Solution NMR studies of acetohydroxy acid synthase I: Identification of the sites of inter-subunit interactions using multidimensional NMR methods

The novel multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been dissected to generate polypeptide fragments. These fragments when cloned, expressed and purified reassemble in the presence of cofactors to yield a catalytically competent enzyme. Structural characterization of AHAS has been impeded due to the fact that the holoenzyme is prone to dissociation leading to heterogeneity in samples. Our approach has enabled the structural characterization using high-resolution nuclear magnetic resonance methods. Near complete sequence specific NMR assignments for backbone H-N, N-15, C-13 alpha and C-13(beta) atoms of the FAD binding domain of ilvB have been obtained on samples isotopically enriched in H-2, C-13 and N-15. The secondary structure determined on the basis of observed C-13(alpha) secondary chemical shifts and sequential NOEs indicates that the secondary structure of the FAD binding domain of E. coli AHAS large Subunit (ilvB) is similar to the structure of this domain in the catalytic subunit of yeast AHAS. Protein-protein interactions involving the regulatory subunit (ilvN) and the domains of the catalytic subunit (ilvB) were studied using circular dichroic and isotope edited solution nuclear magnetic resonance spectroscopic methods. Observed changes in circular dichroic spectra indicate that the regulatory subunit (ilvN) interacts with ilvB alpha and ilvB beta domains of the catalytic subunit and not with the ilvB gamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvB beta and proximal to the intrasubunit ilvB alpha/ilvB beta domain interface. The implication of this interaction on the role of the regulatory subunit oil the activity of the holoenzyme is discussed. NMR studies of the regulatory domains show that these domains are structured in solution. Preliminary evidence for the interaction of ilvN with the metabolic end product of the pathway, viz., valine is also presented.

Fatty acid component of vitamin A ester in sheep liver

Vitamin A, when extracted along with other lipids from sheep liver, had an E1cm.1% value of 14.4, which was raised to 45.57 on removal of the phospholipids by cold acetone. Selective hydrolysis of triglycerides by an extract of acetone-dried sheep pancreas in the presence of HgCl2 as inhibitor of vitamin A esterase, followed by chromatography through alumina gave a product with E1cm.1% value of 276. This on chromatography through magnesium oxide raised the E1cm.1, value to 601.5, representing 64% pure vitamin A ester calculated as palmitate, and the total recovery was 23% of the starting oil. The purified ester preparation, when subjected to reverse-phase chromatography on silicone-impregnated paper, gave a single ultraviolet fluorescent band. The fluorescent band on hydrolysis gave only one fatty acid. This was conclusively identified to be palmitic acid.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea)

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C-4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-C-14]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparision of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

GC-MS study of Artabotrys odoratissimus fatty oil (leaves)

GC-MS study of two fatty oil fractions from Artabotrys odoratissimus (leaves) indicated the presence of fifteen compounds namely, nonanoic acid; methyl phenyl propanoate; decanoic acid; diethyl phthalate; dibutyl phthalate; 2 - amino-3-ethyl biphenyl; 5-methyl-9-phenylnonan-3-ol; hexadeca-2,7,11-triene; 2,6-dimethyl-1-phenylhepta-1-one; 2,5-dimethyltetradecahydrophenenthrene; 1-phenylundecane; 1-isopropyl-4,6-dimethyl naphthalene; 5-(2-butyl phenyl)pent-3-en-2-ol; 1-phenyideca-1-one and 1-phenylundecan-1-one. Some of the compounds are rare occurring and biologically active.

Copolyesters from Soybean Oil for Use as Resorbable Biomaterials

A family of soybean oil (SO) based biodegradable cross-linked copolyesters sourced from renewable resources was developed for use as resorbable biomaterials. The polyesters were prepared by a melt condensation of epoxidized soybean oil polyol and sebacic acid with citric acid (CA) as a cross-linker. D-Mannitol (M) was added as an additional reactant to improve mechanical properties. Differential scanning calorimetry revealed that the polyester synthesized using only CA as the cross-linker was semicrystalline and elastomeric at physiological temperature. The polymers were hydrophobic in nature. The water wettability, elongation at break and the degradation rate of the polyesters decreased with increase in M content or curing time. Modeling of release kinetics of dyes showed a diffusion controlled mechanism underlies the observed sustained release from these polymers. The polyesters supported attachment and proliferation of human stem cells and were thus cytocompatible. Porous scaffolds induced osteogenic differentiation of the stern cells suggesting that these polymers are well suited for bone tissue engineering. Thus, this family of polyesters offers a low cost and green alternative as biocompatible, bioresobable polymers for potential use as resorbable biomaterials for tissue engineering and controlled release.

