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Rational enhancement of enzyme performance in organic solvents. Final technical report, 1992--1996

Description: This research focused on the following: the dependence of enzymatic activity of several model hydrolases in nonaqueous solvents; control of substrate selectivity of the protease subtilisin Carlsberg by the solvent; control of catalytic activity and enantioselectivity of this enzyme in organic solvents by immobilization support; lipase-catalyzed acylation of sugars in anhydrous hydrophobic media; the possibility of accelerating enzymatic processes in organic solvents by certain cosolvents; whether lipase catalysis in organic solvents can be enhanced by introducing interfaces in the in the reaction medium; the structure of proteins suspended in organic solvents; improving enzymatic enantioselectivity in organic solvents; analyzing the plunge in enzymatic activity upon replacing water with organic solvents; and the structural basis for the phenomenon of molecular memory of imprinted proteins in organic solvents.
Date: December 31, 1996
Creator: Klibanov, A.M.
Partner: UNT Libraries Government Documents Department

Immobilized enzymes in organic media: Determinants of water dependence. Interim report

Description: The overall goals of our DOE-funded project are to investigate the critical factors that limit commercial scale applications of enzymes in organic solvents, and to scale-up a process for the production of a precursor to optically active polymers. Initially, we are attempting to quantify the effect of intra-particle dynamics on overall process performance. This involves examination of coupled enzyme kinetics, solubility, wetting and mass transfer effects toward support selection, reactor design/scheme and scale-up. For this study we selected a pure enzyme preparation: a lipase from C. Viscosum. The enzyme has been immobilized on celite and the catalyzed interesterification kinetics in petroleum ether and its dependence on water concentration have been determined. The results are very similar to those obtained in the past with crude Pseudomonas Cepacia. So far we have formulated a model for tripalmitin depletion, based on taking mass balances. A parameter has been included which accounts for the effect of water content inside the celite particle. The initial rate of depletion can be described by Monod-like kinetics. Our experimental data, at 0.003 mg H{sub 2}O per cm{sup 2}, fit a model and parameters presented in the literature. Because of the small size of the support, the relatively low amount of water, and the low reaction rates, our calculations predict that the lipase system at 0.003-0.01 mgH{sub 2}O/cm{sup 2} is reaction rate limited. The mass transfer through the organic phase within the support particles is not limiting. In order to test the dense particles with varying diameter but the same total surface area have been tested and compared to the porous celite.
Date: June 1, 1993
Creator: Nandi, S.; DeFilippi, I.; Bedwell, B. & Zemel, H.
Partner: UNT Libraries Government Documents Department

The fatty liver dystrophy (fld) mutation: Developmentally related alterations in hepatic triglyceride metabolism and protein expression

Description: Fatty liver dystrophy (fld) is an autosomal recessive mutation in mice characterized by hypertriglyceridemia and development of a fatty liver in the early neonatal period. Also associated with the fld phenotype is a tissue-specific deficiency in the expression of lipoprotein lipase and hepatic lipase, as well as elevations in hepatic apolipoprotein A-IV and apolipoprotein C-II mRNA levels. Although these lipid abnormalities resolve at the age of weaning, adult mutant mice exhibit a peripheral neuropathy associated with abnormal myelin formation. The fatty liver in fld/fld neonates is characterized by the accumulation of large triglyceride droplets within the parenchymal cells, and these droplets persist within isolated hepatocytes maintained in culture for several days. To identify the metabolic defect that leads to lipid accumulation, the authors investigated several aspects of cellular triglyceride metabolism. The mutant mice exhibited normal activity of acid triacylglycerol lipase, an enzyme thought to be responsible for hydrolysis of dietary triglycerides in the liver. Metabolic labeling studies performed with oleic acid revealed that free fatty acids accumulate in the liver of 3 day old fld/fld mice, but not in adults. This accumulation in liver was mirrored by elevated free fatty acid levels in plasma of fld/fld neonates, with levels highest in very young mice and returning to normal by the age of one month. Quantitation of fatty acid oxidation in cells isolated from fld/fld neonates revealed that oxidation rate is reduced 60% in hepatocytes and 40% in fibroblasts; hepatocytes from adult fld/fld mice exhibited an oxidation rate similar to those from wild-type mice.
Date: July 1, 1997
Creator: Reue, K.; Rehnmark, S.; Cohen, R.D.; Leete, T.H.; Doolittle, M.H.; Giometti, C.S. et al.
Partner: UNT Libraries Government Documents Department

Immobilized enzymes in organic media: Determinants of water dependence. Progress statement

Description: The overall goals of this project are to investigate the critical factors that limit commercial scale applications of enzymes in organic solvents, and to scale-up a process for the production of a precursor to a specialty polymer. The overall performance of an immobilized enzyme can be influenced by its intrinsic structure and by external factors such as water content, support, pH, etc.. We have investigated the interrelation between support morphology and water content, and its effect on overall enzyme performance. Using a lipase catalyzed inter-esterification reaction as a model, we studied the controlling factors when water content in the organic solvent is such that a micro-aqueous phase is formed. In such an environment it was found that support particle aggregation is the major cause for decline in enzyme activity. We have shown that particle porosity, as well as the use of a particular non-woven fabric as an enzyme support, could alleviate this problem. These findings are being translated into a bioreactor design. We have also studied two {open_quotes}dry{close_quotes} non-aqueous systems, where a water phase is not formed since the water content is below its solubility in the organic solvent. In one of the systems, Subtilisin catalyzed trans-esterification of vinyl acrylate with a chiral alcohol, we have demonstrated that the use of a proprietary fabric support provides a significant boost in enzyme activity. We suggest that this particular fabric with its hydrophilic fibers acts as a lyoprotectant in the process of drying the enzyme. The benefits of this material as an enzyme support and its use in a lab scale bioreactor are being studied. Preliminary experiments have also been performed with a second {open_quotes}dry{close_quotes} reaction. This is the lipase catalyzed synthesis of AlliedSignal`s new product, VEctomer 4010.
Date: August 1, 1994
Creator: Nandi, S.; DeFilippi, I.; Bedwell, B. & Zemel, H.
Partner: UNT Libraries Government Documents Department

