the biocatalytic production turned out to be comparable to silica–sulphuric acid, and moreover led to a better quality of the final product. The products showed suitable cold-flow properties for application at low temperature. A general trend of increasing pour point (−75 °C to −42 °C) and viscosity index (80–208) with increase in alkyl chain of the carboxylic acid from C5 to C18 was observed. The synthesis of TMP-trioleate using the solid acid catalysts and the biocatalyst was compared using the freeware package EATOS (environmental assessment tool for organic synthesis) and showed the enzymatic route to have the least environmental impact.     

A role for hormone-sensitive lipase in the selective mobilization of adipose tissue fatty acids.

The mobilization of fatty acids from rat and human fat cells is selective according to molecular structure, and notably carbon atom chain length. This study aimed at examining whether the release of individual fatty acids from triacylglycerols (TAG) by hormone-sensitive lipase (HSL) plays a role in the selectivity of fatty acid mobilization. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 18 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation degree from 0 to 3 double bond(s), was measured by comparing the composition of non-esterified fatty acids (NEFA) to that of the original TAG. The relative hydrolysis (% in NEFA/% in TAG) differed between fatty acids, being about 5-fold and 3-fold higher for the most (18:1n-7) than for the least (24:0) readily released fatty acid by recombinant rat and human HSL, respectively. Relationships were found between the chain length of fatty acids and their relative hydrolysis. Among 12-24 carbon atom saturated fatty acids, the relative hydrolysis markedly decreased (by about 5- and 3-times for recombinant rat and human HSL, respectively) with increasing chain length. We conclude that fatty acids are selectively released from TAG by HSL according to carbon atom chain length. These data provide insight on the mechanism by which fatty acids are selectively mobilized from fat cells.     

Toxicity of medium chain fatty acids to Penicillium crustosum Thom and their detoxification to methyl ketones

Conversion of medium chain length fatty acids into methyl ketones by some filamentous fungi is important in the development of flavour in the blue mould-ripened cheeses and in the promotion of ketonic rancidity in the lauric acid oils. The effect of fatty acid composition of single acid glycerides, pH, temperature and medium chain length fatty acids on conversion of fatty acids into methyl ketones has been studied with two strains of Penicillium crustosum Thom. Medium chain length fatty acids were fungitoxic. The minimum inhibitory concentration was 3776, 2884, 1723 and 3066 mg/l for the C6:0, 8:0, 10:0 and 12:0 acids, respectively, at pH 7·0 in liquid suspension culture. Evidence is provided to suggest that the cellular site for this conversion is the microbody. Growth of P. crustosum on single acid glycerides involves two distinct physiological processes; the first, a general inhibition of growth, and the second, a detoxification. The fatty acid composition and the slip point of the substrate are of paramount importance in these two processes.     

EFFECTS OF VARIATION IN TEMPERATURE. II. ON THE FATTY ACID COMPOSITION OF EIGHT SPECIES OF MARINE PHYTOPLANKTON1

Eight species of marine phytoplankton showed significant variation in the relative amount of some fatty acids (FAs) in response to variation in temperature. Large changes in relative amounts of certain FAs occurred as a result of a 15° C change in growth temperature. For example, 14:0 increased from ?4% of total FAs at 10° C to > 20% at 25° C for Chaetoceros simplex and Isochrysis aff. galbana but decreased for Phaeodactylum tricornutum. The percentage of the polyunsaturated fatty acid (PUFA) 16:ω1 was consistently greater at 10° C than at 25° C, and the converse was usually true for 16: 4ω3. Calculated over all eight species, there was a modest but significant inverse relationship between the percentage of PUFAs and temperature. Only for Thalassiosira pseudonana was the percentage of either of the PUFAs and nutritionally essential fatty acids (EFAs) also an inverse function of temperature. For T. pseudonana, the percentage of the EFA 22:6ω3 decreased linearly with increasing temperature over the range from 10 to 25° C. For three species, the ratio of unsaturated/saturated FAs was correlated with growth rate when growth rate was controlled by variation in irradiance and temperature. Only for Thalassiosira pseudonana was the ratio of unsaturated/saturated FAs also an inverse function of temperature alone.     

Preparation of long-chain acyl thioesters - thio wax esters - by the use of lipases

Long-chain acyl thioesters have been prepared by lipase-catalyzed thioesterification of lauric, myristic, palmitic and stearic acids with decanethiol, dodecanethiol, tetradecanethiol, hexadecanethiol and octadecanethiol. Lipase from Candida antarctica was more effective than that from Rhizomucor miehei. The extent of thioesterification increased with increasing chain length of the fatty acids and decreasing chain length of the alkanethiols. Lipase-catalyzed transthioesterification of fatty acid methyl esters with alkanethiols was less effective than thioesterification for the preparation of acyl thioesters. © Rapid Science Ltd. 1998     

Reorganization of membrane lipids during fast and slow cold hardening in Drosophila melanogaster

Abstract Rapid cold hardening is a naturally occurring phenomenon in insects that is thought to be responsible for increased cold tolerance during diurnal variations in temperature. The underlying physiological mechanisms are still not fully resolved but, in Drosophila melanogaster (Meigen 1830), rapid cold hardening is accompanied by specific changes in the membrane lipid composition. To further understand the link between rapid cold hardening and adjustments in the membrane lipid composition, the present study investigates how different rates of cooling affect thermotolerance and the composition of phospholipid fatty acids. Female Drosophila are cooled gradually from 25 to 0 °C at 0.01, 0.05, 0.1 or 0.5 °C min^?1, respectively, and, subsequently, phospholipid fatty acid composition and survival after a 1‐h cold shock at ?5 °C is measured. The rapid cold hardening treatments all influence cold tolerance differently so that short and intermediate rapid cold hardening treatments (0.05, 0.1 or 0.5 °C min^?1 cooling rates) increase cold shock survival, whereas the slow cooling treatment (0.01 °C min^?1) decreases survival relative to an untreated control. The intermediate rapid cold hardening treatments (0.05 or 0.1 °C min^?1) induce a similar type of response characterized by an increase in the molar percentage of linoleic acid, 18:2(n‐6), at the expense of 16:0 and 18:1(n‐9), which leads to an increase in the degree of unsaturation. The slowest cooling treatment (0.01 °C min^?1) results in a large increase in cis‐16:1(n‐7) and significant reductions in the saturated phospholipid fatty acids 16:0, 18:0 and the unsaturated 16:1(n‐9) and 18:2(n‐6) fatty acids. These changes cause a slight decrease in the average length of the phospholipid fatty acids and an increase in the overall ratio of unsaturated vs. saturated fatty acids. These findings demonstrate that the rate of cooling is important for both the reorganization of membrane lipids, and for the degree of acquired cold tolerance during rapid cold hardening, and they suggest an important role for rapid cold hardening during diurnal rather than seasonal temperature changes.