Changes in body composition and fatty acid profile during embryogenesis of quillback rockfish (Sebastes maliger)

We investigated developmental changes in the body compositions and fatty acid (FA) profiles of embryos and preparturition larvae of the quillback rockfish (Sebastes maliger). Comparisons of proximate composition data from early-stage embryos with data from hatched preparturition larvae taken from wild-caught gravid females indicated that embryos gain over one-third their weight in moisture while consuming 20% of their dry tissue mass for energy as they develop into larvae. Lipid contributed 60% of the energy consumed and was depleted more rapidly than protein, indicating a protein-sparing effect. Oil globule volume was strongly correlated with lipid levels, affirming its utility as an indicator of energetic status. FA profiles of early embryos differed significantly from those of hatched larvae. Differences in the relative abundances of FAs between early embryos and hatched larvae indicated different FA depletion rates during embryonic development. We conclude that some metabolically important FAs may prove useful in assessing the condition of embryos and preparturition larvae, particularly 20:4n-6, which cannot be synthesized by many marine fish and which is conserved during embryogenesis. Variability in body composition and energy use among rockfish species should be considered when interpreting any measures of condition.

Lipid breakdown in oil sardine (Sardinella longiceps) during frozen storage

The problem of hydrolysis of lipids and consequent accumulation of free fatty acids and development of rancidity due to oxidation of the lipids are major problems in frozen storage of oil sardine (Sardinella longiceps). The course of the phospholipid breakdown, production of free fatty acids and the changes taking place in the major unsaturated fatty acids during frozen storage are described in this paper. The rate of free fatty acid production is faster in the fish, with the higher fat content. Unlike in lean fish, the neutral lipids are found to contribute substantially to the free fatty acid production. The fatty acids most affected during storage are C sub(20:5) and C sub(22:6). The polyene indices were found to decrease during storage. These effects are more pronounced in the fish with the higher fat content.

Freezing characteristics of tropical fishes 1. Indian oil sardine

The influence of different pre-freezing ice storage periods on the biochemical and organoleptic qualities of Indian oil sardines (Sardinella longiceps) in the individual quick frozen (IQF) and block frozen (BF) forms and frozen storage at temperatures of -12°C and -23°C was studied. The shelf-life of the sardines varied between 24 and 2 weeks for samples iced for 0 to 5 days prior to freezing. The deterioration in quality was accompanied by considerable increase in the peroxide value (PV) and free fatty acid (FFA) content and decrease in salt extractability of the proteins. These changes were more rapid at -12°C than at -23°C. BF sardines appeared to be better than IQF samples with respect to the biochemical changes although the differences in overall organoleptic quality were not significant.

Canning of oil sardine (Sardinella longiceps): natural pack

A simple and economic process for canning of oil sardine (Sardinella longiceps) in its own juice having very good organoleptic characteristics has been developed. The process consists in dipping eviscerated, scaled and cleaned fish in brine containing potash alum and citric acid, packing in cans, exhausting and seaming without addition of any filling medium and heat processing.

Determination of proximate composition, shelf life and fatty acid profiles of breaded kilka (Clupeonella cultriventris) during frozen storage

