published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

The effects of oxygen, malonate, and acetate on the conversion of radioactively-labeled Coenzyme A thioesters of lauric and stearic acids to unsaturated fatty acids by 15,000 x g supernates of Penicilliumlehrysogenum were studied. Following the termination of reactions, the incubation products were saponified, acidified, extracted, and methylated. The resulting methyl esters were purified, separated, identified, and collected. Radioactivities of each fatty acid fraction were determined and converted to percentage of total radioactivity recovered.Desaturase activity was virtually non-existent in the anaerobic incubations indicating that this organism utilized the aerobic pathway of desaturation. The absence of acetate and malonate from incubation mixtures of either laurate or stearate markedly decreased the overall desaturase activity and the amount of linoleate produced. The failure of labeled laurate to be converted to long-chain unsaturated fatty acids in the absence of malonate indicates that chainelongation in this organism proceeds by the malonate pathway. The data presented suggest that laurate may be directly desaturated and then elongated to oleate instead of following the typical aerobic pathway. / Department of Biology

The elongation and desaturation of fatty acids was investigated by studying the fate of 1-14C lauric, myristic, and stearic acids added to submerged cultures of Aspergillus niger. The mycelium produced oleic and linoleio acids from 1-14C lauric and 1-14C steario acids and to only a slight extent from 1-14C myristic acid. Stearic acid was the principal labeled saturated fatty acid produced when laurio acid was the substrate; both palmitic and steario acids were produced in reduced amounts from myristic acid. Myristic acid has been reported to be a poor precursor for long chain fatty acids in Penicillium chrysogenum and Torulopsos bombicoli well. The absence of label in fatty acids shorter than the added substrates indicated that oxidation followed by do novo synthesis did not occur. Pariodate-permanganate oxidation data verified that do novo synthesis did not occur.When either 1-14C lauric acid or 1-14C myristic acid was the substrate, Schmidt decarboxylation data of the saturated fatty acids longer that the substrate indicated that the terminal acetate unit of the substrate was removed and used to elongate palmitio acid to steario acid. The rapid incorporation. of label into long chain fatty acids supports this conclusion. When 1- 4C steario acid was the substrate, decarboxylation data of the saturated fatty acids longer than stearicindicated that the terminal acetate unit of the substrate was removed and used to produce fatty acids with chain lengths longer than stearic acid.Ball State UniversityMuncie, IN 47306

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published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Particulate and solubilized preparations of phosphatidate (PA) phosphatase (EC 3.1.3.4) and dia-cylglycerol acyltransferase (DGAT, EC 2.3.1.20) from microspore-derived (MD) cultures of Brassica napus L. cv Topas were characterized. The activity of solubilized PA phosphatase decreased by about 50% following storage for 24 h at 4 degrees celsius, whereas the activity of DGAT decreased by 30%. Bovine serum albumin increased the stability of both enzymes. Both preparations were enriched in the target enzyme and thus, may be useful in studies of regulation with limited influence by the other Kennedy pathway enzymes. Solubilized PA phosphatase was shown to dephosphoryolate a number of phosphate-containing compounds and showed a preference for dioleoyl-PA and dipalmitoyl-PA over other forms of PA tested. Microsomal PA phosphatase from MD embryos was partially dependent on Mg2+ and partially inhibited by the thioreactive agent, N-ethylmaleimide (NEM). The partial sensitivity to NEM suggest that MD embryos of B. napus may contain forms of PA phosphatase involved in glycerolipid synthesis and signal transduction. NEM-sensitive and NEM-insensitive PA phosphatase activity was found in microsomes of a cell suspension culture of B. napus L. cv Jet Neuf. PA phosphatase, solubilized from MD embryos, was partially purified using ammonium sulfate fractionation followed by anion exchange chromatography. PA phosphatase was resolved into two distinct peaks following anion-exchange chromatography. The peaks contained both NEM-sensitive and NEM-insensitive PA phosphatase activity. Following gel filtration, solubilized PA phosphatase displayed a minimum apparent Mr of about 40 000. Antibodies raised against partially purified preparations of PA phosphatase and DGAT from MD embryos of B. napus L. cv Topas were used in the development of immunochemical probes for these enzymes. Inhibitory anti-PA phosphatase antibodies were developed. Attempts were also made to identify a sub-class of antibodies which could interact with both denatured and native DGAT. / xviii, 137 leaves : ill. ; 28 cm.