Raman Spectroscopic Analysis of Biochemical Changes in Individual Triglyceride-Rich Lipoproteins in the Pre- and Postprandial State

Description: Individual triglyceride-rich lipoprotein (TGRL) particles derived from human volunteers are non-destructively analyzed by laser tweezers Raman microspectroscopy and information on their composition and distribution is obtained. The Raman signature of single optically trapped very low-density lipoproteins (VLDL), a subclass of TGRL, which play an important role in cardiovascular disease, exhibits distinct peaks associated with molecular vibrations of fatty acids, proteins, lipids, and structural rearrangements of lipids. Our analysis of pre- and postprandial VLDL exhibits the signature of biochemical changes in individual lipoprotein particles following the consumption of meals. Interaction of VLDL with endothelium leads to the breakdown of complex triacylglycerols and the formation of a highly ordered core of free saturated fatty acids in the particle. A particle distribution analysis reveals trends in the degree to which this process has occurred in particles at different times during the postprandial period. Differences in particle distributions based on the different ratios of polyunsaturated to saturated fats in the consumed meals are also easily discerned. Individual lipoprotein particles hydrolyzed in-vitro through addition of lipoprotein lipase (LpL) exhibit strikingly similar changes in their Raman spectra. These results demonstrate the feasibility of monitoring the dynamics of lipid metabolism of individual TGRL particles as they interact with LpL in the endothelial cell wall using Raman spectroscopy.
Date: September 13, 2004
Creator: Chan, J; Motton, D; Rutledge, J; Keim, N & Huser, T
Partner: UNT Libraries Government Documents Department

Cultured human astrocytes secrete large cholesteryl ester- andtriglyceride-rich lipoproteins along with endothelial lipase

Description: We cultured normal human astrocytes and characterized their secreted lipoproteins. Human astrocytes secreted lipoproteins in the size range of plasma VLDL (Peak 1), LDL (Peak 2), HDL (Peak 3) and a smaller peak (Peak 4), as determined by gel filtration chromatography, nondenaturing gradient gel electrophoresis and transmission electron microscopy. Cholesterol enrichment of astrocytes led to a particular increase in Peak 1. Almost all Peak 2, 3 and 4 cholesterol and most Peak 1 cholesterol was esterified (unlike mouse astrocyte lipoproteins, which exhibited similar peaks but where cholesterol was predominantly non-esterified). Triglycerides were present at about 2/3 the level of cholesterol. LCAT was detected along with two of its activators, apolipoprotein (apo) A-IV and apoC-I. ApoA-I and apoA-II mRNA and protein were absent. ApoJ was present equally in all peaks but apoE was present predominantly in peaks 3 and 4. ApoB was not detected. The electron microscopic appearance of Peak 1 lipoproteins suggested partial lipolysis leading to the detection of a heparin-releasable triglyceride lipase consistent with endothelial lipase. The increased neuronal delivery of lipids from large lipoprotein particles, for which apoE4 has greater affinity than does apoE3, may be a mechanism whereby the apoE {var_epsilon}4 allele contributes to neurodegenerative risk.
Date: December 1, 2003
Creator: Yang, Lin; Liu, Yanzhu; Forte, Trudy M.; Chisholm, Jeffrey W.; Parks, John S. & Shachter, Neil S.
Partner: UNT Libraries Government Documents Department

Mechanism of lipid lowering in mice expressing human apolipoprotein A5

Description: Recently, we reported that apoAV plays key role in triglycerides lowering. Here, we attempted to determine the mechanism underlying this hypotriglyceridemic effect. We showed that triglyceride turnover is faster in hAPOA5 transgenic compared to wild type mice. Moreover, both apoB and apoCIII are decreased and LPL activity is increased in postheparin plasma of hAPOA5 transgenic mice. These data suggest a decrease in size and number of VLDL. To further investigate the mechanism of hAPOA5 in hyperlipidemic background, we intercrossed hAPOA5 and hAPOC3 transgenic mice. The effect resulted in a marked decreased of VLDL triglyceride, cholesterol, apolipoproteins B and CIII. In postprandial state, the triglyceride response is abolished in hAPOA5 transgenic mice. We demonstrated that in response to the fat load in hAPOA5XhAPOC3 mice, apoAV shifted from HDL to VLDL, probably to limit the elevation of triglycerides. In vitro, apoAV activates lipoprotein lipase. However, apoAV does not interact with LPL but interacts physically with apoCIII. This interaction does not seem to displace apoCIII from VLDL but may induce conformational change in apoCIII and consequently change in its function leading the activation of lipoprotein lipase.
Date: January 15, 2004
Creator: Fruchart-Najib, Jamila; Bauge, Eric; Niculescu, Loredan-Stefan; Pham, Tatiana; Thomas, Benoit; Rommens, Corinne et al.
Partner: UNT Libraries Government Documents Department