The present study aimed production of a new product with various texture and sensory properties in chase of the impetus for increasing human consumption considering suitable resources of Kilka fish in Caspian Sea. Following deheading, gutting, and brining, common Kilka were battered in two different formulations, i.e. simple batter and tempura batter, via automated predusting machinery and then, they were fried through flash frying for 30 seconds at 170°C in sunflower oil after they were breaded with bread crumbs flour. The products were subjected to continuous freezing at -40°C and were kept at -18°C in cold storage for four months once they were packed. Chemical composition (protein, fat, moisture, and ash), fatty acid profiles (29 fatty acids), chemical indices of spoilage (peroxide value, thiobarbituric acid, free fatty acids, and volatile nitrogen), and microbial properties (total bacteria count and coliform count) were compared in fresh and breaded Kilka at various times before frying (raw breaded Kilka), after frying (zero-phase), and in various months of frozen storage (phases 1, 2, 3, and 4). Organoleptic properties of breaded Kilka (i.e. odor, taste, texture, crispiness, cohesiveness of batter) and general acceptability in the phases 0, 1, 2, 3, and 4 were evaluated. The results obtained from chemical composition and fatty acid profiles in common Kilka denoted that MUFA, PUFA, and SFA were estimated to be 36.96, 32.85, and 29.12 g / 100g lipid, respectively. Levels of ù-3 and ù-6 were 7.6 and 1.12 g / 100 gr lipid, respectively. Docosahexaonoic acid (20.79%) was the highest fatty acid in PUFA group. ù-3/ù-6 and PUFA/SFA ratios were 7.6 and 1.12, respectively. The high rates of the indices and high percentage of ù-3 fatty acid in common Kilka showed that the fish can be considered as invaluable nutritional and fishery resources and commonsensical consumption of the species may reduce the risk of cardiovascular diseases. Frying breaded Kilka affected overall fat and moisture contents so that moisture content in fried breaded Kilka decreased significantly compared to raw breaded Kilka, while it was absolutely reverse for fat content. Overall fat content in tempura batter treatment was significantly lower than that of simple batter treatment (P≤0.05). Presence of hydrocolloids, namely proteins, starch, gum, and other polysaccharides, in tempura batter may prohibit moisture evaporation and placement with oil during frying process in addition to boosting water holding capacity through confining water molecules. During frying process, fatty acids composition of breaded Kilka with various batters changed so that rates of some fatty acids such as Palmitic acid (C16:0), Stearic acid (C18:0), Oleic acid (C18:1 ù-9cis), and linoleic acid (C18:3 ù-3) increased considerably following frying; however, ù-3/ù-6, PUFA/SFA, and EPA+DHA/C16:0 ratios (Polyan index) decreased significantly after frying. ù-3/ù-6, PUFA/SFA, and EPA+DHA/C16:0 ratios in tempura batter treatment were higher than those of simple batter treatment which is an indicator of higher nutritional value of breaded Kilka with tempura batter. Significant elevations were found in peroxide, thiobarbituric acid, and free fatty acids in fried breaded Kilka samples compared to raw samples which points to fat oxidation during cooking process. Overall microorganism count and coliform count decreased following heating process. Both breaded Kilka samples were of high sanitation quality at zero-phase according to ICMSF Standard. The results acquired from organoleptic evaluation declared that odor, cohesiveness, and general acceptability indices, among others, had significant differences between the treatments (P≤0.05). In all evaluated properties, breaded Kilka with tempura batter in different phases gained higher scores than breaded Kilka with simple batter. During cold storage of various treatments of breaded Kilka, total lipid content, PUFA, MUFA, ù-3, ù- 3/ù-6, PUFA/SFA, Polyen index decreased significantly. The mentioned reductions in addition to significant elevation of spoilage indices, namely peroxide, thiobarbituric acid, and free fatty acids, during frozen storage, indicate to oxidation and enzymatic mechanism activity during frozen storage of breaded Kilka. Considering sensory evaluation at the end of the fourth month and TVB-N contents exceeded eligible rate in the fourth month, shelf life of the products during frozen storage was set to be three months at -18°C. The results obtained from statistical tests indicate to better quality of breaded Kilka processed with tempura batter compared to simple batter in terms of organoleptic evaluation, spoilage indices, and high quality of fat in various sampling phases.