Streptomyces coelicolor produces fatty acids for both primary metabolism and for production of the components of natural products such as undecylprodiginine. Primary metabolism makes the longer and predominantly branched-chain fatty acids, while undecylprodiginine utilizes shorter and almost exclusively straight chain fatty acids. The first step in fatty acid biosynthetic process is catalyzed by FabH (β-ketoacyl synthase III), which catalyzes a decarboxylative condensation of an acyl-CoA primer with malonyl-acyl carrier protein (ACP). The resulting 3-ketoacyl-ACP product is reduced by NADPH-dependent FabG into 3-hydroxyacyl-ACP, which is dehydrated by FabA to form enoyl-ACP. The NADH-dependent FabI (InhA) completes the cycle. Subsequent rounds of elongations in the pathways are catalyzed by the condensing enzyme FabF. For undecylprodiginine biosynthesis in S. coelicolor, homologues of the condensing enzymes (FabH and FabF) and the ACP (FabC) are encoded by redP, redR and redQ respectively in the red gene cluster. The genes encoding 3-ketoacyl-ACP reductase (FabG), 3-hydroxyacyl-ACP dehydratase (FabA), and enoyl-ACP reductase (FabI), are putatively shared between fatty acid and undecylprodigine biosynthesis, since the corresponding genes are not present within the red gene cluster of S. coelicolor. RedP is proposed to initiate biosynthesis of undecylprodiginine alkane chain by condensing an acetyl-CoA with a malonyl-RedQ, in contrast to FabH which process a broad range of acyl-CoA with malonyl-FabC. The 3-keto group of the resulting 3-ketoacyl-RedQ is then reduced to provide butyryl-RedQ, presumably by the type II FAS enzymes FabG, FabA and FabI. These enzymes would not differentiate between straight and branched-chain substrates, and have equal preference for FabC and RedQ ACPs. RedR would then catalyze four subsequent elongation steps with malonyl-RedQ, with appropriate 3-keto group processing after each step. The proposed role and substrate specificities of condensing enzymes RedP and FabH have not been investigated in S. coelicolor. The genes encoding FabG, FabA, and FabI have not been characterized in Streptomyces. Analysis of the S. coelicolor genome sequence has revealed the presence of one fabI gene (SCO1814, encoding an enoyl-ACP reductase), and three likely fabG genes (SCO1815, SCO1345, and SCO1346, encoding β-ketoacyl-ACP reductase). In the current study the substrates specificities of both RedP and FabH were determined from assays using pairings of two acyl-CoA substrates (acetyl-CoA and isobutyryl-CoA) and two malonyl-ACP substrates (malonyl-RedQ and malonyl-FabC) (FabC is a dedicated ACP for fatty acid biosynthesis and RedQ for undecylprodiginine biosynthesis in S. coelicolor). For RedP, activity was only observed with a pairing of acetyl-CoA and malonyl-RedQ. No activity was observed with isobutyryl-CoA consistent with the proposed role for RedP and the observation that acetyl CoA-derived prodiginines predominate in S. coelicolor. Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. In contrast to RedP, FabH was active with all pairings but demonstrated the greatest catalytic efficiency with isobutyryl-CoA using malonyl-FabC. Lower catalytic efficiency was observed using an acetyl-CoA and malonyl-FabC pairing consistent with the observation that in streptomycetes, a broad mixture of fatty acids are biosynthesized, with those derived from branched chain acyl-CoA starter units predominating. Diminished but demonstrable FabH activity was also observed using malonyl-RedQ, with the same preference for isobutyryl-CoA over acetyl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can also play a role in prodiginine biosynthesis, producing branched alkyl chain prodiginines. The identification and characterization of both enzymes FabG and FabI was also carried out. A series of straight and branched-chain β-ketoacyl and enoyl substrates tethered to either NAC or ACP were synthesized and used to elucidate the functional role and substrate specificity of these enzymes. Kinetic analysis demonstrates that of the three S. coelicolor enzymes, SCO1815 and SCO1345 have NADPH-dependent β-ketoacyl-reductase activity, in contrast to SCO1346, which has NADH-dependent β-ketoacyl-reductase activity. Spectrophotometric assays revealed that all three FabGs are capable of utilizing both straight and branched-chain β-ketoacyl-NAC substrates. These results are consistent with FabGs role in fatty acid and undecylprodiginine biosynthesis, wherein it processes branched-chain for primary metabolism as well as straight-chain products for undecylprodiginine biosynthesis. LC/MS assays demonstrate that these FabG enzymes do not discriminate between primary metabolism ACP (FabC) and secondary metabolism ACP (RedQ) (except for SCO1345, which does not have any activity with RedQ). This relaxed substrate specificity allows these enzymes to process 3-ketoacyl-FabC substrates for fatty acid biosynthesis as well as 3-ketoacyl-RedQ substrates for undecylprodiginine biosynthesis. Similar to FabG, spectrophotometric and LC/MS assays were also carried out to elucidate the functional role and substrate specificity of S. coelicolor FabI. The kinetic analyses demonstrate that SCO1814 has NADH-dependent enoyl-ACP reductase activity. Spectrophotometric and LC/MS assays demonstrated that FabI does not differentiate between straight and branched-chain substrates, and has equal preference for FabC and RedQ ACPs. These observations provide experimental support for the hypothesis that these enzymes are shared and process the intermediates in the elongation cycle of both fatty acid and undecylprodiginine biosynthesis. In summary, these studies have demonstrated the activity of enzymes RedP, FabH, FabG and FabI (InhA) previously uncharacterized in S. coelicolor and clarified their role in fatty acid and undecylprodiginine biosynthesis.