Supply of Astaxanthin and its combinations through live feed (Moina micrura) enrichment affects the growth, survival and fatty acid profile of Macrobrachium rosenbergii larvae

A study was carried out with three replicates to determine the effects of feeding Moina micrura enriched with astaxanthin alone (M1) or astaxanthin in combination with either vitamin E (M2), vitamin D (M3) or Cod Liver oil (M4) on the growth, survival and fatty acid composition of giant fresh water prawn Macrobrachium rosenbergii (de Man) larvae. Growth rate was expressed as the time taken to the settlement of 95% post larvae. Maximum growth, the lowest time taken to the 95% PL settlement (38.5±0.50 days), was observed in larvae fed with M3 Moina. The highest survival rate (66.0±1.00%) was observed in those fed with M4 Moina and the second highest survival (61.0±1.00%) and growth rates (40.0±0.00 days) were shown with M2 Moina. The minimum values for both growth (42.5±0.50 days) and survival (33.0±1.50%) were observed in the group fed un-enriched Moina. Results also showed that the survival of prawn larvae increased as the quantities of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased in the dietary Moina. The highest levels of EPA (5.57±0.21%), DHA (3.50±0.21%) and highest total Highly Unsaturated Fatty Acids (HUFA) (13.87±0.68%) were seen in the Moina fed on astaxanthin and Cod Liver Oil (CLO). The results of the study showed that the nutritive quality of Moina, with respect to important fatty acids, can be increased by enrichment and will influence the growth, survival and the fatty acid composition of fresh water prawn larvae fed on them.

The study of Indian shrimp larvae feeding by enriched rotifer with vitamin C and unsaturated fatty acid (epa, dha) and its effects on growth factors, survival rate and environmental stresses

In this experiment, the feeding of Indian white shrimp larvae by unenriched rotifers (treatment 1) and enriched with highly unsaturated fatty acid (treatment 2) and highly unsaturated fatty acid along with vitamin C (treatment 3) on the growth factors, survival and resistance against salinity and formalin stress tests were studied and their differences with control treatment including newly hatched Artemia nauplii is compared. In this the study four treatments in a completely randomized design with 3 replicates per treatment were used. Farming of shrimp larvae of Zoea II to postlarvae 5 was done in 20 liter plastic bucket. Present results indicated that growth factors and survival rate of stage Zoea II to postlarvae 1 in treatments 1, 2 and 3 improve rather than control in which this case was due to optimal size rotifer rather than Artemia nauplii. Also, treatments 2 and 3 feeding with oil liver cod emulsion enriched rotifer have the highest concentration of DHA (mg/g DW) and the ratio DHA/EPA in which due to have shown the highest growth factors and a significant difference (P<0.05) with treatments 1 and control. The highest survival at stage PL1 were observed in treatment 3 that was enriched with ascorbyl palmitate in which have to the synergistic properties of vitamin C rather than treatments 2, 1 and control and showed a significant difference (P<0.05). But in stage PL5 the highest amount of growth and survival rates were related to control treatment which showed a significant difference (P<0.05) with other treatments that control has higher size rather than treatments 1, 2 and 3. Also, among experiment treatments that the two treatments 2 and 3 due to enrichment had higher growth and survival rates compared with treatment 1 in which their differences have also been significant (P<0.05). In the case of stress tests, results indicated that the highest survival rate has been reported when specimens were offered a diet containing high levels of highly unsaturated fatty acids with vitamin C. So that in stage PL1 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3. As for the second, treatment 2 showed a significant difference (P<0.05) with treatment 3. It is worth mentioning that treatment 3 showed a higher survival rate compared to treatment 2 due to the synergistic properties of vitamin C. The difference between these two treatments with treatment 1 and control was also significant. No significant difference was observed in formalin stress test 100 ppm in this stage between treatments 3 and 2 which shows the highest survival rate. But their difference with treatments 1 and control was significant (P<0.05). Also, in stage PL5 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3 which showed no significant difference (P<0.05) with control treatment. While their difference in the amount of survival rate with treatment 1 and 2 was significant (P<0.05). In this stage, the highest observed survival rate in formalin stress test 100 ppm included treatments control, 3 and 2 among which there were no significant differences (P<0.05). While the difference between these three treatments with treatment 1 was significant.

Effect of dietary linolenic acid/linoleic acid ratio on growth performance, hepatic fatty acid profiles and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco

The present study aimed to evaluate the effect of dietary linolenic acid (LNA)linoleic acid (LA) ratio on growth performance, hepatic fatty acid profile and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco. Six isonitrogenous and isolipidic diets were formulated to contain incremental levels of LNA from 0 to 5% at the expense of corn oil (rich in LA), resulting in six dietary treatments with LNA to LA ratios ranging from 0.35 to 14.64. The experiment continued for 7 weeks. Best growth and feed intake were obtained in the fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). In contrast, feed conversion ratio was the lowest for fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). Dietary LNA to LA ratios significantly influenced viscerosomatic index and hepatosomatic index (P<0.05), but not condition factor (P>0.05). Body composition was also significantly influenced by dietary LNA to LA ratios (P<0.05). Generally, liver FA compositions reflected dietary FA profiles. Declining LA and increasing LNA contents in liver were observed with the increasing dietary LNA/LA ratios (P<0.05). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased with the increasing LNA to LA ratios, suggesting that yellow catfish could elongate and desaturate C18 polyunsaturated fatty acids into highly unsaturated fatty acids. As a consequence, the n-6 fatty acids (FA) declined, and total n-3 FA and n-3/n-6 ratios increased with the dietary ratios of LNA/LA (P<0.05). Dietary LNA to LA ratios significantly influenced several enzymatic activities involved in liver intermediary metabolism (P<0.05), such as lipoprotein lipase, hepatic lipase, pyruvate kinase, succinate dehydrogenase, malic dehydrogenase and lactate dehydrogenase, suggesting that dietary LNA/LA ratios had significant effects on nutrient metabolism in the liver. To our knowledge this is the first demonstration of the effects of dietary LNA to LA ratios on the enzymatic activities of liver in fish, which provides information on diet quality and utilization, and can also be used as an indicator of the nutritional status of this fish. (C) 2009 Elsevier B.V. All rights reserved.

Determination of phthalate acid esters plasticizers in plastic by ultrasonic solvent extraction combined with solid-phase microextraction using calix[4]arene fiber

Ultrasonic solvent extraction combined with solid-phase microextraction (SPME) with calix[4]arene/hydroxy-terminated silicone (C[4]/OHTSO) oil coated fiber was used to extract phthalate acid esters (PAEs) plasticizers in plastic, such as blood bags, transfusion tubing, food packaging bag, and mineral water bottle for analysis by gas chromatography (GC). Both extraction parameters (i.e. extraction time, extraction temperature, ionic strength) and conditions of the thermal desorption in a GC injector were optimized by analysis of eight phthalates. The fiber shows wonderful sensitivity and selectivity to the tested compounds. Owing to its high thermal stability (380 degreesC), the carryover effect that often encountered when using conventional fibers can be reduced by appropriately enhancing the injector temperature. The method showed linear response over two to four orders of magnitude with correlation coefficients (r) better than 0.996, and limits of detection (LOD) ranged between 0.006 and 0.084 mug l(-1). The relative standard deviation values obtained were less than or equal to 10%. bis-2-Ethylhexyl phthalate (DEHP) was the sole analyte detected in these plastics and recoveries were in the ranges 95.5-101.4% in all the samples. (C) 2004 Elsevier B.V. All rights reserved.

MoNi/gamma-Al2O3 Catalyst: Preparation and Catalytic Activity for Acetic Acid Hydrotreating

MoNi/gamma-Al2O3 catalysts were prepared by the impregnation method. The catalyst samples were characterized by XRD and TPR. The effects of Mo promoter content and the catalyst reducing temperature Oil hydrotreatment activity of the catalyst were studied under 200 degrees C and 3 MPa hydrogen pressure using acetic acid as the model compound. The XRD results indicate that the addition of Mo promoter is beneficial to the uniformity of nickel species on the catalyst and decreases the Interaction between nickel species and the support Which results in the decrease the of NiAl2O4 spinel formation. The addition of Mo promoter also decreases the reducing temperature of the catalyst. After the catalyst of 0.06 MoNi/gamma-Al2O3 being reduced Under the atmosphere of H-2/N-2(5/95, V/V), nickel oxide was reduced to Ni-0. The reaction was promoted obviously upon the addition of the MoNi/gamma-Al2O3 catalyst reduced at 600 degrees C. The Mo-modified Ni/gamma-Al2O3 catalyst reduced at 600 degrees C displayed the highest activity during the reaction, the conversion of acetic acid reached the highest point of 33.2%. The products included ethyl acetate and water.