No description available.

Call number: LD2668 .T4 1986 T39 / Master of Science / Animal Sciences and Industry

Kam, Shuk Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 124-134). / Abstracts in English and Chinese. / Abstract --- p.ii / Abstract in Chinese --- p.iv / Acknowledgements --- p.vi / List of Figures --- p.xi / List of Tables --- p.xii / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Soybeans as Food --- p.1 / Chapter 1.1.1 --- Backgrounds --- p.1 / Chapter 1.1.2 --- Soybean Composition --- p.1 / Chapter 1.1.3 --- Diseases Prevention of Soybean Consumption --- p.3 / Chapter 1.1.4 --- Traditional Soyfoods --- p.3 / Chapter 1.2 --- Sufu --- p.5 / Chapter 1.2.1 --- Historical Information and Synonyms --- p.5 / Chapter 1.2.2 --- Features --- p.5 / Chapter 1.2.3 --- Manufacturing Techniques --- p.5 / Chapter 1.2.4 --- Types and Varieties of Sufu --- p.10 / Chapter 1.2.5 --- Compositional Changes during Fermentation and Ripening --- p.11 / Chapter 1.2.5.1 --- Proteins and Amino Acids --- p.11 / Chapter 1.2.5.2 --- Fats and Free Fatty Acids --- p.13 / Chapter 1.2.5.3 --- Carbohydrates --- p.14 / Chapter 1.2.5.4 --- Isoflavones --- p.15 / Chapter 1.2.6 --- Volatile Flavor Compounds --- p.15 / Chapter 1.3 --- Accelerated-Ripened Sufu --- p.17 / Chapter 1.4 --- Objectives of Project --- p.18 / Chapter Chapter 2 --- Contribution of Lipid to the Fatty Acids and Ethyl Esters in Model Plain Sufu --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and Methodology --- p.23 / Chapter 2.2.1 --- Sufu Preparation --- p.23 / Chapter 2.2.1.1 --- Preparation of Tofu --- p.23 / Chapter 2.2.1.2 --- Preparation of Inoculum --- p.23 / Chapter 2.2.1.3 --- Spore Count in Spore Suspension --- p.24 / Chapter 2.2.1.4 --- Preparation of Pehtzes --- p.25 / Chapter 2.2.1.5 --- Brining and Ripening --- p.26 / Chapter 2.2.1.6 --- Sampling --- p.26 / Chapter 2.2.1.7 --- Free Fatty Acid Analysis --- p.26 / Chapter 2.2.1.7.1 --- Extraction --- p.26 / Chapter 2.2.1.7.2 --- Gas Chromatography-Mass Spectrometry Analysis (GC-MS) for Free Fatty Acid Analysis --- p.27 / Chapter 2.2.1.7.3 --- Compounds Identification and Quantification --- p.28 / Chapter 2.2.1.8 --- Ethyl Ester Analysis --- p.29 / Chapter 2.2.1.8.1 --- Extraction --- p.29 / Chapter 2.2.1.8.2 --- Gas Chromatography-Mass Spectrometry Analysis (GC-MS) for Ethyl Ester Analysis --- p.29 / Chapter 2.2.1.8.3 --- Compounds Identification and Quantification --- p.30 / Chapter 2.2.1.9 --- Enzymatic Activities --- p.30 / Chapter 2.2.1.9.1 --- Enzyme Extracts --- p.30 / Chapter 2.2.1.9.2 --- Lipase Activity Measurement --- p.31 / Chapter 2.2.1.9.3 --- Lipoxygenase Activity Measurement --- p.32 / Chapter 2.2.1.10 --- Determination of Peroxide Value --- p.33 / Chapter 2.2.1.11 --- pH Value Determination --- p.34 / Chapter 2.2.1.12 --- Moisture Content --- p.34 / Chapter 2.2.1.13 --- Statistical Analysis --- p.34 / Chapter 2.3 --- Results and Discussions --- p.35 / Chapter 2.3.1 --- Change of Free Fatty Acids with Sufu Processing Stage --- p.35 / Chapter 2.3.2 --- Change in Ethyl Esters with Sufu Processing Stage --- p.41 / Chapter 2.3.3 --- Activity of Lipase in the Sufu Enzyme Extracts --- p.47 / Chapter 2.3.4 --- Activity of Lipoxygenase in the Sufu Enzyme Extracts --- p.50 / Chapter 2.3.5 --- Lipid Oxidation determined by Peroxide Value --- p.50 / Chapter 2.3.6 --- pH Value Change during Sufu Production --- p.54 / Chapter 2.3.7 --- Moisture Content during Sufu Production --- p.56 / Chapter 2.3.8 --- Overall Discussions --- p.58 / Chapter 2.3.8.1 --- Lipolysis and Ester Synthesis --- p.58 / Chapter 2.3.8.2 --- Lipid Oxidation --- p.58 / Chapter 2.4 --- Conclusion --- p.61 / Chapter Chapter 3 --- A Study on Ripening Model Systems of Sufu --- p.63 / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Materials and Methodology --- p.68 / Chapter 3.2.1 --- Partial Purification Lipase from Mucor hiemalis --- p.68 / Chapter 3.2.1.1 --- Inoculum --- p.68 / Chapter 3.2.1.2 --- Culture --- p.68 / Chapter 3.2.1.3 --- Protein Precipitation --- p.68 / Chapter 3.2.1.4 --- Gel Filtration Column Chromatography --- p.69 / Chapter 3.2.1.5 --- Enzyme Assay --- p.69 / Chapter 3.2.1.6 --- Lipase Activity Confirmation --- p.70 / Chapter 3.2.1.7 --- Protein Determination --- p.70 / Chapter 3.2.2 --- Model Studies of the Formation of Free Fatty Acids and Ethyl Esters --- p.70 / Chapter 3.2.2.1 --- "A System with Lipid, Alcohol, and Lipase" --- p.70 / Chapter 3.2.2.2 --- A System with Different Lipase Concentrations --- p.71 / Chapter 3.2.2.3 --- A System with an Exogenous Fatty Acid --- p.71 / Chapter 3.2.3 --- Characterization of the Crude Lipase from Mucor hiemalis Culture on the Formation of Free Fatty Acids and their Ethyl Esters --- p.72 / Chapter 3.2.3.1 --- Effect of a Phospholipid --- p.72 / Chapter 3.2.3.2 --- Effect of Ethanol Concentration --- p.72 / Chapter 3.2.3.3 --- Effect of Sodium Chloride Concentration --- p.72 / Chapter 3.2.3.4 --- Effect of initial pH --- p.73 / Chapter 3.2.4 --- Orthogonal Design Experiment L9 (33) --- p.73 / Chapter 3.2.5 --- Free Fatty Acids Identification and Quantification --- p.76 / Chapter 3.2.5.1 --- Extraction --- p.76 / Chapter 3.2.5.2 --- Gas Chromatography-Mass Spectrometry Analysis (GC-MS) --- p.76 / Chapter 3.2.5.3 --- Compounds Identification and Quantification --- p.77 / Chapter 3.2.6 --- Ethyl Esters Identification and Quantification --- p.77 / Chapter 3.2.6.1 --- Extraction --- p.77 / Chapter 3.2.6.2 --- Gas Chromatography-Mass Spectrometry Analysis (GC-MS) --- p.78 / Chapter 3.2.6.3 --- Compounds Identification and Quantification --- p.78 / Chapter 3.2.7 --- Statistical Analysis --- p.79 / Chapter 3.3 --- Results and Discussions --- p.80 / Chapter 3.3.1 --- Lipase Partial Purification --- p.80 / Chapter 3.3.2 --- Lipase Activity Confirmation --- p.80 / Chapter 3.3.3 --- Model Studies on the Formation of Free Fatty Acids and Ethyl Esters --- p.84 / Chapter 3.3.3.1 --- "A System with Lipid, Alcohol and Lipase" --- p.84 / Chapter 3.3.3.2 --- A System with Different Lipase Concentrations --- p.84 / Chapter 3.3.3.3 --- A System with an Exogenous Fatty Acid --- p.89 / Chapter 3.3.3.4 --- Summary --- p.92 / Chapter 3.3.4 --- Characterization of the Crude Lipase from Mucor hiemalis Culture on the Formation of Free Fatty Acids and their Ethyl Esters Formation --- p.92 / Chapter 3.3.4.1 --- Effect of a Phospholipid --- p.92 / Chapter 3.3.4.2 --- Effect of Ethanol Concentration --- p.96 / Chapter 3.3.4.3 --- Effect of Sodium Chloride Concentration --- p.103 / Chapter 3.3.4.4 --- Effect of initial pH --- p.109 / Chapter 3.3.5 --- Orthogonal Design Experiment L9 (33) Optimizing the Ethyl Esters Formation --- p.114 / Chapter 3.4 --- Conclusion --- p.118 / Chapter 4 Overall Conclusions --- p.120 / References --- p.124